key: cord-283301-adjjkqt2
authors: Awolade, Paul; Cele, Nosipho; Kerru, Nagaraju; Gummidi, Lalitha; Oluwakemi, Ebenezer; Singh, Parvesh
title: Therapeutic significance of β-glucuronidase activity and its inhibitors: A review
date: 2020-02-01
journal: Eur J Med Chem
DOI: 10.1016/j.ejmech.2019.111921
sha: 
doc_id: 283301
cord_uid: adjjkqt2

The emergence of disease and dearth of effective pharmacological agents on most therapeutic fronts, constitutes a major threat to global public health and man’s existence. Consequently, this has created an exigency in the search for new drugs with improved clinical utility or means of potentiating available ones. To this end, accumulating empirical evidence supports molecular target therapy as a plausible egress and, β-glucuronidase (βGLU) – a lysosomal acid hydrolase responsible for the catalytic deconjugation of β-d-glucuronides has emerged as a viable molecular target for several therapeutic applications. The enzyme’s activity level in body fluids is also deemed a potential biomarker for the diagnosis of some pathological conditions. Moreover, due to its role in colon carcinogenesis and certain drug-induced dose-limiting toxicities, the development of potent inhibitors of βGLU in human intestinal microbiota has aroused increased attention over the years. Nevertheless, although our literature survey revealed both natural products and synthetic scaffolds as potential inhibitors of the enzyme, only few of these have found clinical utility, albeit with moderate to poor pharmacokinetic profile. Hence, in this review we present a compendium of exploits in the present millennium directed towards the inhibition of βGLU. The aim is to proffer a platform on which new scaffolds can be modelled for improved βGLU inhibitory potency and the development of new therapeutic agents in consequential.

The world today is embattled with an increasing paucity of effective therapeutic agents or regimen for many pathological conditions, as well as the menace of drug resistance and adverse effects of available drugs [1] . As a result, smooth and efficient clinical practice is rigidly stymied, while global public health, social security and man's life expectancy are seriously threatened and trickles to a disquieting edge [2] . Likewise, the burdens of developing new therapeutic agents to ameliorate the status quo has become heavier on all stakeholders in drug research.

In this regard, molecular target therapy is fast becoming a spearhead in the search for new drugs with improved therapeutic effects. Amongst many targets explored, glycosyl hydrolases (GHs) are notable due to their role in many important biological processes. Their principal function is to catalytically cleave the glycosidic bond of glycans thereby eliciting different physiological responses. Therefore, inhibitors of this class of enzymes have enjoyed intense research and development owing to their potentials as antiviral, anticancer and antidiabetic agents as well as therapeutic agents for some genetic disorders [3e5] .

GHs have been classified using different indices [6] . For example, based on substrate specificity, those cleaving O-or Sglycosides are grouped into EC 3.2.1 class, while hydrolases of Nglycosides belong to EC 3.2.2 class. Advancements in genomic science have also enabled classification into GH families based on their amino acid sequence similarities [7] . This system further groups GH families into clans, given the improved conservation of protein fold than the sequence [8] . Accordingly, the reviewed enzyme, b-glucuronidase (EC 3.2.1.31) is classified into GH family 1, 2, 30, 79, 154 and GH-A clan. b-glucuronidase (bGLU) is mainly a lysosomal hydrolase widely distributed in mammalian tissues, body fluids and microbiota; but significantly retained in the endoplasmic reticulum [9] . The enzyme is also found in plants, fishes, insects and molluscs.

Specifically, human bGLU belongs to GH family 2. It is a 332 kDa ellipsoidal and homotetrameric glycoprotein with each 75e78 kDa monomer containing 651 amino acid residues (Fig. 1a) . The monomer precursor is synthesized initially on membrane-bound ribosomes and suffers C-terminal proteolytic processing of 18 amino acid propeptide en route or after their transport to the lysosomes [10e13]. X-ray crystallography of the protein structure reveals a dihedral symmetry for the tetramer with two identical monomers in the asymmetric unit arising from disulphide-linked dimers. Each monomer contains three structural domains (Fig. 1b) . The first domain has a barrel-like structure with a jelly roll motif; the second domain exhibits a geometry identical to immunoglobulin constant domains; while the third C-terminal domain forms a TIM barrel motif (b/a) 8 [14] . The active sites of human bGLU ( Fig. 1c) viz. catalytic acid Glu451 (proton donor), catalytic nucleophile Glu540 (carbonium ion stabilizer), Asp207 (plausible role as Glu540) and Tyr504 (unclear catalytic role), are all housed in the third domain and in each of the four catalytic centres of the tetramer [14, 15] . Moreover, the enzyme has an optimal activity at acidic pH~4.5, corresponding to its lysosomal environment and thermally stable up to 70 C [10] ; although hyperthermophilic variants exists in other media [16] . bGLU is encoded by the GUS gene. A deficiency arising from mutations in this encoding gene is associated with atherosclerosis [17] and lysosomal storage disease e Sly syndrome or mucopolysaccharidosis type VII [18] .

On the other hand, bacterial bGLU, which is expressed in human gut microbiota and most strains of Escherichia coli shows 45% sequence similarity with human bGLU. Also, it has a bacterial loop containing 17-amino acid residues not found in human bGLU, an optimal activity at neutral pH and active site catalytic residues as Glu413 (catalytic acid) and Glu504 (catalytic nucleophile) [19] .

Consistent with the activities of lysosomal GHs, bGLU deconjugates b-D-glucuronides to their corresponding aglycone and b-Dglucuronic acid via an S N 2 reaction and "configuration retaining" mechanism ( Fig. 2) . The catalytic mechanism is conceived to proceed as follows; catalytic glutamic acid residue Glu451 (or Glu413 in bacterial ortholog) protonates exocyclic glycosidic oxygen of glucuronide (1) hence releasing the aglycone via a putative oxocarbenium ion-like transition state (2) . 'Back-side' nucleophilic attack by glutamate ion Glu540 (or Glu504 in bacterial ortholog) e the catalytic nucleophile, stabilizes the transition state and results in glucuronyl ester intermediate (3) with an inverted configuration. Finally, hydrolysis through an inverting attack of water molecule on the anomeric centre releases Glu540 to form b-D-glucuronic acid (4) and a concurrent overall retention of substrate configuration [14,15,19e21 ].

Due to the increased expression of bGLU in necrotic areas and other body fluids of patients with different forms of cancer such as breast [22] , cervical [23] , colon [24] , lung [25] , renal carcinoma and leukaemia [26] , compared to healthy controls, the enzyme is proffered as a reliable biomarker for tumour diagnosis and clinical therapy assessment [27] . This overexpression is also a potential diagnostic tool for other disease states such as urinary tract infection [28] , HIV [29] , diabetes [30] , neuropathy [31] and rheumatoid arthritis [32] . In this vein, empirical data update on clinical applications of bGLU for these and other disorders is provided on BRENDA database [33] .

bGLU activity is also harnessed in prodrug monotherapy. In normal body systems, drugs and other xenobiotics are detoxified via glucuronidation, an S N 2 conjugation reaction and important pathway in phase II metabolism, catalysed by UDP- glucuronosyltransferases (UGTs). The resulting usually less active glucuronide metabolite is readily excreted by renal clearance due to increased polarity or sometimes via biliary clearance [34] . However, elevated levels of bGLU activity reverts this process through deglucuronidation, which hydrolyses the phase II metabolites to their active forms (Fig. 2) . Hence, glycosidation of a drug to give its glucuronide enhances selective release of the active form at necrotic sites via bGLU-mediated deglucuronidation thus improving the drug's therapeutic potential [35] .

bGLU's postulated ability to increase T Regulator cells (TReg) is also applied in low-dose immunotherapy (LDI) for managing allergic diseases [36, 37] , Lyme disease [38] and other chronic conditions. While it's hydrolytic activity on glucuronide conjugates is harnessed in forensic analysis [39] and assessment of microbial water quality [40] .

Nonetheless, enterobacterial bGLU deconjugation of drug and xenobiotic glucuronides in the gastrointestinal (GI) tract has been implicated in colonic genotoxicity [41] and certain drug-induceddose-limiting toxicities. For example, the GI toxicity of anticancer drug Irinotecan (CPT-11) [42] , enteropathy of non-steroidal antiinflammatory drug (NSAID) Diclofenac [43] , tissue inflammation and hepatoxicity.

Furthermore, bGLU is deemed a potential molecular target for;

(1) anticancer chemotherapy considering its role in tumour growth and metastasis [44, 45] . (2) Neonatal jaundice treatment due to its high expression in breast milk and role in enterohepatic bilirubin circulation (hyperbilirubinemia) [46, 47] . (3) Diabetes mellitus management consequent to the positive correlations between the disease state and enzyme activity level as well as associated periodontitis [48, 49] . (4) Anti-inflammatory agents development owing to its pro-inflammatory role following significant release from degranulated mast cells and neutrophils [50, 51] . Expectedly, inhibition of bGLU markedly alleviated these pathological conditions and their adverse effects hence improving regimens' efficacy.

Based on the foregoing, we extrapolate that the development of potent, specific and non-cytotoxic inhibitors of bGLU is imperative to improving the clinical efficacy of therapeutic agents and effective disease management while bearing in mind the physiological significance of both human and bacterial orthologs of the glycosyl hydrolase. However, the fate of these inhibitors rests on their inhibition constants (K i ), since GHs are generally characterized by high rate enhancement (k cat /k uncat > 10 17 -fold). Also, accumulating evidence suggests the dependence of inhibitory potency on the ability to mimic the highly enzyme-stabilized transition state of an enzymatic reaction (K i z 10 À20 M) en route to catalytic product [21, 52, 53] . Considering the proven and encouraging potentials of enzyme inhibition and molecular target therapy in drug development, and in continuation of our exploits and expositions thereon [54e58], herein we present a comprehensive review of research undertakings in the present millennium (2000e2019) directed towards the development of potent inhibitors of bGLU that are either natural products or synthetic scaffolds. Apropos, before discussing the different inhibitors, this article will first highlight the potentials of bGLU activity as a diagnostic tool within the defined period.

However, therapeutic application in prodrug monotherapy and enzyme replacement therapy (ERT) will not be covered as these have been excellently treated in other reviews [59e61].

Hitherto, our search of extant literature revealed that, although there exists a plethora of scholarly research on potential inhibitors of bGLU activity, no review article is exclusively devoted to the subject matter. The aim of this review is therefore to bring to light those bioactive frameworks bestowed with promising bGLU inhibitory potency. Our principal goal is to intimate the reader on key structural features of reviewed molecules crucial to their inhibitory activities and toxicity profiles, while establishing the comprehensive relationships existing between reported molecules.

The availability of safe, easy to use, consistent, less-invasive and cost-effective tool for early diagnosis of diseases or appraisal of therapeutic interventions is of uttermost importance in clinical medicine. Since most disease states are accompanied by elevated levels of specific enzymes in the diseased milieu (tissues, plasma and other body fluids), quantification of enzymes' activity levels is seen as a reliable biomarker of either disease status, severity, effects, susceptibility or exposure [62e64]. Moreover, the substrate specificity and selective quantification of enzymes in the presence of other biomolecules makes them a tool of choice thereto [65] . A review of bGLU activity as a biomarker of some physiologically important conditions is hereby presented together with a concise summary in Table 1 .

Periodontal disease is a group of inflammatory disorders triggered by host's immune response to the actions of virulent subgingival plaque bacteria biofilms which activates the release of polymorphonuclear leukocytes and macrophages into the gingival crevice. This leads to gingivitis e an inflammation of periodontal tissues and distortion of periodontal histology that is reversible with improved oral hygiene; or, subsequent tissue destruction, alveolar bone resorption and tooth loss if left unattended i.e. periodontitis [66, 67] . Therefore, a reliable tool to ascertain disease status, severity, risk or efficacy of administered therapy is highly desirous to clinicians.

However, conventional diagnosis involving the measurement of periodontal clinical parameters such as probing depth (PD), clinical attachment level (CAL), gingival index (Gg-I), bleeding on probing (BOP) and alveolar bone loss (ABL), suffers from intrinsic limitations. They only define the status of patient's periodontium at the time of examination and not periodontal disease susceptibility or risk [68] . Thus, since periodontitis is characterized by an influx of inflammatory mediators and corresponding enzymes into the gingival sulcus, the quantification of neutrophil-derived bGLU activity in gingival crevicular fluid (GCF) or GCF's outflow into the oral cavity and subsequent less invasive estimation of bGLU activity in saliva is considered a reliable biomarker for periodontal disease diagnosis.

To this effect, the relationship between salivary bGLU activity and periodontal clinical parameters (PD, CAL and Gg-I) was investigated in subjects with different stages of periodontal disease [69] . The mean PD and Gg-I, number of sites with PD ! 5 mm and total number of white blood cells, blood neutrophils and monocytes all showed highly significant correlations with enzyme's activity, while CAL had a weaker correlation. Using logistic regression modelling and the presence of at least 1 or 4 sites with PD ! 5 mm as disease criterion, bGLU activity showed promising potentials as a tool for periodontal disease screening or assessment of therapeutic intervention. The study also observed smoking status to be insignificant on enzyme activity. However, in a similar study, only PD, CAL and lymphocyte count exhibited positive correlation with salivary enzyme activity while no significant relationship was observed for Gg-I [70] . Recently, subjects with chronic generalized periodontitis were also found to have significant increase in bGLU activity (8-fold) compared to normal ones. Although, a reduction in enzyme activity persisted in smokers regardless of periodontal status [71] . Table 1 Reported potential applications of bGLU activity as a biomarker. [112] 284** AChE activity Chronic exposure (W) Acute exposure (N) Elevated in 16.5% and 60% of subjects with chronic and acute exposure respectively [113] NS: Not specified; W: weak correlation; N: No correlation; PI: Russell periodontal index; *threshold to distinguish culture positive from culture negative BALF; ** acute exposure (5), chronic exposure (230) .

The efficacy of therapeutic intervention using amoxicillin and metronidazole to downregulate amplified neutrophil activity was studied in 14 patients with aggressive periodontitis [72] . Treatment involved seven consecutive days of antibiotic administration with concurrent scaling, root planning and surgical therapy and a total of 36 months posttreatment evaluation period. Subsequently, a markedly downregulated neutrophil activity with approximately 50% inhibition of bGLU activity in GCF was observed. Periodontal health was also restored and maintained during posttreatment evaluations. In another study, bGLU activity was posited as a better biomarker compared to alkaline phosphatase for evaluating the response to non-surgical periodontal therapy in patients with different stages of periodontal disease [73] . Taken together, these results articulate the potentials of bGLU activity as an indication of PD, tissue inflammation or destruction as well as a biomarker of neutrophil influx, disease risk, susceptibility, status, or severity for timely diagnosis of the inflammatory disorder. However, administration of doxycycline hyclate in 16 subjects with aggressive periodontitis was inefficient on salivary bGLU activity [74] . Surprisingly, an increase in enzyme activity was found even after 2 months of treatment in 12 patients and a decrease in 4. Although, the authors concluded bGLU concentration only facilitated the detection of periodontal inflammation and not worthy as biomarker of susceptibility, their contrasting result is linkable to periodontal pretreatment of subjects prior to examination and short treatment time using doxycycline.

Empirical evidence affirms the role of inflammatory mediators and signalling pathways in the pathogenesis of insulin resistance and b-cell dysfunction in diabetes mellitus [75e77]. In parallel, these inflammatory mediators e.g. cytokines and MMPs are also produced in periodontal tissues [78e82]; hence, leading to compromised glycaemic control after accessing systemic circulation. The susceptibility to periodontitis is therefore heightened in persons with diabetes or a history of the hormonal imbalance and vice versa. Putatively, an effective therapy for one affords an improved management of the other [83e85].

Accordingly, bGLU activity was found to be significantly elevated in the saliva of patients with chronic periodontitis and diabetes compared to nondiabetic ones [86] . A significant correlation to bGLU activity was observed for PD and CAL and not Gg-I in nondiabetic subjects with periodontitis; whereas, these periodontal parameters were similar in diabetics. The increased disease burden was also established when bGLU activity was measured in the sera of patients with both diabetes and periodontitis [87] . Compared to controls, enzyme activity was 9-fold higher in diabetic subjects with periodontitis and only 2-fold higher in diabetic subjects without periodontitis. This difference was attributed to damaged lysosomal membrane and consequent enzyme leakage into the cytosol.

The quantification of bGLU activity in neutrophil leukocytes exposed to bacteria stimuli has shown that diabetic patients with chronic periodontitis have strikingly higher enzyme activity compared to nondiabetics burdened with periodontitis and healthy subjects [88] . Using discriminant analysis, the study established that bGLU activity has a diagnostic potential with great accuracy in distinguishing healthy subjects from diseased ones. bGLU activity stimulated by nonopsonized Staphylococcus aureus showed strongest correlation with the intensity of periodontal parameters compared to opsonized zymosan and prodigiosan, while the highest enzyme activity was stimulated by opsonized prodigiosan.

The strength of association between salivary bGLU activity, periodontitis and type 2 diabetes mellitus has been examined in dentate patients with different stages of periodontal disease, diabetic patients and edentulous patients [89] . In all subjects, diabetic status contributed significantly to bGLU activity, while periodontal status had greater influence on enzyme activity. Higher enzyme activity was also found in nondiabetic dentate patients with periodontitis compared to edentulous controls. Overall, compared to IL-1b, bGLU activity level was more reliable as biomarker of disease severity for periodontitis than it was for the presence of diabetes. In a predating study [90] , GCF bGLU activity also correlated strongly with PD, CAL and BOP regardless of diabetic status. Lower enzyme activity was seen in diabetic subjects compared to those with periodontitis. The results suggested a lower release of bGLU in response to systemic inflammation (diabetes) due to reduced deficiency in neutrophil activity, in contrast to amplified activity in response to local inflammation (periodontitis).

Despite landmark developments in oncology, the high rates of morbidity and mortality and increased medical costs associated with all forms of cancer coupled with patients' psychological trauma on disease diagnosis has remained a major threat to global public health. The successful disease management and sustained wellbeing of affected individuals is however subject to early disease diagnosis and constant evaluation of administered therapy. This in turn relies on efficient tumour markers to ascertain disease risk or status i.e. early stage or metastatic cancer [91] . Although a vast number of biomarkers have been identified for cancer diagnosis, only few have gained clinical approval due to inconsistencies and false positives in their utility [92] . A successful biomarker is that which will not only be specific and selective but will also predict treatment response, while differentiating lethargic and aggressive tumours.

The aetiology of cancer is known to be closely associated with inflammatory pathways and oxidative stress, which jointly create microenvironments favouring neoplasia [93, 94] . Hence, in the tumour milieu, an increase in extracellular activity of lysosomal exoglycosidases responsible for the catalytic degradation of glycoconjugates occur, due to malignancy-mediated cell-death and/or lysosomal damage. In this vein, available practical data supports the overexpression of bGLU in extracellular fluids and tissues around tumour sites as a prime factor in cancer aetiology [24] ; thus, suggesting the enzyme's viability as cancer biomarker [95] .

For example, bGLU activity was 2-folds higher in the blood samples of 21 patients with colorectal adenocarcinoma compared to healthy controls [96] . Based on cell maturity and clinical grading, enzyme activity was highest in subjects with low or moderately differentiated cells and in subjects with tumours infiltrating surrounding tissues and organs or visceral peritoneum respectively.

Estimation of serum bGLU activity in this case proved to be 80% sensitive and 82% specific in distinguishing diseased and healthy subjects. Likewise, enzyme activity was elevated by 6-fold in the peritoneal fluids of patients with ovarian or endometrial cancer compared to women with infertility used as controls [97] . The stronger correlation between cancer stage and bGLU activity, b-galactosidase and a-mannosidase, reiterated the improved clinical viability of bGLU activity as biomarker of gynaecologic tumour status and severity. However, bGLU activity was equally elevated in the peritoneal fluid of patients with pelvic inflammation hence compromising its application for differential diagnosis of gynaecologic cancer and pelvic inflammatory disease.

Bacterial peritonitis is a life-threatening inflammation of the peritoneum e the tissues lining of the inner abdominal walls. As with other inflammatory conditions, cellular damage and polymorphonuclear leukocytes activity increases the level of lysosomal enzymes in the extracellular space via enzyme leakage through the cellular membrane. Thus, quantification of these enzymes provides a potential diagnostic platform.

In fact, bGLU activity in the peritoneal fluid of patients with culture positive bacterial peritonitis was 9 and 33-fold greater compared to that of patients with acute mesenteric lymphadenitis and controls respectively [98] . Peritoneal fluid-bGLU activity measurement in this case holds greater clinical potential compared to b-galactosidase and a-mannosidase for early disease diagnosis and evaluating patient's response to treatment.

Similarly, considering the current global prevalence of antimicrobial resistance, timely diagnosis of bacteria-mediated meningeal inflammation (bacterial meningitis) is crucial to the outcome of therapeutic intervention. It has been shown that bGLU activity is elevated in cell-free cerebrospinal fluid (CSF) of bacterial meningitis patients early in the disease pathogenesis, even when traditional laboratory parameters such as number of CSF cells, CSF-blood glucose ratio and protein concentration indicated normal status [99] . CSF-bGLU as biomarker was superior to these traditional parameters for early and sensitive prediction of patient's response to antibiotic treatment.

CSF-bGLU activity in neonates and infants has also been studied as biomarker for differential diagnosis of sterile CSF pleocytosis due to urinary tract infection (UTI) or meningitis [100] . Median bGLU activity in CSF of patients showed significant difference without overlapping in each disease state i.e., bacterial meningitis (168), viral meningitis (26.5) and UTI with sterile CSF pleocytosis (44.1); median enzyme activity was lowest (19.1) in febrile subjects without CSF pleocytosis used as controls. This proffers an unambiguous diagnosis of each condition with 100% sensitivity and specificity. In contrast, broad overlapping was found with classic CSF laboratory parameters viz., CSF cell number, neutrophil number, protein concentration and CSF-blood glucose ratio.

Recently, a case-control study concluded that bGLU activity in bronchoalveolar lavage fluid (BALF) is clinically useful as biomarker of bacterial lung infection [101] . In BALF samples from 92 children, enzyme activity was significantly higher in patients with positive BALF bacterial culture (Cþ) compared to those with culture negative BALF (Ce). 43 nmol 4-methylumbelliferone (4MU)/ml/h was identified as optimum activity value, which allowed differential sample screening (i.e. Cþ from Ce) with 84.8% sensitivity and 78.3% specificity. Moreover, receiver operating characteristics (ROC) curve analysis established the superiority and higher prognostic value of bGLU activity for bacterial lung infection compared to % polymorphonuclear cell count, human leukocyte elastase, IL-8 and TNFa.

Repeated exposure to organophosphorus compounds (OP) in pesticides is responsible for the lethal poisonings seen in agricultural and veterinary workers; particularly, those in developing countries. OP exerts their fatal effects by inhibiting acetylcholinesterase (AChE) in nervous tissues, leading to muscarinic and nicotinic effects with central nervous disturbance [102] . On the other hand, bGLU is retained in liver microsomal endoplasmic reticulum by forming non-covalent binding complexes with its accessory 64 kda glycoprotein, egasyn, a carboxylesterase isozyme [103, 104] . Liver intake of OP however cleaves microsomal bGLUegasyn complex thus elevating the level of bGLU in plasma to consequently making it an alternative biomarker for OP pesticide poisoning diagnosis [105e107].

A cross-sectional study of pest control workers [108] and plastic greenhouse workers [109] , established that plasma bGLU activity was more sensitive as biomarker of OP poisoning compared to butyrylcholinesterase (BuChE) and acid phosphatase (AcP). The enzyme's activity was higher in subjects with increased exposure than those with low exposure and controls. bGLU activity correlated significantly with BuChE and AcP activities. Likewise, in a case-control study, serum bGLU activity was significantly increased in patients with severe poisoning compared to mildly affected patients and controls [110] . The difference in enzyme activity between the latter groups was insignificant. Nonetheless, mildly exposed subjects have been reported to show elevated bGLU activity than severely exposed and control subjects, which suggests the enzyme's utility for diagnosing lowlevels of OP exposure. For instance, a study [111] , observed the order of serum bGLU activity based on poisoning severity as mild > severe > moderate poisoning, while the order after 12 and 24 h of admission was mild > severe z moderate poisoning; strong correlation also persisted between serum bGLU and BuChE activities. Similar results were also obtained in a recent cross-sectional study [112] . Therein, moderately exposed subjects showed higher enzyme activity than the highly exposed, while non-significant statistical difference persisted between control and highly exposed groups. Notably, bGLU activity correlated well with diabetes propensity, lipid profile, liver function and BuChE but not AChE.

In another cross-sectional study, significant difference in plasma bGLU activity only exists between controls and subjects with chronic exposure (1e45 years) to OP [113] . Activity level was similar in controls and patients with acute poisoning; although 3 out of the 5 examined acutely poisoned patients showed increased level of plasma bGLU activity. However, a case report of an acute OP self-poisoned patient reached contrasting conclusion [114] . The opposing result can be linked to sample size and limited data on patient's medical history. Moreover, the reduced susceptibility of bGLU-egasyn complex to OP in humans is another primal factor [115] . In contrast to murine bGLU-egasyn interaction, binding in humans is independent of the C-terminal 18 amino acids propeptide in bGLU and esterase active site of egasyn. Rather, it involves the 51 amino residues in bGLU internal segment i.e. residues 228 to 279.

The progress and momentous achievements in drug discovery cannot be isolated from the wealth of chemical entities i.e. natural products, gifted to man by nature. Many successful molecular candidates of pharmaceutical drug discovery programmes are indebted to the presence of natural product-derived/inspired fragments in their scaffolds [116] . Therefore, the chemical space of natural products (both plants and microbes) has continued to be a much-researched depository for therapeutically significant molecules. In this section, we review some application of natural products including isolated compounds, whole plant extracts and natural product-inspired molecules as potent inhibitors of bGLU. The structures of selected inhibitors (IC 50 5 mM) are presented in Table 2 at the end of the section.

The clarification [117, 118] that D-saccharic acid-1,4-lactone (5, Fig. 3 ) (saccharolactone or D-glucaric acid-1,4-lactone) is the active and non-toxic bGLU inhibitor responsible for the strong inhibitory potency found with saccharate solutions has precipitated an increased interest in the compound. Despite the poor stability at physiological pH, D-saccharic acid-1,4-lactone (D-SAL) has been explored extensively for its therapeutic significance. Although an IC 50 value of 3.6 mM was reported in the pioneering work [117] , a value ca. 40 mM is recurrent in literature. Nevertheless, at a concentration of 1 mM, D-SAL completely inhibited the hydrolytic action of human liver-derived bGLU on quercetin glucuronides [119] , while over 90% inhibition of bGLU in breast milk was recorded at 10 mM [120] . Also, using urine samples of male Sprague-Dawley rats, D-SAL was identified via metabolomics strategy as one of the therapeutic constituents in LiuWeiDiHuang pills, a famous traditional Chinese prescription for cancer treatment and prevention [121] . Whereas, in a 6-days cumulative study, intraperitoneal pre or cotreatment of female Wistar rats with 3 mg/ml, 10 mg/ml or 10 mg/0.5 ml D-SAL reduced the severity of CPT-11 induced smallintestine mucosal damage assessed by the number of apoptotic cells or mitotic figures, compared to CPT-11 treated controls [122] . Damage reduction was independent of treatment schedule.

The synthetic and natural precursors of D-SAL i.e., 2,5-di-Oacetyl-D-glucaro-1,4:6,3-dilactone (D-SDL, 6, Fig. 3 ) and D-glucurono-g-lactone (D-GL, 7) respectively, have also potently inhibited bGLU activity in male Fischer rats thereby providing chemopreventive effects against azoxymethane-induced colon carcinogenesis [123] . Diet supplementation with 0.5 or 2% D-SDL for 5 weeks significantly reduced aberrant crypt foci formation (i.e. preneoplastic lesions) by over 48.6 and 55.3% respectively, compared Table 2 Most active natural product-derived bGLU inhibitors with IC 50 to azoxymethane-controls. D-GL did not afford any significant reduction in colonic tumour incidence during this initiation phase. In addition, 32 weeks treatment with D-SDL or high dose (2%) of D-GL, after 3-weeks subcutaneous injections of 15 mg/kg azoxymethane, provided over 70% inhibition of colon carcinogenesis during the post-initiation phase. It was suggested that D-SDL is a blocking agent which may inhibit pre-neoplasia during the initiation phase, while D-GL is only active during post-initiation of colon carcinogenesis. The increased hydrolytic stability of D-SDL to D-SAL in vivo might also be responsible for these observed phenomena [124] .

In another study, D-SAL was more therapeutically efficient compared to its natural precursor (D-GL), for reducing epidermal hyperplasia (lethargic tumour promoter) and inflammation in 7,12dimethylbenz(a)anthracene (DMBA)-induced complete skin carcinogenesis of SENCAR mice [125] . Pre and cotreatment of murine models with D-SAL for 4-weeks (twice weekly) by topical administration (0.5e4 mg) or dietary treatment (0.5 and 1%), both significantly reduced epidermal hyperplasia and inflammation by up to 57% of DMBA-controls in a dose-dependent manner. D-SAL also inhibited the initiation of carcinogenesis by reducing DMBAinduced oxidative DNA damage (C-8 hydroxylation of guanine) and mutations in codon 61 of Ha-ras gene, by up to 78%. In contrast, D-GL only inhibited epidermal hyperplasia with topical treatment and inflammation by dietary treatment (5% in diet); albeit with inferior potency compared to D-SAL.

Interestingly, another lactone-based bGLU inhibitor (8, Fig. 3 ), exhibited 8-fold superior potency compared to D-SAL [126] . Isolated from the ethyl acetate extracts of Aspergillus terreus, an endophytic fungus initially isolated from marine alga Laurencia ceylanica, butyrolactone (8) with IC 50 ¼ 6.2 mM possesses 16-fold stronger potency than its prenylated isomer (9) . bGLU in breast milk is believed to be involved in neonatal jaundice and hyperbilirubinemia, by increasing serum bilirubin levels via enterohepatic bilirubin circulation in breastfed newborns, in contrast to those fed with infant formulas [47] . Therefore, the suppression of bGLU-mediated deglucuronidation has been proffered as a practicable regimen. L-aspartic acid (10, Fig. 3 ) in casein hydrolysate formulas was identified as the active bGLU inhibitor responsible for the lower levels of neonatal jaundice observed in newborns receiving such formulae [127] . At 100 mM, the natural amino acid showed~86% inhibition of bGLU that is, 100-fold more potent than D-isomer. Moreover, in a randomized and double-blind clinical trial involving 64-newborns, supplementing breastfeeding in the first week of life with 6 doses of L-aspartic acid (180 mg/5 ml of water/day) was more potent against bGLU activity than higher concentrations of enzymatically hydrolysed casein (infant formula containing the inhibitor) or whey/casein (routine formula lacking the inhibitor) [128] . L-aspartic acid supplementation at minimal aliquot concentration significantly lowered transcutaneous bilirubin levels (25% lower than control), leading to higher faecal bilirubin excretion and reducing neonatal jaundice, with no adverse effects.

Flavonoids are evidently an indispensable class of natural products due to their ubiquity in the vegetal domain and their therapeutic significance. Found in a variety of plant parts (leaves, flowers, stems, nuts, seeds etc.), they perform important functions especially plant's growth and protection against pathogenic invasion. This intrinsic property encourages their utility as a major constituent of different local medicinal formulations and diets, while their acceptable toxicity profile and physiologic tolerance further presents them as druggable subjects. Flavonoids are typified by the C6eC3eC6 ring system that is, their basic structural skeleton consists of two benzene rings (A and B) linked by heterocyclic pyran ring C (Fig. 4) . Variations on the chromane core (ring A and C) and attachment position of ring B due to biosynthetic origins lead to classification into flavones, flavanols, flavanones, flavonols, isoflavones, neoflavonoids, anthocyanins and chalcones [129] . This ring system and the presence of hydroxyl units has availed flavonoids with different pharmacological properties such as antidiabetic, antioxidant, antimicrobial, antiplasmodial, antiproliferative and particularly enzyme inhibition [130, 131] .

Luteolin (11, Fig. 4 ), a dietary 3 0 , 4', 5, 7-tetrahydroxyflavone has been reported as a viable chemopreventive and anticarcinogenic agent plausibly by inhibiting bacterial bGLU-mediated enterohepatic circulation of colonic carcinogens [132] . The 30-weeks cumulative study using male Wister rats showed that pre or cotreatment with luteolin by intragastric gavage per os (p.o.) at 0.1, 0.2 or 0.3 mg/kg body weight/day, significantly reduced bacterial bGLU activity thereby suppressing 1,2-dimethylhydrazine (DMH)induced colon adenocarcinomas in a dose-dependent manner compared to DMH-controls. Although 0.2 and 0.3 mg kg À1 day À1 produced similar result. Luteolin supplementation also reduced tumour size from 2 cm to 0.25 and 0.50 cm during the initiation and post-initiation stages respectively, as well up to 90% reduction in tumour incidence. In addition, luteolin (11), its 7-O-glycoside (12), apigenin-7-O-glycoside (13) and catechin (14) were identified as key constituents in the leaf extracts of Pistacia terebinthus responsible for the high E. coli bGLU inhibitory potency [133] . At the highest test concentration (8.2 mg/ml), the leaf extracts exhibited 97.2% inhibition of bGLU activity, corresponding to an IC 50 value of 2.11 mg/ml.

In another exploit, 32 natural flavonoids were evaluated for their inhibitory strengths against E. coli bGLU [134] . It was established that luteolin (11) , similar flavones e baicalein (15), scutellarein (16) and its glucuronidated analogue scutellarin (17) , as well as dietary and ubiquitously occurring flavonol e quercetin (18) , are superior inhibitors (IC 50 ¼ 5.76e29.64 mM) compared to reference inhibitor D-SAL (IC 50 ¼ 36.07 mM); isoflavones and dihydroflavones displayed weaker inhibition. Overall, luteolin (11) and scutellarein (16) emerged the most potent flavone-based E. coli bGLU inhibitors with IC 50 ¼ 8.68 and 5.76 mM respectively. SAR analysis (Fig. 4 ) revealed the importance of 5,6,7-trihydroxy (pyrogallol) unit to bacterial bGLU inhibition, as unsubstitution or replacement of a hydroxy unit with methoxy or glycosyl unit led to significant loss of inhibitory activity. O-methylation at positions-6, 7 and 4 0 and installing OH unit at position-3 was also detrimental to potency, whereas the presence of hydroxy unit at C-4' favoured potency. In addition, molecular docking studies of luteolin and scutellarein in the active site of E. coli bGLU showed hydrogen bonding (H-b) interactions of phenolic OH units at C-5 and C-7 with catalytic acid Glu413 and Arg562 as well as hydrophobic contacts with Ser360 and Leu361 in the bacterial loop.

Furthermore, subjecting the methanolic extracts of edible flower and pedicel of aquatic rhizomatous herb e Nymphaea pubescens (water lily) to bioactivity-guided fractionation established the superior (3-fold) bacterial bGLU inhibitory potency of crude flower extracts compared to pedicel extracts and silymarin e a marketed natural bGLU inhibitor [135] . Kaempferol (19) was subsequently identified as one of the active metabolites with promising activity; IC 50 ¼ 36.47 mM and 76-fold superior to silymarin. Interestingly, the lower activity of kaempferol (19) compared to flavonoids (11, 15e18) affirmed the SAR results described earlier.

Amidst 21 constituents found in commonly used Chinese herbal medicines, two prenylflavonoids, Sanggenon C (20, Fig. 5 ) and Kuwanon G (21) emerged as the most potent broad-spectrum inhibitors (>70% inhibition; IC 50 ¼ 12.5 and 7.4 mM respectively) of whole human gut bacterial bGLU (consisting of eight bacterial isolates), compared to reference compound amoxapine (7.3% inhibition) [136] . Compound 20 exhibited improved potency against recombinant bGLU from E. coli (EcoGUS) and S. pasteuri (SpasGUS), IC 50 ¼ 0.40 and 0.33 mM respectively, compared to compound 21 (IC 50 ¼ 1.6 and 0.98 mM respectively). However, compound 21 was a stronger inhibitor of bGLU from three representative bacterial isolates (E. coli, E. fergusonni and S. pasteuri) compared to compound 20. Additionally, molecular docking studies of 20 and 21 in the binding pockets of EcoGUS and SpasGUS revealed both flavonoids bind (via their hydroxy groups) in the allosteric site and not the active site of the recombinant enzymes. The higher number of H-b interactions formed by compound 21 supported the overall increased potency compared to compound 20.

The realization that pro-inflammatory mediators such as bGLU, released from degranulated mast cells or neutrophils, play significant roles in inflammatory disorders has sparked increased interest in the search for potent inhibitors of such processes as antiinflammatory drug candidates. In this regard, dihydroxychalcones 22 and 23 (Fig. 6 ) isolated from the roots of Hypericum geminiflorum holds therapeutic promise [137] . Compounds 22 and 23 potently inhibited the release of bGLU from degranulated rat neutrophils with IC 50 values of 5.80 and 6.60 mM respectively, better than the reference inhibitor trifluoperazine. However, only compound 23 showed moderate activity against the enzyme's release from degranulated rat peritoneal mast cells with IC 50 ¼ 70.0 mM.

Conversely, an isomer of luteolin e norartocarpetin 24 (Fig. 6) and flavonoid-like mornigrol D 25 (Fig. 6) , both isolated from the barks of Morus nigra (black mulberry), were moderately potent bGLU inhibitors [138] . At 100 mM, compounds 24 and 25 showed 67.7% and 65.9% inhibition respectively against the release of bGLU from activated rat neutrophils. However, chiral flavonoid alkaloids isolated from the ethanolic extracts of Scutellaria moniliorrhiza and subsequently separated using chiral HPLC, e scumonilines 26e29 (Fig. 6) , are better drug candidates compared to compounds 24 and 25, but similar to compounds 22 and 23 [139] . Scumonilines 26e29 displayed~63% inhibition at 10 mM against bGLU release from activated rat neutrophils with IC 50 values of 5.21, 5.85, 5.47 and 5.16 mM respectively; better than reference inhibitor ginkgolide B (IC 50 ¼ 6.63 mM). Compounds 26e29 were also more potent lead molecules compared to both glucuronate esters 30e34 (Fig. 6 ) isolated from similar Scutellaria regeliana [140] and chiral egeninebased alkaloids 35e37 isolated from the rhizomes of Corydalis decumbens [141] . Compounds 30e34 showed 43.7e47.1% inhibition at 10 mM, whereas compounds 35e37 were more inferior inhibitors at the same concentration with 32.4e41.3% inhibition.

Evidently due to its phycocyanin-rich content and potent reduction of zymosan-induced damage to knee joint histological architecture, edible blue-green microalgae e spirulina is deemed a therapeutically viable anti-arthritic agent [142] . An analysis of the synovial fluid of female OF1 mice knee joints after 4-days intraarticular injection of zymosan, followed by 8-days oral administration of spirulina water-suspension at 100 mg/kg and 400 mg/kg, showed 78.7% and 89.2% inhibition of bGLU activity respectively.

Subsequently, arthritic parameters such as tibial articular cartilage destruction, erosion of bone structure, articular tissue inflammation, loss of general joint architecture and pannus formation, were all markedly reduced in spirulina fed mice compared to those receiving zymosan only. These anti-inflammatory and anti-arthritic effects were comparable to reference drug Triamcinolone, a Tuber extracts of Arisaema tortuosum (Whipcord Cobra Lily) has shown moderate anti-inflammatory effect via bGLU inhibition in a dose-dependent manner [143] . Conceivably, the presence of quercetin, rutin and lectin, identified through chromatographic profiling, facilitated maximum inhibition of 92.9% at 100 mg/ml, superior to reference inhibitor salicylic acid. Employing a similar approach with ginger rhizomes [144] , only 6-Gingerol (38, Fig. 7) was identified with 85% inhibition of bGLU at 1 mM, comparable to salicylic acid (82% inhibition). It is also noteworthy that constituents profiling of essential oils extracted from the leaves of seven local varieties of Piper betle L. identified eugenol (39) with an IC 50 value of 616.68 mg/ml similar to 794.62 mg/ml of silymarin; although the essential oils were only moderate bGLU inhibitors with IC 50 ! 5.26 mg/ml [145] . However, metabolomics profiling of the leafy shoots-methanolic extracts of three Swertia species viz. S. chirayita, S. decussata and S. bimaculate, presented S. chirayita as the strongest inhibitor of bGLU and xanthones as the most active metabolites responsible for the Swertia species' potency [146] . The C-2-glycoside of norathyriol, mangiferin (40) emerged as the most active and therapeutically promising xanthone with IC 50 ¼ 38 mM and 50-fold more potent than silymarin. Shikunshito-Kamiho (SKTK), a traditional oriental formulation and Fenugreek seeds (FGS), an Indian spice, are natural ethnotherapeutic agents with potentials identical to luteolin (11) i.e., attenuation of colon carcinogenesis via potent inhibition of bacterial bGLU activity [147, 148] . 5-weeks oral administration of SPF male ICR mice's diet supplemented with 20 mg/kg or 60 mg/kg SKTK water extracts, after 10 weeks subcutaneous injection of DMH (weekly), equipotently reduced bGLU activity during and after treatment by 14.9% and 21.3% of controls (DMH alone) respectively. Tumour incidence in colon, measured by the number of aberrant crypt foci was also significantly reduced from 21.6 (distributed in sigmoid and descending colons) in controls, to 6.9 and 6.4 (distributed in rectum) at 20 mg/kg and 60 mg/kg doses respectively. Similarly, diet supplementation with FGS significantly reduced intestinal bGLU activity, leading to a marked reduction in tumour incidence from 93.3% in male Wister rats receiving DMH alone for 15 weeks to 16.6% in rats receiving DMH þ 2 g/kg FGS water extracts for 30-weeks cumulative period. Flavonoid, saponin and fibre rich content of FGS were presumed to be responsible for its anticarcinogenic activity.

The hepatoprotective activity of Chinese medicinal formulation e Reduohanxiao-tang, used in the treatment of stroke and liver diseases has also been attributed to its ability to inhibit bacterial bGLU activity in vivo [149] . Water extracts of this local formulation and two of its ingredients viz. rhizomes of Pueraria thunbergiana and Scutellaria baicalensis potently inhibited the activities of E. coli and rat liver-derived bGLU with IC 50 values ranging from 0.35 to 1.42 mg/ml. Subsequently, oral administration of these extracts at 100 mg/kg alleviated CCl 4 -induced liver injury measured by significant reduction in serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic acid dehydrogenase (LDH) levels, compared to controls. The superior hepatoprotective effect of Pueraria thunbergiana was accredited to isoflavone daidzein (41, Fig. 7) , an aglycone metabolite of main components puerarin and daidzin by intestinal bacteria. Daidzein inhibited E. coli and rat liver bGLU with IC 50 ¼ 0.41 and 0.50 mg/ml respectively whereas puerarin and daidzin were inactive.

Fascinatingly, structurally similar tectorigenin (42), an aglycone metabolite of 7-O-glycoside e tectoridin (43), isolated from the flowers of Pueraria thunbergiana, exhibited better inhibitory potency (IC 50 ¼ 0.30 mg/ml) than its congeners [150] . 50 mg/kg intraperitoneal pre-treatment of male ICR mice with tectorigenin or 100 mg/kg oral administration of tectoridin provided better hepatoprotection than dimethyl diphenyl bicarboxylate (DDB) and daidzein, by significantly lowering serum AST, ALT and LDH levels relative to CCl 4 -treated controls. The prodrug behaviour of tectoridin, identical to puerarin and daidzin above, was also established by its absence in the serum after 250 mg/kg oral administration (only tectorigenin was detected) and its failure to provide desired hepatoprotection after 100 mg/kg intraperitoneal administration.

The triterpenoid 18b-glycyrrhetinic acid (44, Fig. 8 ) also known as enoxolone, is the aglycone metabolite by human intestinal bacteria responsible for the hepatoprotective activity exhibited by saponin e glycyrrhizinic acid 45 [151] . Glycyrrhizinic acid (45) is isolated from the rhizomes of Glycyrrhiza uralensis e the sweetening agent in SKTK. It exists as a natural glucuronide conjugate of 18b-glycyrrhetinic acid (44) hence making it (45) a substrate for bGLU-mediated hydrolysis for consequent release of active inhibitor 44. In vitro evaluation against E. coli and rat liver bGLU revealed stronger potency of glycyrrhizinic acid (IC 50 ¼ 12.15 and 97.21 mM respectively) compared to 18b-glycyrrhetinic acid (IC 50 ¼ 509.90 Fig. 7 . Natural bGLU inhibitors profiled from plant extracts and ethnomedicinal preparations. and 42.49 mM respectively). Moreover, at 100 mg/kg doses, oral treatment with glycyrrhizinic acid or intraperitoneal treatment with 18b-glycyrrhetinic acid, protected the hepatocytes of male Wistar rats against CCl 4 -induced liver injury evident by the significant reduction in AST, ALT and LDH levels compared to CCl 4controls. Intraperitoneal administration of glycyrrhizinic acid also did not provide hepatoprotection akin to tectoridin (43) . In addition, structurally similar emodinol (46) , isolated from the roots of perennial herb Paeonia Emodi showed stronger E. coli bGLU inhibition in vitro with IC 50 ¼ 63 mM compared to 18b-glycyrrhetinic acid [152] .

Microbial biotransformation of drugs and/or their precursors to new molecules with modulated pharmacological profile is considered an invaluable tool in drug design and development [153] . The natural product class e steroids, are key substrates in this regard [154] . Accordingly, naturally occurring androstane steroid hormone, 5-dehydroepiandrosterone (47, Fig. 8 ) and its Macrophomina phaseolina metabolites were examined for their bGLU inhibitory potentials [155] . The inferior potency/inactivity of metabolites compared to parent 5-dehydroepiandrosterone (IC 50 ¼ 77.9 mM) suggests that having an alkene unit at position-5 and cyclopentanone unit in the molecular architecture is crucial to inhibitory potency. Conversely, the synthetic anabolic steroid, dianabol and its metabolites by filamentous fungus Cunninghamella elegans or Macrophomina phaseolina were inactive against bGLU; except metabolite (48) with IC 50 ¼ 60.7 mM [156] . The E-oxime derivative (49) of an inactive metabolite however showed decent inhibitory potency with IC 50 ¼ 49.0 mM, equipotent with D-SAL and 2-fold superior than its Z-isomer. Nonetheless, the therapeutic safety of these dianabol derived bGLU inhibitors is questionable considering the increased risk of hepatotoxicity associated with oral administration of 17a-alkylated anabolic steroids [157, 158] .

The fascinating ability of prebiotics and probiotics to positively influence the composition and behaviour of human intestinal microbiota, has been continuously explored with considerable success to improve human health [159e161]. Precisely, lactic acid bacteria (LAB) probiotics can selectively utilize non-digestible oligosaccharide prebiotics as carbon source to produce active metabolites, which modulates or stimulates the activities of certain intestinal bacteria hence, eliciting important physiological response and conferring health benefit [162] .

Consequently, LAB e Lactobacillus acidophilus CSG afforded stronger inhibition of intestinal bacteria (including E. coli) producing bGLU thus providing hepatoprotection to male ICR mice, compared to Lactobacillus brevis HY7401 and Bifidobacterium Iongum HY8001 [163] . When anaerobically cocultured with E. coli (HGU-3), L. acidophilus CSG also potently inhibited bGLU productivity of E. coli compared to other LABs. As a result, oral treatment of the murine models with 500 mg/kg of L. acidophilus CSG alleviated CCl 4 -induced hepatotoxicity by lowering AST and ALT levels to 66% and 57% respectively, of CCl 4 control group. Whereas, for t-BHPinduced hepatotoxicity, AST and ALT levels were lowered to 62% and 48% respectively, better than reference hepatoprotective agent DDB. Similarly, Lactobacillus plantarum CFR 2194 was the most effective strain of Lactobacilli metabolizing fructooligosaccharide prebiotics to short chain fatty acids, thereby altering bGLU productivity of E. coli [164] . Lactic acid (50, Fig. 9 ) and especially nbutyric acid (51) were identified as the major short chain fatty acid metabolites in the culture filtrate of Lactobacillus plantarum CFR 2194 and fructooligosaccharides responsible for the observed decrease in bGLU activity. 

The most abundant and relatively stable thiosulfinate, S-methyl methanethiosulfinate (52, Fig. 9 ), found in Chinese chive (Allium tuberosum Rottier), has exhibited strong inhibitory potency (IC 50 ¼ 3.60 mM) against E. coli bGLU [165] . Compared to other disulphides viz., dimethyl, allyl methyl, and diallyl disulphides, compound 52 is more useful for alleviating drug induced toxicities or providing hepatoprotection. In parallel, a naturally occurring acetal isolated from red macroalga Neodilsea yendoana, isogloiosiphone B (53), possesses similar potential, although with weaker potency (IC 50 ¼ 57.0 mM) compared to compound 52 [166] .

The adverse role of inflammatory pathways and corresponding mediators including lysosomal enzymes during the pathogenesis of myocardial infarction again implicates bGLU in the cardiovascular disease. In a study which examined the protective effects of thymol (54, Fig. 9 ) on inflammation in isoproterenol-induced myocardial infarction using male albino Wistar rats [167] , the therapeutic benefit of inhibiting the release of bGLU, other lysosomal enzymes and pro-inflammatory cytokines was evident in the restoration of near-normal myocardial histology and function, compared to diseased controls. Rats pre and cotreated with thymol by intragastric gavage at 7.5 mg/kg body weight/day for 7 days and subcutaneous injection of 100 mg/kg isoproterenol for 2 days (day 6 and 7), showed significantly reduced levels of cardiac troponin-T (a cardiotoxicity biomarker) and high-sensitive C-reactive protein in their sera, compared to those treated with isoproterenol only. Most importantly, the activities of lysosomal enzymes i.e. bGLU, bgalactosidase, cathepsin B and D in serum and heart were also potently inhibited to near-normal. These anti-inflammatory effects of thymol afforded a well-protected myocardium with stable histological architecture.

Polyhydroxylated piperidine and pyrrolidine alkaloids commonly referred to as iminosugars (Fig. 10a) , are a noble class of sugar mimics in which the pyrano or furano endocyclic oxygen has been replaced with a basic nitrogen atom [168] . This class also include isoiminosugars (i.e. 1-azacarbasugars or 1-N-iminosugars) having a methylene unit in place of endocyclic oxygen and the anomeric carbon replaced with nitrogen. Although these ring alterations render iminosugars metabolically inert, their protonated forms mimic the pyranosyl or furanosyl unit in GH substrates, especially the putative oxocarbenium ion-like transition state (2, Fig. 2 ) in GH catalysed hydrolysis [21, 169] . This in turn facilitates their recognizability by GHs and other carbohydrate-recognizing proteins for corresponding enzyme inhibition. Since the isolation of nojirimycin (55, first iminosugar) and siastatin B (56, first isoiminosugar) from Streptomyces cultures in 1966 and 1974 respectively [170, 171] , these sugar mimics have been a recurring subject of intensive research owing to their extensive biological activities and therapeutic applications elicited majorly through potent inhibition of GHs [172] . The strongest iminosugar-based inhibitors of bovine liver-derived bGLU reported so far (Fig. 10b) , with promising antitumor potentials are siastatin B derivatives 57, 58 and 59 with IC 50 ¼ 65, 62 and 68 nM respectively [173] . Therefore, this section reviews iminosugars and their analogues as natural productinspired bGLU inhibitors, without undue repetitions of other monographs [4, 21, 168, 169] covering them.

Uronic analogues of nojirimycin bearing glycaro-1,5-lactams or the bicyclic analogues with imidazole and tetrazole units (i.e. tetrahydrotetrazolopyridine-5-carboxylates and imidazopyridine-5carboxylates respectively), were prepared to examine the effect of sugar-acid configuration and presence of lactam, tetrazole or imidazole moieties on inhibitory potency against bovine liver bGLU [174, 175] . Evidenced by the most potent inhibitor 60 ( Fig. 11 ) with K i ¼ 12 nM, gluco-configured units, are stronger bGLU inhibitors compared to galacto-and manno-analogues, due to their better mimicry of glucuronic acid (4). The imidazole ring also conferred stronger inhibitory potency than tetrazole or lactam units whereas glycarolactams and tetrazoles shared similar potencies; except galacto-tetrazole (64) with~200-fold inferior potency to galactolactam (62) . Further, although sugar configuration at C-4 was found to be ineffective on bGLU inhibition for glycaro-1,5-lactams 61 and 62, it was very significant for tetrazole and imidazole derivatives. Gluco-tetrazole (63) was 300-fold better than galacto-tetrazole (64), while gluco-imidazole (60) was 600-fold more potent than galacto-imidazole (65) and 1200 superior to manno-imidazopyridine (66) , which differs at C-2. Moreover, sugar configuration of acid moiety at C-5 also had significant influence on potency. Lconfigured units 67, 68 and 69 were 20-50-fold weaker than their D-isomers 63, 62 and 61 respectively. More importantly, the 2-fold increased potency of gluco-imidazole (60) compared to gluco-tetrazole (63) was attributed to stronger interaction of gluco-imidazole (60) with catalytic nucleophile Glu540 compared to glucotetrazole (63) . Interaction with catalytic acid Glu451 was compromised due to protonation of the imidazole ring in the zwitterionic form.

Conversely, similar bicyclic molecules of nojirimycin with cyclic carbamate, urea or guanidine pharmacophores are weaker bGLU inhibitors [176] . In all, only cyclic carbamates 70e73 (Fig. 12) showed moderate inhibitory activity against bovine liver bGLU.

Cyclic carbamates with carboxylic acid unit at C-5 of nojirimycin ring were also better inhibitors than their hydroxymethyl analogues. Thus, compounds 71 and 73 were the most potent overall with IC 50 ¼ 218 and 259 mM respectively. It is noteworthy that bicyclic indolizidine iminosugar 74, with hydroxymethyl unit is also a weak inhibitor (<50% inhibition at 1 mM), although it exhibits desirable selectivity (7-fold) for E. coli bGLU [177] . Surprisingly, carbasugar 75 shared a similar fate with IC 50 ¼ 170 mM against E. coli bGLU, even though it is not an iminosugar [178] . Taken together, these results partly suggest that ability to mimic D-glucuronic acid favours bGLU inhibitory activity.

Accordingly, glucuronic acid analogue of naturally occurring hemiaminal calystegine B 2 (76, Fig. 13 ), uronic-noeurostegine (77) , was synthesized in 27-steps from levoglucosan [179] . Compound conferred with improved selectivity for E. coli bGLU. This is partly due to their improved lipophilic balance for bacterial cell penetration afforded by their C-alkyl unit and improved chemical stability of CeC bond at the pseudoanomeric C-1 centre [180] . Liminosugar C-glycoside, (À)-adenophorine (80, Fig. 14a ), an enantiomer of natural iminosugar C-glycoside (þ)-adenophorine (81), was prepared together with it analogues via skeletal rearrangement of corresponding azepanes [181] . However, compound 80 and analogues were found to be weak but selective inhibitors of E. coli bGLU. The C-propyl analogue, compound 82 with IC 50 ¼ 586 mM showed total selectivity for the bacterial ortholog. It was also superior to compound 80, its azepane precursor 83, and other iminosugars in the study which showed moderate selectivity with 3.1e18.1% inhibition at 1 mM. However, increasing the lipophilicity of azepane scaffold 84 via C-2 or N-alkylation, while tuning the configuration of alkyl and hydroxy substituents at C-2 and C-6 respectively (Fig. 14b) , birthed E. coli bGLU inhibitors with markedly increased potency and highly conserved selectivity [182] . SAR analysis revealed that a combination of (6S)eOH unit and N-alkylation with hexyl, nonyl or dodecyl produced stronger inhibitors and their potency increased with increasing chain length. In contrast, (2R, 6R)-C-butyl and (2S, 6R)-Cnonyl derivatives, compounds 85 and 86 respectively, were the most active C-alkylated analogues with IC 50 ¼ 261 and 27 mM respectively. Compound 85 was stronger than its N-alkylated analogue, while compound 86 showed a compromised selectivity (IC 50 ¼ 97 mM against bovine liver bGLU). Overall, highly lipophilic compound 87, having (6S)eN-dodecyl framework was the strongest inhibitor with IC 50 ¼ 3.30 mM, 3-fold superior potency to (6S)e N-nonyl analogue 88 (IC 50 ¼ 10.0 mM) and absolute selectivity for E. coli bGLU. Furthermore, substituent type and configuration at C-6 also influenced the inhibitory potencies of noeuromycin azepane analogues [183] . (6S)-configured compound 89 and 90 were weakly potent, compared to their inactive (6R)-configured analogues.

Compound 89 (IC 50 ¼ 139 mM) was 4-fold more potent than its 6hydroxymethylated analogue compound 90.

Incorporating the acetamido unit in Siastatin B (56), while retaining the iminosugar C-glycoside scaffold via similar skeletal rearrangement of azepanes, afforded L-configured C-glycosides of 1-deoxynojirimycin (91) with weak inhibitory potencies [184] . Sugar configuration at C-1 and C-2 (Fig. 14d) , significantly influenced selectivity for GHs. Hence, (2R, 3R)-configured molecules were selective inhibitors of E. coli bGLU but inactive against bovine liver bGLU, a-N-acetylgalactosaminidase and b-N-acetylgalactosaminidase. Whereas, (2S, 3S)-configuration infused selectivity for b-N-acetylgalactosaminidase. Consequently, (2R, 3R)-configured compound 92 with a rigid benzyl substituent emerged as the best E. coli bGLU inhibitor in the series with IC 50 ¼ 90.7 mM compared to 37.2% inhibition at 1 mM of compound 93 with (2S, 3S)-configuration and butyl unit. The authors attributed these inferior activities to stereochemical mismatch between the L-configured units and Dconfigured substrates of GHs. We also conceive that the presence of hydroxymethyl and not carboxylic unit (to mimic glucuronic acid) might also be responsible for the weak inhibitory potencies. This was indeed the case for 3,4,5-trihydroxypipecolic acids, uronic analogues of 1-deoxynojirimycin [185] . D-isomers were stronger than corresponding L-isomers, while the most active bGLU inhibitors were those with better mimicry of glucuronic acid viz. Dgluco and D-galacto configured units; compounds 94 and 95 respectively (Fig. 15 ). Compound 94 showed 3-fold selectivity for bovine liver bGLU (IC 50 ¼ 70 mM), whereas compound 95 displayed an exclusive inhibition (IC 50 ¼ 86 mM).

Azoles are a prominent class of heterocycles with at least one nitrogen atom in their 5-membered aromatic ring. Due to their structural rigidity and amazing physicochemical properties, which confers highly coveted and broad pharmacological activity spectrum and therapeutic potentials, they have remained a targeted scaffold of many synthetic protocols. Members of this class include pyrazoles, imidazoles, thiazoles, triazoles, oxadiazoles, thiadiazoles and tetrazoles together with their benzo analogues i.e., indoles, benzimidazoles, benzothiazoles and benzotriazoles. These compounds also enjoy increased hydrogen bonding (H-b) capability furnished by ring N and/or O atoms for biomolecular targets binding. Consequently, this section is an overview of reported bGLU inhibitors bearing one or more azole nuclei. The critical focus is on the most potent inhibitor(s) in a given series, the pharmacological profile, structure-activity relationship (SAR) and molecular docking analysis.

bGLU inhibitory potency (IC 50 ¼ 1.20e44.16 mM) on a set of imidazolylethylaryl carboxylates compared to reference inhibitor D-SAL (IC 50 ¼ 48.38 mM) [186] . Compound 96 (Fig. 16 ) emerged as the most potent bGLU inhibitor (IC 50 ¼ 1.20 mM) with 40-fold superior potency to D-SAL. SAR analysis supported by in silico studies articulated that compounds with electron-donating groups (EDGs) displayed inferior inhibitory activities compared to those with electron-withdrawing groups (EWGs). Moreover, molecular hybridization with indole nucleus resulted in a potent bGLU inhibitor compound 97 with IC 50 ¼ 2.10 mM and 23-fold increased potency than D-SAL. Compound 96 and 97 displayed strong H-b interaction of indole NH unit and p-p interaction with active site residues of bGLU.

In vitro screening of imidazolopyridines ( Fig. 17 ) for their bGLU inhibitory potentials presented dihydroxy-substituted derivatives as promising inhibitors of enzyme activity [187] . (Fig. 19) . However, replacing OH unit with more lipophilic MeO or EtO groups led to significant loss of activity. Molecular docking analysis also disclosed the importance of iminic nitrogen and proximity of thiazole nitrogen to catalytic acid Glu451 for strong inhibitory potency. Compound 102 with adjacent OH units had a favourable fit into bGLU catalytic pocket for stronger interactions with amino acid residues Glu540, Glu451 and Tyr508.

However, installing pyren-1-ylmethylenehydrazinyl moiety on thiazole skeleton improved bGLU inhibitory potency [190] . Com- with Br, Me and MeO substituents were weaker inhibitors [191] . As a result, compound 108 with IC 50 ¼ 2.16 mM (Fig. 21) , was the most potent inhibitor. It was 2 and 22-fold stronger than compound 109

and D-SAL respectively. Docking studies revealed that the free amino NH unit in compound 108 formed strong H-b interaction with OH unit of catalytic acid Glu451. The dichloro substituents also substituted phenyl units at position-2 of benzimidazole core [192] . Activity was dependent on the presence and position of Cl and OH substituents hence 3,4-dichlorophenyl analogue 110 (Fig. 22 ) was found with strongest inhibitory potency (IC 50 ¼ 6.33 mM). Interestingly, introducing 5,7-dichloro unit on benzimidazole nucleus gave remarkable results as previously inactive molecules gained significant activity [193] . Compound 111 emerged as the most potent inhibitor overall with IC 50 ¼ 4.48 mM and 11-fold stronger than D-SAL. Moreover, replacing an OH unit of the dihydroxy substituent with MeO led to significant or total loss of activity. F, Me, NO 2 , naphthyl and anthracenyl units also gave inactive compounds. [194] .

Molecules containing EWGs NO 2 , Cl and F groups were superior in the series (Fig. 23) . Thus, the best activity was found with ortho- Akin to imidazole-indole hybrid compound 96 with IC 50 ¼ 2.10 mM (Fig. 16) , molecular hybridization of benzimidazole with amide bond bioisostere, 1,3,4-oxdiazole, resulted in an isopotent hybrid 113 ( Fig. 24 ) with IC 50 ¼ 2.14 mM [195] . Again, OHsubstituted derivatives were stronger inhibitors compared to those containing F, Cl and NO 2 , Me and MeO substituents. Docking studies revealed that the molecular hybrids adopted a linear configuration in the active site of bGLU. The oxadiazole nucleus and central-phenyl ring of compound 113 formed strong H-b interaction with NH 2 unit of Asn484 and phenolic OH of Tyr508 respectively. Compound 114 (IC 50 ¼ 3.14 mM) on the other hand interacted with catalytic acid Glu451 and Tyr508 via its central phenyl and phenol rings respectively.

In another comparative study using 113 as lead but replacing benzimidazole with benzohydrazone unit (Fig. 25) , the importance of benzimidazole nucleus to bGLU inhibitory activity was established by the pronounced loss of activity [196] . Nevertheless, OH-substituted derivatives and benzenetriol compound 115 emerged as the strongest inhibitor with IC 50 ¼ 7.14 mM. Inhibitory potency was dependent on substituent's position in the order; para ˃ meta ˃ ortho for OH, NO 2 and Cl groups, while the presence of Me or MeO groups gave inactive compounds. Molecular docking studies showed that inhibitory activity correlated strongly with the strength of H-b interaction hence the improved potency seen with OH-substituted derivatives. Notably, the benzenetriol OH unit on compound 115 formed H-b interactions with Glu540, Asp207, Tyr508, His385, and Asn450, while the benzohydrazide unit interacted similarly with Tyr508, Glu540 and Tyr504. In like manner, replacing the pharmacophores in compound 115 i.e. hydroxyl for methoxy and imine for more polar sulfonamide unit (Fig. 26) , led to stronger bGLU inhibitory potency for the resulting oxadiazole-benzenesulfonamides [197] . The most potent inhibitors were compounds 116 and 117 with IC 50 ¼ 2.40 and 6.34 mM respectively; compound 116 showed similar potency as compound 113. Inhibitory activity was improved for parasubstituted derivatives, while EWGs produced stronger inhibitors than EDGs. Binding interactions in the active site of bGLU especially H-b interaction of benzamide NH unit with Glu451 established the improved activity of the sulfonamides compared to benzohydrazones. Compound 116 formed H-b interaction via sulfonyl oxygens with Asp207 and Asn450, while the benzamide oxygen interacted in similar fashion with Tyr508. Additional ionic bonding between NO 2 groups in compound 117 and Glu451 also accounted for the improved potency.

Molecular hybridization (Fig. 27) inhibitor D-SAL respectively (Fig. 28) . In silico studies of these benzothiazoles revealed that they adopted a linear conformation which allowed appropriate fit into the binding groove of bGLU.

Ortho-OH unit on compound 120 formed strong H-b interactions with Glu451 while the para-OH unit provided H-b interaction with Asp207. The enzyme-inhibitor complex was stabilized in the active site through hydrophobic interactions of benzenetriol ring with indole nucleus of Trp587, His385, Asn484, Tyr504, His509, Arg600, and Lys606. Most importantly, all the active inhibitors were noncytotoxic in a cytotoxicity assay using mouse embryo fibroblasts (3T3-L1) and Wistar rat hepatocytes (CC-1). Benzothiazole and benzimidazole are bioisosteres due to their structural and pharmacological similarities; therefore, bioisosteric replacement of one for the other has been consistently explored in drug development. Accordingly, bioisosteric replacement of the benzimidazole moiety in compound 113, to give new benzothiazole-oxadiazole hybrids resulted in equally potent bGLU inhibitors [200] . The most potent compound 122 (Fig. 29 ) with IC 50 ¼ 2.16 mM, was equipotent with compound 113. Fascinatingly, compound 123 (IC 50 ¼ 4.38 mM) was also isopotent with similarly substituted compounds 121 and 111. Further, docking studies of compounds 122 and 123 showed that H-b interactions of phenolic OH units with active site residues viz., Glu451, Tyr508, and Asp207, favoured their strong inhibitory potency. Moreover, compound 122 formed hydrophobic interactions with Tyr504 and Lys606 via its thiazole core and benzene ring respectively. The reduced potency of compound 123 was established in the strength of these interactions as well as the absence of additional van der Waal contacts of benzothiazole ring in compound 122 with catalytic residues Glu451 and Glu540. In silico pharmacokinetic properties modelling of these inhibitors predicted good cell permeability and solubility. The compounds were also non-cytotoxic to 3T3-L1 and CC-1 cell lines.

Following a similar molecular development strategy leading to compound 122, new benzothiazole-hydrazone hybrids were assembled by deleting the 2-oxadiazolylphenol fragment in compounds 122 and 115 (Fig. 30) [201] . Compared to the parent compound series, the new hybrids had marked reduction in potency. The recurring pattern of reduced or abolished activity also persisted with Me and MeO substituents. Nevertheless, compound 124 was 3.2.1.7. Triazole. The triazole ring also affords potent bGLU inhibitors. In vitro evaluations of 1,2,4-triazole-Schiff bases [202] presented isatin-containing compound 125 (Fig. 31) as the strongest inhibitor with IC 50 ¼ 2.50 mM and 19-fold superior activity than D-SAL. Cl, OH and NO 2 substituents at ortho-positions also gave appreciable activities whereas Me, MeO, cumyl and other aryl derivatives were inactive. Docking simulations of compound 125 in the active site of bGLU revealed strong H-b interaction between isatin NH and catalytic residues Glu540 and Tyr504. The phenyl ring also provided p-anion and p-p interactions with Glu451 and Tyr504 respectively while van der Waal contacts with Lys606, Tyr508, Val410 and Asn450 stabilized the inhibitor-bGLU complex.

However, amide bond bioisostere, 1,2,3-triazole, is a stronger bGLU inhibitor compared to its 1,2,4-triazole isomer. Molecular hybridization with carbazole [203] delivered eight compounds having IC 50 < 3 mM (Fig. 32) . Compound 126 (IC 50 ¼ 0.55 mM) was the most potent hybrid in the series and 83-fold stronger than D-SAL. All the carbazole-1,2,3-triazole tethers were non-cytotoxic to 3T3 cells.

3.2.1.8. Indole-based hybrids. A library of indole analogues containing the hydrazone unit in compound 115 have been examined for bGLU inhibition [204] . Evinced by the increased potency, the indole pharmacophore delivers stronger bGLU inhibitors compared to benzothiazole and oxadiazole. For instance, the most potent inhibitor 127 with IC 50 ¼ 0.50 mM (Fig. 33) , was 100-fold more potent than D-SAL and 42-fold stronger than similarly substituted oxadiazole variant from compound 115 library. Compounds 128 (IC 50 ¼ 2.40 mM) and 129 (IC 50 ¼ 2.50 mM) were also 3-fold and 43fold stronger respectively than their corresponding oxadiazole variants. Moreover, substitution at ortho and para-positions gave stronger inhibitors compared to meta-position, while potency in monosubstituted analogues followed the order; F ˃ OH ˃ Cl ˃ pyridyl ˃ NO 2 ˃ MeO. The excellent potency of compound 127 was afforded by strong H-b interaction of position-5-OH with Glu451 and Glu540 as well as position-4-OH with Tyr508. Indole and benzohydrazone NH units formed H-b interactions with Asn502 whereas the ligandreceptor complex was stabilized by p-alkyl interaction of indole ring with Trp528. Expectedly, the reduced activity of compound 128 was seen in its weaker interactions in the active site of bGLU. Based on the pharmacological significance of thiadiazole pharmacophore and the promising inhibitory activity of indole hybrid compound 127, hybrids of indole-thiadiazole were assembled as new class of bGLU inhibitors [205] . Generally, thiadiazole nucleus infused stronger inhibitory potencies on the resulting hybrids compared to hydrazone unit. Dihydroxy substituted compound 130 emerged as the strongest inhibitor with IC 50 ¼ 0.50 mM and equipotent as compound 127. Ortho-substitution was more beneficial to potency than para and meta-substitutions, while inhibitors' strength followed the order; OH ˃ F ˃ Me ˃ pyridyl ˃ Cl ˃ NO 2 (Fig. 34) . However, bioisosteric replacement attempt with oxadiazole proved that thiadiazole unit is preferred for stronger inhibition [206] . The strongest potency remained with 2,3-dihrdoxy substituted derivative 131 (IC 50 ¼ 0.90 mM), although 2-Cl derivative had an outlying potency.

Bazedoxifene (Fig. 35) is a novel indole-based inhibitor of IL-6/ GP130 for the management of triple negative breast cancer [207] . It is also a selective estrogen receptor modulator administered as co-drug with conjugated estrogens (Duavee) in estrogen replacement therapy for the prevention of postmenopausal osteoporosis. Albeit, the high ratio of circulating estrogen metabolites and parent estrogen aided by bGLU deglucuronidation activity in the gut is considered a risk factor for postmenopausal estrogen receptor positive (ERþ) breast cancer. Accordingly, a study on the therapeutic utility of this combination drug in an ovariectomized mouse model showed significant reduction in faecal bGLU activity without altering faecal microbiota diversity [208] . The study articulated the significance of bacterial bGLU inhibition to improving the outcome of long-term administered estrogens for postmenopausal women or breast cancer patients.

Consistent with the excellent inhibitory potencies of indolebased compounds, a library of bis-indoles (Fig. 36) have been reported as strong bGLU inhibitors [209] . Once more, the importance of (di)hydroxy substitution to potency was prominent. Compound 132 emerged as the most active molecule with IC 50 ¼ 1.62 mM and 30-fold superior to D-SAL. In silico studies suggested that H-b donor-acceptor potential of OH units on compound 132 significantly influenced ligand-receptor interaction. Ortho-OH formed strong H-b with amino acid residues Asp207 and Tyr508, while para-OH interacted similarly with His385. Arene-arene interaction of indole ring with Tyr504 also aided the favourable binding to bGLU. In another study [210] , both compound 132 and its 2,3dihydroxy compound 133 (IC 50 ¼ 1.2 mM) were the strongest inhibitors of bacterial bGLU; albeit with moderate cytotoxicity against 3T3 mouse fibroblasts.

The presence of N-phenyl substituted thiosemicarbazide unit however introduced marked increase in bGLU inhibition of 1Hbisindoles [211] . Inhibitory potency for monosubstituted analogues followed the order F z CF 3 ˃ Cl ˃ Br ˃ Me ˃ MeO and ortho-substitution produced the best result (Fig. 37) . Accordingly, 2-F to Glu540, Asn450, Lys606, Trp528. Tyr508 also formed H-b interaction with amide NH and arene-arene interaction with phenyl ring of tolyl unit.

Chalcones are both a class of naturally occurring flavonoid family and synthetically obtainable compounds with extensive therapeutic applications [213] . The presence of a highly reactive a,b-unsaturated ketone unit and delocalized electrons makes them coveted precursors for many synthetic manipulations and pharmacological explorations.

The pro-inflammatory role of bGLU in inflammatory disorders motivated the design of a series of (di)hydroxychalcones (Fig. 39) , to inhibit the release of bGLU from stimulated rat mast cells and neutrophils [214e216]. It was established that hydroxychalcones were stronger and selective inhibitors of neutrophil-derived bGLU over mast cell-derived bGLU. The best inhibitors were compounds 139 (IC 50 ¼ 0.6 mM; ˃ 160-fold selectivity) and 140 (IC 50 ¼ 1.3 mM; 7fold selectivity). SAR analysis showcased the importance of a,b- Compound 141 (Fig. 40 ) has emerged as the most potent inhibitor with 75% inhibition at 10 mM and only 3-fold stronger than standard salicylic acid, amongst a series of chalcone-Mannich adducts [217] . Adopting a similar molecular design [218] , a library of chalcone-benzamides were found with slightly improved potency and SAR analysis established the preference of EWGs over EDGs for better enzyme inhibition. Compound 142 containing similar 3-Br substituent as compound 141, emerged as the most potent molecule (84.68% inhibition at 1 mM); 3-fold stronger potency than salicylic acid (24.77% at 1 mM). It was also non-cytotoxic in CCK-8 assay in contrast to 2-fold increased cytotoxicity of equally potent 3-CF 3 analogue. Notably, activity modulation by substituting trimethoxy unit for indole was unsuccessful [219] , leading to prominent reduction in percentage inhibitory activity of the chalcones (˃ 8-fold).

Structural development of a coumarin (chromen-2-one) compound 143 (Fig. 41 ) recovered from virtual screening delivered a set of moderately potent inhibitors of bGLU activity [220] . Superior activity compared to D-SAL (IC 50 ¼ 48.40 mM) was recorded compounds 144e147 with IC 50 values of 9.90, 11.70, 21.40 and 34.20 mM respectively. In another attempt [221] , despite different molecular tunings including polar OH and Cl groups, the coumarin pharmacophore remained inactive against bacterial bGLU. Conversely, structurally isomeric flavenone (chromen-4-one) bearing a pyranose appendage, showed stronger inhibition of bacterial bGLU [210] . Compound 148 (Fig. 42) , the most active inhibitor thereof with IC 50 ¼ 4.50 mM and 10-fold superior potency to D-SAL, was totally non-cytotoxic against 3T3 mouse fibroblasts. In silico studies further disclosed the importance of pyranose ring in H-b interactions with active site residues Glu503, Asp161 and Glu413.

Fascinatingly, introducing 1,3,4-oxadiazole pharmacophore to a chromen-4-one scaffold (Fig. 43) , led to significant increase in potency [222] . The new hybrids exhibited consistent trend in their potency based on substituent type and position in the order; F > Cl > Me > NO 2 > Pyridyl > MeO and ortho > para > meta respectively.

Thus On the other hand, azacoumarins (1H-quinolin-2-ones) having a basic nitrogen atom in place of endocyclic coumarin oxygen, are conferred with stronger and selective inhibitory potencies against bacterial bGLU. Plausibly, their admirable activity is provided by the ability to mimic or interfere the oxocarbenium ion-like transition state akin to iminosugars; thus, allowing the alleviation of druginduced toxicities and prevention of colon carcinomas. Renowned examples in this class of bGLU inhibitors are the strongly potent azacoumarins 151 and 152 (Fig. 44) , identified from high-throughput screening [19, 223] . The compounds exhibited in vitro IC 50 values of 0.28 and 0.37 mM respectively, over 1000-fold selectivity for E. coli bGLU and 100-fold increased potency than reference inhibitor glucaro-d-lactam 61. The inhibitors also maintained their potency in living bacterial cells with EC 50 ¼ 0.018 and 0.028 mM. Other similarly active compounds (IC 50 (once daily), alleviated Irinotecan (CPT-11) induced diarrhoea evident by a protected GI epithelium of BALB/cJ mice compared to bloody diarrhoea due to damaged tissues and glandular structure in those receiving CPT-11 alone [19] . Pre-treating C57BL/6J mice with 10 mg of inhibitor 151 before intraperitoneal administration of ulcerogenic dose of NSAIDs e diclofenac (60 mg/kg), indomethacin (10 mg/kg) and ketoprofen (100 mg/kg), also successfully protected the mice models against NSAID-induced small intestine mucosal damage [224, 225] . Moreover, compound 151 reduced all enteropathy parameters by inhibiting GI bacterial bGLU-mediated hydrolysis of NSAID-acyl glucuronide in a concentration-dependent manner (IC 50 z 0.164 mM). Interestingly, the compound did not alter drug's systemic exposure, hepatobiliary excretion of glucuronides and GI microbiota diversity. Although it possesses a short half-life (˂1 h) and pan-cytochrome P450-mediated poor bioavailability (21%). However, an attempt to reduce the inhibitor's serum exposure for increased GI residence by replacing ethoxy unit with hydroxyl or morpholinyl groups led to inferior pharmacological profile compared to parent compound 151 [226] . Further, compound 152 exerted 2-fold reduction in the severity of diclofenacinduced anastomotic leakage, without affecting drug's plasma concentration in Wistar rats receiving 0.8 mg of the inhibitor and 3 mg/kg of diclofenac [227] . In contrast, rats receiving diclofenac only suffered severe leakage thus suggesting the inhibitor's potential clinical utility to improve anastomotic healing. 

Emerging from the screening protocol which birthed compound 151, three piperazine-containing compounds 153, 154 and 155 ( Fig. 45) showed promising inhibitory potentials against bacterial bGLU [228] . Though with slightly weaker inhibitory strengths (IC 50 

The therapeutic significance of fused pyridinone-furans as antiinflammatory drug candidates via inhibition of neutrophil-derived bGLU has been reported [234] . Compounds bearing MeO and CF 3 substituents on ring B (Fig. 47) showed over 70% inhibition at 1 mM with no cytotoxic effects (<32% at 10 mM) in CCK-8 assay as compared to reference salicylic acid (25% inhibition). Replacing ring B with furan, thiophene or pyridine however gave poor inhibitors (<45% inhibition). Compound 158 was the most potent with 75% inhibition.

The inhibitory potencies of dihydropyrimidinone carboxylates ( Fig. 48) , against bovine liver-derived bGLU has reiterated that small polar substituents are necessary for strong enzyme inhibition [235] . Appreciable potency (IC 50 ˂ 20 mM) was exclusive to CF 3 ˃ F ˃ OH ˃ thienyl ˃ Cl substituted compounds in that order. Whereas, MeO, Br, benzyloxy, furanyl, aliphatic alkyl or aryl units either reduced potency when co-substituted with polar F or OH groups, gave inferior activity with monosubstitution or rendered the molecule totally inactive. Therefore, the most active compound 159

with IC 50 ¼ 9.38 mM, found favourable fit in the active site of bGLU to form strong H-b interactions with key residues. NH of Asp207 was both a H-b donor and acceptor to pyrimidinone carbonyl oxygen and free NH at position-1 respectively. Carboxylate carbonyl oxygen interacted similarly with Arg600 whereas position-3 NH interacted with catalytic residues Glu451 and Tyr508. However, pyrimidinetriones (barbituric acid) [236, 237] , exhibited inferior potencies compared to standard D-SAL (IC 50 ¼ 45.75 mM), despite their similar binding interactions as compound 159 with active site residues and non-cytotoxicity to 3T3 cells.

In a predating study, the structural homolog of pyrimidinone, quinazolinone [238] , exhibited a unique trend in inhibitory potency against E.Coli bGLU with significant tolerance for lipophilic alkoxy groups, although poor activity with methyl substituent persisted. The most active compound 160 (Fig. 49) Intriguingly, replacing alkoxy units with more polar OH, NO 2 and Cl substituents led to significant reduction in potency. Inhibitory potency therefore followed the order; MeO ˃ EtO ˃ N(Me) 2 ˃ NO 2 ˃ OH ˃ Cl and ortho ˃ para ˃ meta. Importantly, the quinazolinone-based inhibitors were all non-cytotoxic to 3T3 cells. It is also noteworthy that a quinazolinone compound (Table SI) from Ref. [223] , also showed strong inhibitory potency (IC 50 ¼ 1.90 mM) against bacterial bGLU.

The quinoline nucleus can be regarded as a household name in medicinal chemistry, due to its inexhaustible pharmacological application and occurrence in many clinically important natural products and synthetic molecules. The desirable pharmacokinetic profile and ease of accessibility with wide substrate tolerance for different synthetic protocols, also encourages its utility in countless synthetic explorations. Research endeavours aimed at quinolinebased bGLU inhibitors are therefore presented in this section.

Quinoline-3-carbohydrazides [239] , prepared analogously as hydrazone tethers above also holds promising inhibitory activities. Similarly, OH unit at ortho-position yielded stronger inhibitors than F > Cl > NO 2 > Pyridyl > thiophenyl > furanyl z Me > MeO, in that order. Moreover, ortho-substitution consistently gave superior inhibitors compared to para-position, while meta-substitution conferred the weakest potency. 2 to 3-fold reduction in potency persisted when OH unit is substituted for MeO unit (Fig. 50) . Hence, compound 162 (IC 50 ¼ 2.11 mM) and 163 (IC 50 ¼ 2.60 mM) emerged as the most potent inhibitors overall. Docking studies revealed that OH unit at position-4 of quinoline ring fostered important H-b interaction with catalytic acid Glu451, while carbonyl unit of hydrazone moiety formed H-b interaction with NH of Tyr504.

In another quest for new anti-inflammatory agents, quinolinefuran hybrids (Fig. 51) were designed as inhibitors of bGLU release from activated neutrophils [240] . SAR analysis, established that 2-(furan-2-yl)quinolines (Type A) were stronger inhibitors of inflammatory mediators than tricyclic furo[2,3-b]quinolines (Type B). Whereas, formyl unit favoured potency compared to acetyl, oxime, methyl oxime as well as both a,b-unsaturated butan-2-one and its saturated variant. Therefore, non-cytotoxic compound 164

(IC 50 ¼ 5.0 mM) was the strongest inhibitor of bGLU release with 3fold superiority to reference trifluoperazine. Acetyl isomer compound 165, also had similar non-cytotoxic and strong inhibitory potency (IC 50 ¼ 7.5 mM).

Akin to azacoumarins (1H-quinolin-2-ones) 151 and 152, new quinoline-pyrazole conjugates were prepared via hit development protocol of compound 166 (Fig. 52) identified from highthroughput screening, for the inhibition of intestinal bacterial bGLU's hydrolytic activity on SN-38G fo alleviate CPT-11 druginduced toxicity [241, 242] . Interestingly, all the resulting derivatives showed good selectivity for E. coli bGLU except 2-Cl, 3-OH, 3-Me and 4-NH 2 substituted derivatives. The trend in inhibitory potency, based on substituent's position varied in the order; meta < ortho < para, while the presence of strongly hydrophilic OH, NO 2 and NH 2 units at para-position was detrimental to potency. following oral administration to BALB/cJ mice for 5 consecutive days compared to compound 168 (40%). Whereas, pharmacokinetic profiling of compound 168 revealed its lower plasma concentration and increased GI residence for improved intestinal bGLU inhibition compared to azacoumarin compound 151. Most importantly, oral co-administration of compounds 167 or 168 at 3 mg kg À1 day À1 for 10 consecutive days with 50 mg kg À1 day À1 intraperitoneal injection of CPT-11, protected BALB/cJ mice models against CPT-11 drug-induced intestinal mucosal injury, without altering drug's therapeutic efficacy. Furthermore, mechanistic studies established that the presence of electronegative and electroneutral surface charges on E. coli active site pocket and the protonation of N-1 and N-5 atoms avails a pH-dependent and selective inhibition for the compounds (Fig. 52) . Consequently, compounds 167 and 168 are clinically viable agents for protecting against GI E. coli bGLUmediated mucosal damage and preventing the release of chemical carcinogens crucial to the development of precancerous lesions for colon carcinogenesis.

The 200-fold increased inhibitory potency of benzohydrazideeN-cyanoethyl tethers [243] , compared to parent compounds articulates the significance of a balanced molecular lipophilicity to bGLU inhibition. O-alkylation with benzyl, benzoyl or tosyl groups slightly influenced potency for OH-substituted units; benzylation produced the best result (Fig. 53) . Hence, 169

(IC 50 ¼ 1.60 mM) and 170 (IC 50 ¼ 2.20 mM) were the most active conjugates in the series. Similarly, phenoxyacetohydrazones [244] have displayed 2 to 5-fold improved potency compared to D-SAL (IC 50 ¼ 48.40 mM). Compounds 171 and 172 (Fig. 54 ) emerged as the The desirable therapeutic significance of fused thienothiophenes has stimulated the synthetic exploration of a thienothiophene skeleton 173 (Fig. 55) for bacterial bGLU inhibition [245] .

From the resulting thienothiophene library with interesting structural diversity, only compound 174 showed inhibitory activity against bacterial bGLU with IC 50 value of 0.9 mM; 51-fold superior potency to D-SAL. Notably, the predicted pharmacokinetic properties using Molinspiration and Osiris calculations suggested that the inhibitor possesses good bioavailability and no toxicity risk as bacterial bGLU inhibitor.

In vitro study of phenyl disulphideebenzenesulfonamide tethers [246] , disclosed compounds 175 and 176 (Fig. 56 ) as potent inhibitors of bGLU (IC 50 ¼ 2.20 and 3.34 mM respectively), analogous to similarly substituted oxadiazole-benzenesulfonamide compounds 116 and 117 (Fig. 28) . Interestingly, bulkier phenyl disulfides 175 and 176 shared similar potency with lower molecular mass thiosulfinate 52, Fig. 9 (IC 50 ¼ 3.60 mM). However, substitution at ortho-position gave stronger inhibitors than para-and meta-positions, in contrast to compounds 116 and 117 series. Docking studies further revealed that the strong inhibitory potency of compound 175 is aided by the H-b interactions of sulfonamide NH and oxygen with Tyr205 and Asp207 respectively.

The efficacy of urea unit for bGLU inhibition was also examined using a library of NO 2 substituted N-phenyl ureas [247] . Therefrom, appropriate positioning of polar NO 2 unit was important to inhibitory potency of examined molecules. Compound 177 (Fig. 57) , emerged as the only inhibitor with promising activity with IC 50 value of 3.38 mM. However, thiourea analogues bearing meta-Cl substituent on N-phenyl ring [248] , were stronger inhibitors compared to those with unsubstituted N-phenyl ring or NO 2 substituted ureas (Fig. 58 ). Akin to compound 141, thioureas containing piperazine unit also exhibited improved potency compared to their morpholine and piperidine analogues, while N-alkylation of piperazine with N-(o or p-methoxyphenyl) or N-(2-pyridyl), 

Extant empirical data identified bacterial bGLU as a chelating agent, since enzyme activity was totally lost in the presence of Cu 2þ , Hg 2þ and Ag þ metal ions and restored in the presence of other chelating agents e Versene and cysteine [249] . Therefore, metal complexes with coordinatable metal centres exhibit significant bacterial bGLU inhibitory activities. To this end, Pd(II) complexes containing aniline and triphenylphosphine ligands were synthesized [250] . Para-chloroaniline derived metal complex 182 (Fig. 59) , displayed the strongest activity against bacterial bGLU with 98.5% inhibition and IC 50 ¼ 15.40 mM. It was also 2-and 5-fold superior to meta-chloroaniline derived and N-methyl substituted metal complexes respectively. Similarly, N-heterocyclic carbene (NHC) complexes of Au(I) [251] , as well as bis (NHC) complexes of Au(I) and Au(III) [252] , containing hydroxy, chloride, acetoxy and acetonyl ligands were equally potent bacterial bGLU inhibitors with IC 50 ranging from 0.14 to 2.60 mM. The strongest Au(I) NHC complex 183 (IC 50 ¼ 0.14 mM) was 327-fold more potent than reference D-SAL and 7-fold superior to strongest bis (NHC) complexes 184. Nonetheless, undesirable cytotoxicity against 3T3 cells discomfits the therapeutic significance of these metal complexes.

Parallel to iminosugars 60e74, the inhibitory potencies of structurally similar glycopolymers (Fig. 60 ) was dependent on the presence of carboxyl unit, type and configuration of sugar unit. Hence, glycopolymers with D-glucaric pendants linked to the polymer frame at C-1 (185), were stronger inhibitors compared to glycopolymers linked at C-5 (186) or those bearing D-gluconic (187) [253], D-mannaric (188) [254] and shorter chain D,L-xylaric (189) and L-tartaric (190) [255] pendants. However, these glycopolymers only show >60% inhibition at millimolar concentrations.

Patents and patent applications disclosing potent inhibition of bGLU particularly bacterial bGLU and the therapeutic significance thereof, have been documented. We therefore review in this section, patents applications filed in the present millennium relevant to bGLU inhibition. We have also included the only biomarker application of the enzyme filed within the period [256] . However, some of the patents and applications [257e263] emanated from published papers [19, 210, 228, 229, 241, 262] , reviewed in previous sections; therefore, only their summary is presented in Table 3 .

Considering the limitations associated with traditional clinical assessments for periodontal diseases diagnosis and the improved reliability of bGLU as a biomarker, a simple method requiring less expertise has been claimed to quantify elevated levels of the enzyme in the saliva of diseased patients relative to healthy ones, as a biomarker of disease risk and status [256] . The invention involves adding known b-D-glucuronide substrates (4-methylumbelliferone or phenolphthalein b-D-glucuronides) to a sample of patient's saliva in bGLU activity assay and thereafter quantifying the amount of aglycone produced via fluorometry, colorimetry or spectrophotometry. The addition of a labelled polyclonal or monoclonal antibody specific for bGLU to saliva sample and subsequent estimation of the amount of labelled antibody forming bGLU-antibody complex was also claimed, as well as a test-kit for claimed methods.

Bacterial bGLU-mediated hydrolysis of steroid glucuronides on the skin leads to body odours hence inhibitors of the process were developed and applied in deodorant and antiperspirant formulations [264] . The invention claimed that deodorant formulations with described compounds only inhibits bGLU activity without altering the natural composition of skin microbiota; in contrast to bacteriostatic or bactericidal mode of action of prior arts. Amongst the claimed inhibitors of different class, the strongest inhibition of E. coli bGLU was found with galactaric acid (99% inhibition at 0.1% test concentration in water), as well as zinc glycinate and zinc gluconate; 99% and 97% inhibition respectively at 0.25% test concentrations.

In the same vein, potent inhibitors of bacterial bGLU activity on urine have found non-therapeutic application in hygiene and sanitary products for suppressing the generation of urine odour [265] . At 0.1 wt% in dipropylene glycol, all the claimed inhibitors viz. macrocyclic ketones, ketones, macrocyclic lactones, macrocyclic oxalactones, esters, aldehydes, alcohols, ethers and terpenes, showed over 60% inhibition of E. coli bGLU. Macrocyclic ketones were stronger inhibitors than others. Consequently, the invention's most active inhibitor was compound 191 with 100%, 99.9% and 95.8% inhibition at 0.1, 0.01 and 0.001 wt% respectively. It was also effective after 48 h in suppressing the increase in urine odour when incorporated into different hygiene and sanitary products.

Phenoxy thiophene sulfonamides [266] were designed as adjuvants to eliminate the dose-limiting GI toxicity (diarrhoea) of CPT-11, through potent inhibition of bacterial bGLU-mediated deconjugation of active metabolite's glucuronide (SN-38G) in the intestine; thus, improving the drug's therapeutic efficacy. The synthesis of 76 bGLU inhibitors and 18 analogues of BRITE-355252 (compound 192) , the most active compound (IC 50 ¼ 20 nM), together with their therapeutic application was claimed. SAR study of compound 192 analogues revealed that inhibitory potency was markedly dependent on N-piperazinyl pendant at meta-position of phenoxy ring. Over 500-fold reduced inhibitory potency occurred with N-methylation (akin to compound 156), 15-fold reduction when appended at para-position and total loss of activity when removed. Uninstalling the chloro unit on thiophene ring also slightly diminished potency by 5-fold. Whereas, replacing naphthyl with substituted phenyl units afforded inhibitors with similar potencies i.e. IC 50 < 50 nM (Table SI) ; as a result, 193 was equipotent with 192.

bGLU is a physiologically important lysosomal glycosyl hydrolase with appreciable therapeutic potentials such as biomarker for disease diagnosis, endogenous bioactivator in prodrug monotherapy and enzyme replacement therapy. The enzyme's role in cell proliferation and inflammation also renders it a potential target for anticancer and anti-inflammatory drugs development respectively. Moreover, since a significant number of commercially available drugs are metabolised by glucuronidation, hydrolytic activity (i.e. deglucuronidation) by bGLU in human intestinal microbiota has been linked to colon carcinogenesis and genotoxicity as well as drug-induced dose-limiting toxicities of anticancer agents and NSAIDs, which thwarts their therapeutic potential and clinical utility. Therefore, potent inhibition of bGLU holds enormous clinical importance.

Generally, our literature survey found that a significant number of natural products-derived inhibitors display moderate inhibition of bGLU, and increased potency is found with inhibitors containing a flavonoid skeleton. The physiological tolerance, acceptable toxicity and favourable pharmacodynamic profiles of these natural inhibitors, posits them as worthy scaffolds warranting witty molecular developments. In addition, iminosugars distinguish themselves as a unique class of natural product-inspired molecules with promising potentials regardless of their usually multiple synthetic steps. Notable amongst these are the nojirimycin analogues and iminosugar C-glycosides, both conferred with highly selective inhibition of bacterial bGLU. However, the inhibitory potency of these carbohydrate mimics relies on several factors such as sugar configuration, mimicry of glucuronic acid substrate and correct lipophilic balance. Hence, to harness their remarkable potential, ingenious manipulations of the synthetic medicinal chemist interested in exploring their chemical space is expressly required.

On the other hand, purely synthetic inhibitors show fascinating diversity in their inhibitory potencies and pattern, due to the plethora of pharmacophoric enrichments possible for a single molecular design. Based on the foregoing, a comprehensive list of potent synthetic inhibitors (IC 50 5 mM) in this review is provided in Table SI of supporting information. Evinced by their nanomolar IC 50 values, quinoline-pyrazoles (167 and 168), piperazinecontaining compounds (156 and 157; 192 and analogues) as well as the indole nucleus, are conferred with the strongest inhibition of bGLU activity overall. The order of potency for hydrazone tethers was found as; bisindole ˃ indole > quinoline > thiazole ˃ benzothiazole z oxadiazole. Whereas, NO 2 , Cl, particularly F and OH substituents at ortho-or para-positions usually produce stronger bGLU inhibitors compared to their meta-substitutions.

Taken together, these suggests that the presence of polar groups with strong hydrogen bonding potential is crucial to inhibitory potency, while an indiscriminate increase in molecular lipophilicity is detrimental to strong bGLU inhibition. Consequently, we envisage that the model bGLU inhibitor will be that which is appositely tuned in its lipophilicity and polarity for easy cell penetration, favourable fit into the binding/catalytic pocket of bGLU for energetically stable binding and strong enzyme inhibition. We also perceive that rationale molecular hybridization of those active class of natural products and synthetic molecules will furnish stronger inhibitors of bGLU with improved selectivity and acceptable toxicity profile.

Furthermore, we found that due to the mixture of electroneutral and electronegative spots on the surface of bacterial bGLU binding pocket at physiological pH, in contrast to the electropositive spots for human bGLU, the presence of a protonatable N-atom confers improved selectivity for bacterial bGLU inhibition. This was particularly true for piperazine based inhibitors. Therefore, we believe further probing of this phenomenon will allow structureactivity guided molecular tuning to afford stronger bGLU inhibitors.

To the best of our knowledge, mechanistic studies on the behaviour of these inhibitors is scanty in literature and only found in two articles describing gluco-imidazole (60) and piperazine (156) based inhibitors. This investigative approach provides insight to the behaviour of these inhibitors in the catalytic cycle of bGLU thereby allowing deeper understanding of important structural features crucial to inhibitory activity beyond SAR and molecular docking studies. Finally, we hope this review will inspire medicinal chemists to direct their search light towards this molecular target, for the development of new therapeutic agents with improved efficacy and betterment of human health in consequential.

The authors declare no conflict of interest. 

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DMH: 1,2-dimethylhydrazine D-SAL: D-saccharic acid-1,4-lactone D-SDL: 2,5-di-O-acetyl-D-glucaro-1,4:6,3-dilactone D-GL: D-glucurono-g-lactone GCF: Gingival crevicular fluid GH: Glycosyl hydrolase GI: Gastrointestinal Gg-I: Gingival index GPCR: G protein-coupled receptors H-b: Hydrogen bonding IC 50 : Half maximal inhibitory concentration IL-1b: Interleukin-1 beta IL-8: Interleukin-8 k cat : Rate constant of catalysed reaction kDa: kilodalton K i : Inhibition constant k uncat : Rate constant of uncatalyzed reaction LAB

Glucuronide conjugate of SN-38 TNF-a: Tumour necrosis factor-a UDP: Uridine-5 0 -diphosphate UGT: UDP-glucuronosyltransferase

The National Research Foundation (NRF) of South Africa is appreciated for KIC grant and other financial support.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2019.111921.