Structure-Based Virtual Screening of Human β-Glucuronidase Inhibitors

Glycosidases (GH) are vital for almost all living organisms
(exceptions are some Archaeans and a few unicellular parasitic
eukaryotes) playing diverse and different roles...


Glycosidase Family of Enzymes
Glycosidases (GH) are vital for almost all living organisms (exceptions are some Archaeans and a few unicellular parasitic eukaryotes) playing diverse and different roles. Considering the diversity of reactions, they catalyze, amino acid sequence and folding, glycosidases have been classified in many different categories [1].

Beta-Glucuronidases
Beta-glucuronidase (β-glucuronidase) enzyme (EC3.2.1.31) is a glycosidase that have role in the hydrolysis of glycosaminoglycans at specific glucuronide site [2]. These specific enzymes are found in plants, bacteria and vertebrates. Intestinal β-glucuronidase is categorized as one of the key enzymes for the treatment of numerous intestinal diseases. In invertebrates, β-glucuronidase from mollusk sp (Ampullaria and Helix pomatia) are used in the hydrolysis of steroids in order to assess urinary conjugate cortisol [3]. Besides temperatures, the enzymes have a positive response from acidic to neutral pH. At 75 °C temperature and pH above 8, the activity of enzyme is lost [3]. Other key roles of β-glucuronidase includes carbohydrate processing.
They also show functional importance and are used in the hydrolysis of various steroids, bioassay for plant hormones, prodrug therapy [4] and as biomarker [5] and are present in extracellular fluid during the inflammation [6]. In human plasma, β-glucuronidase serves as a biomarker for the identification of human exposure to organophosphate [4]. The deficiency of this enzyme can lead to autosomal recessive disorders which is mucopolysaccharidosis VII, commonly known as Sly syndrome [4]. Genetic studies have revealed that β-glucuronidase gene

Structure of Beta-Glucuronidases
Human beta-glucuronidase is found to be as 80 kDa in monomeric form (653 amino acids residues) and its proteolysis cleaves 18 amino acid residues from the carboxy terminal end leaving it as 78 kDa monomer. It exists in homotetrameric form with 332 kDa while carrying various notable structural changes, including beta barrel type, also known as a jelly-roll barrel, and the TIM barrel [8]. The crystal structure of the enzyme has been solved by X-ray crystallography. The specific site for the transport of lysosomal β-glucuronidase is mannose 6-phosphate receptor (MPR) [9].

Chromosomal Location of Beta-Glucuronidases GAUSB Gene
Its cytogenetic location is 7q11. 21

Beta-glucuronidase and Diseases
In 2000, Sperker et al. addressed the least investigated issue regarding the excess release of beta-glucuronidase in pancreatic cancer [10]. This fatal pancreatic cancer is world's 5th cause of death. This study illustrated the excess release of enzyme by carcinoma cells as compared to normal cells. It is also up regulated in different pathological diseases like urinary tract infection and renal diseases. It also causes transplant rejection, epilepsy and cancers i-e., breast and larynx cancer. Moreover, it is involved in the bone related disorders like inflammatory joint disorder and rheumatoid arthritis, and several hepatic diseases, and later acquired immunodeficiency syndrome which is linked to the overexpression of beta-glucuronidase [11]. In many tumors and inflammatory areas, β-glucuronidase activity is high and the enzyme is secreted into the extracellular space [10]. Since then, the researchers started focusing the inhibition of beta-glucuonidase enzyme through the development of various inhibitors mainly nitrogen heterocyclic compounds such as indole, triazoles and benzothiazole [12].

Computational Screening
Virtual screening (VS) is a powerful computer-based technique which has widely been used for the identification of promising compounds such as specific inhibitors that are capable of binding with known molecular target structures. VS is considered as an imperative technique, for new inhibitors and molecular drug discovery, depending upon the growing demand of protein and nucleic acid structure. Virtual screening evaluates numerous compounds all at once, two significant steps should be well understood and perceived for this purpose. Primary concern is to find docking technique which will enable us to acquire our desired configuration [13]. To justify a docking method, various experiments of redocking are accomplished, where separation of whole series of known complexes are completed followed by the process of redocking. The secondary concern is to assure whether this docking strategy will be able to replicate the detected binding mode. Through these validation studies, nearly 10 degree of torsional freedom was found in drug like molecules of recent version of Auto-Dock. Furthermore, next step is to ensure experimentally that the compounds do bound at their predicted binding energy, allowing precise grading of compounds [13].
Solely standard deviation of 2-3 Kcal/mol, provides efficiency of energy prediction through computational based docking algorithms and Auto Dock which is insufficient for promising ranking though. The compound enrichment process is implied where predicted compounds bind tightly inferring to give away real solid bonding upon testing experimentally. In this methodology of two-step docking, Auto-Dock yields more for virtual screening, because at the start of screening interaction map is calculated only once where further working on docking simulation takes place.
Expansion of Auto Dock can be accomplished through various map modification methods. Prior to docking, grid maps are modified for contemporary chemical or physical property insertions, Example are maps for predicting covalently linked complexes, covalent maps; maps for bridging water position prediction, water maps; for de novo design of ligand, mutable atom approach; and for modelling protein flexibility, energy weighted averages [14].

Docking Based Screening (DBVS)
The workflow chart for docking-based virtual screening (DBVS) presented below functions normally using targeted structure for input, which can be modelled experimentally or on computer basis together with small-molecules containing compound library which is either purchased or synthesized ( Figure 2). Proper assignment of protonation, tautomeric, and stereoisomeric states are also the preparations required for both target and compound libraries. Using docking program, every compound of the library is docked into its target binding site virtually. An optimum amount of complementarity of steric and physicochemical properties by computationally modelling the ligand-target interaction are achieved using this program. Subsequently, a mathematic algorithm "Scoring Function" is signed to assess the accuracy level among target and the docked compound followed by ranking and selection, which is put through by binding score calculation, only some of them from a group of top-ranked compounds were selected to perform experimental assay [13,14].   [15]. The similar chemical structures of standard inhibitor for beta-glucuronidase (GusB) [18] from ZINC and PubChem were downloaded which were 500 [15]. Subsequently, all the structures were drawn using a computational tool Chem Draw and saved as cdx files. structure is available, then it is capable for molecular docking for the process of identification for the drug or inhibition quality [21].

Standard Precision (SP) and Extra Precision (XP) Docking:
It is fundamentally a structural sampling process in many docked structures to find the one with the potential, which is subsequently subjected to experimental NMR spectroscopic and X-ray crystallographic analysis. The sampling process for structures runs on genetic algorithm that stimulate Annealing, Monte-Carlo simulation, the distance geometry and miscellaneous methods,

Result and Discussion
To find the potent inhibitor of beta-glucuronidase, 6 Figure 3).   Same interactions were found in the most potent inhibitor of the series which is 1,3-benzothiazol-6-yl-(4-piperidin-1-ylphenyl) diazene. All the interaction of this compound are showed in the given 3D structure which is generated through the discovery studio.
The resulting image depicts the position of the compound inside the active pocket of beta-glucuronidase. Specificity pocket formed by the residues Trp528, Asn484, Ser485, Asn482, His509 and Tyr508 occupies by the benzothiazole ring ( Figure 4).

Conclusion
Beta-glucuronidase inhibition offers potential therapeutic target for the management of digestive tract related diseases.
Keeping this in view, the present study was designed to select and identify potential scaffolds from various classes of compounds followed by beta-glucuronidase inhibition and computational studies. Compounds identical to cognate ligand of human betaglucuronidase were retrieved from PubChem database and library was generated. The pharmacophore method was applied for the screening of potent inhibitor of beta-glucuronidase. From total 500 compounds, 9 compounds were selected and filtered out applying the above-mentioned method. The compounds were finally scrutinized based on binding energy and interactions with the active site residues. The highest binding energy was found to be for PubChem ID 128635, among benzothiazole based compounds as -32.21. Physicochemical parameters of compounds, selected after pharmacophore generation were used for the prediction of ADME profile.