Carbonic anhydrase IX is a tumor-associated membrane-bound metallo-enzyme which catalyzes the reversible hydration of carbon dioxide (CO) to bicarbonate (HCO) and proton (H) ions. It is a hypoxia-inducible enzyme and plays a critical role in tumor pH homeostasis favoring tumor cell invasiveness and drug resistance. Over expression of CAIX is documented in cancers of breast, lung, kidney, colon/rectum, etc. Chemical inhibition of CAIX activity has proven to be an effective therapeutic modality towards targeting cancer. Hence, in this study, we intend to identify potential molecules from NCI (National Cancer Institute) and Maybridge databases implementing high-throughput virtual screening. CAIX co-crystallized with acetazolamide (a known inhibitor of CAIX) (PDB ID: 3IAI) was used for reference-guided docking protocol. The potential inhibitors among the coupled data sets were finalized based on Glide docking score, Prime/MMGBSA scoring, significant intermolecular interactions, ADMET (absorption, distribution, metabolism and excretion, toxicity) prediction and stability of complex formation, molecular dynamics simulation, and comparative analysis. By this study, we propose NSC_93618, NSC_170253, NSC_93618, JFD03677, SEW06488, and BTB09372 to be highly significant, as all these compounds were found to qualify as potential leads surpassing all the stringent filtering process. However, NSC_93618 was found to be the most potential, as it featured with higher complex stability with strong bonded interactions, binding affinity synonymous to acetazolamide. Hence, these proposed compounds shall prove to be effective in targeting CAIX towards modulating carcinogenesis.
Lysyl oxidase (LOX) is a copper dependent amine oxidase which catalyses the cross linking of collagen and elastin towards the maturation of extracellular matrix. The expression and activity of LOX is known to vary under pathological conditions such as tumorigenesis, hyperhomocysteinemia, copper deficiency diseases, pseudoexfoliation syndrome and proliferative diabetic retinopathy. Despite the implication of LOX in many diseases, there is inadequate information about its structure. Therefore, we describe a molecular model of Human Lysyl Oxidase (LOX) with optimal copper orientation in the catalytic cavity for induced fit docking studies with potential modulators. The predicted model was found to be highly plausible as per the stereochemistry checks. Further, Molecular Dynamics (MD) studies also inferred the stability of the predicted structure. We performed Induced Fit Docking (IFD) of LOX modulators to the predicted structure and also validated the molecular interactions in implicit solvent model by calculating Molecular Mechanics Generalized Born Surface Area (MMGBSA). The IFD results strongly reveal that aspartic acid residues in the catalytic cavity as the key players in establishing interactions with small molecules. The insights from this study will aid in better exploration of the structure-function relationship of LOX.
Fatty acid synthase (FASN, EC 2.3.1.85), is a multi-enzyme dimer complex that plays a critical role in lipogenesis. This lipogenic enzyme has gained importance beyond its physiological role due to its implications in several clinical conditions-cancers, obesity, and diabetes. This has made FASN an attractive pharmacological target. Here, we have attempted to predict the theoretical models for the human enoyl reductase (ER) and β-ketoacyl reductase (KR) domains based on the porcine FASN crystal structure, which was the structurally closest template available at the time of this study. Comparative modeling methods were used for studying the structure-function relationships. Different validation studies revealed the predicted structures to be highly plausible. The respective substrates of ER and KR domains-namely, trans-butenoyl and β-ketobutyryl-were computationally docked into active sites using Glide in order to understand the probable binding mode. The molecular dynamics simulations of the apo and holo states of ER and KR showed stable backbone root mean square deviation trajectories with minimal deviation. Ramachandran plot analysis showed 96.0% of residues in the most favorable region for ER and 90.3% for the KR domain, respectively. Thus, the predicted models yielded significant insights into the substrate binding modes of the ER and KR catalytic domains and will aid in identifying novel chemical inhibitors of human FASN that target these domains.
Human fatty acid synthase (hFASN), a homo dimeric lipogenic enzyme with seven catalytic domains, is an important clinical target in cancer, metabolic syndrome and infections. Here, molecular modelling and docking methods were implemented to examine the inter-molecular interactions of thioesterase (TE) domain in hFASN with its physiological substrate, and to identify potential chemical inhibitors. TE catalyses the hydrolysis of thioester bond between palmitate and the 4' phosphopantetheine of acyl carrier protein, releasing 16-carbon palmitate. The crystal structure of hFASN TE in two inhibitory conformations (A and B) were geometry-optimized and used for molecular docking with palmitate, orlistat (a known FASN inhibitor) and virtual screening against compounds from National Cancer Institute (NCI) database. Relatively, low binding affinity was observed during the complex formation of palmitate with A (-.164 kcal/mol) and B (-.332 kcal/mol) forms of TE, when compared with orlistat-docked TE (A form: -5.872 kcal/mol and B form: -5.484 kcal/mol), clearly indicating that the native inhibited conformation (crystal structure) was unfavourable for substrate binding. We used these orlistat dual binding modes as positive controls for prioritizing the ligands during virtual screening. From 2, 31,617 molecules in the NCI database, 916 high-scoring compounds (hit ligands) were obtained for A-form and 4582 for B-form of the TE-domain, which were then ranked according to glide docking score, XP H bond score, absorption, distribution, metabolism and excretion and binding free energy (Prime/MM-GBSA). Consequently, two top scoring ligands (NSC: 319661 and NSC: 153166) emerged as promising drug candidates that may be tested in FASN-over-expressing diseases.
Fatty acid synthase (FASN, UniProt ID: P49327) is a multienzyme dimer complex that plays a critical role in lipogenesis. Consequently, this lipogenic enzyme has gained tremendous biomedical importance. The role of FASN and its inhibition is being extensively researched in several clinical conditions, such as cancers, obesity, and diabetes. X-ray crystallographic structures of some of its domains, such as β-ketoacyl synthase, acetyl transacylase, malonyl transacylase, enoyl reductase, β-ketoacyl reductase, and thioesterase, (TE) are already reported. Here, we have attempted an in silico elucidation of the uncrystallized dehydratase (DH) catalytic domain of human FASN. This theoretical model for DH domain was predicted using comparative modeling methods. Different stand-alone tools and servers were used to validate and check the reliability of the predicted models, which suggested it to be a highly plausible model. The stereochemical analysis showed 92.0% residues in favorable region of Ramachandran plot. The initial physiological substrate β-hydroxybutyryl group was docked into active site of DH domain using Glide. The molecular dynamics simulations carried out for 20 ns in apo and holo states indicated the stability and accuracy of the predicted structure in solvated condition. The predicted model provided useful biochemical insights into the substrate–active site binding mechanisms. This model was then used for identifying potential FASN inhibitors using high-throughput virtual screening of the National Cancer Institute database of chemical ligands. The inhibitory efficacy of the top hit ligands was validated by performing molecular dynamics simulation for 20 ns, where in the ligand NSC71039 exhibited good enzyme inhibition characteristics and exhibited dose-dependent anticancer cytotoxicity in retinoblastoma cancer cells in vitro.
Carbonic anhydrase IX (CAIX) is a membrane-bound enzyme associated with tumor hypoxia and found to be over expressed in various tumor conditions. Targeting CAIX catalytic activity is proven to be efficient modality in modulating pH homeostasis in cancer cells. Proteoglycan-like (PG) region is unique to CAIX and is proposed to serve as an antenna enhancing the export of protons in conjunction with facilitated efflux of lactate ions via monocarboxylate transporters. Moreover, the PG region is also reported to contribute to the assembly and maturation of focal adhesion links during cellular attachment and dispersion on solid supports. Thus, drug targeting of this region shall efficiently modulate pH homeostasis and cell adhesion in cancer cells. As the PG region is intrinsically disordered, the complete crystal structure is not elucidated. Hence, in this study, we intend to sample the conformational landscape of the PG region at microsecond scale simulation in order to sample the most probable conformations that shall be utilized for structure-based drug design. In addition, the sampled conformations were subjected to high-throughput virtual screening against NCI and Maybridge datasets to identify potential hits based on consensus scoring and validation by molecular dynamics simulation. Further, the identified hits were experimentally validated for efficacy by in vitro and direct enzymatic assays. The results reveal 5-(2-aminoethyl)-1,2,3-benzenetriol to be the most promising hit as it showed significant CAIX inhibition at all levels of in silico and experimental validation.
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