Role In Inhibitor Binding Research Articles

Structure Based in Silico Screening Revealed a Potent Acinetobacter Baumannii Ftsz Inhibitor From Asinex Antibacterial Library.

The superbug Acinetobacter baumannii is an increasingly prevalent pathogen of the intensive care units where its treatment is challenging. The identification of newer drug targets and the development of propitious therapeutics against this pathogen is of utmost importance. A drug target, cell division enzyme (FtsZ), involved in A. baumannii cytokinesis is a promising avenue for antibacterial therapy. Structure based virtual screening illustrated a lead-like molecule from Asinex antibacterial library to have the best binding affinity for the FtsZ active pocket. Computational pharmacokinetics predicted the compound to have the safest pharmacokinetics profile, thus maximizing the chances of the molecule reaching the market with enhanced efficacy and lesser toxicity. Molecular dynamics simulations in an aqueous environment revealed the flexibility of protein loop regions, and upward extension followed by the backward movement of the inhibitor N, N-dimethylpyridazin-3-amine ring on its axis. The active pocket residue Thr310 demonstrated to play significant role in inhibitor binding. The binding free energy predicted by MM/GBSA and MM/PBSA reflected system stability with a total value of -62.15 kcal/mol and -10.60 kcal/mol, respectively. The absolute binding free energy estimated by WaterSwap was -16 kcal/mol that validates and affirms complex stability. The inhibitor represents a promising scaffold as a lead optimization for the FtsZ enzyme.

High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity

Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 μM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.

MD simulations reveal alternate conformations of the oxyanion hole in the Zika virus NS2B/NS3 protease.

Recent crystallography studies have shown that the binding site oxyanion hole plays an important role in inhibitor binding, but can exist in two conformations (active/inactive). We have undertaken molecular dynamics (MD) calculations to better understand oxyanion hole dynamics and thermodynamics. We find that the Zika virus (ZIKV) NS2B/NS3 protease maintains a stable closed conformation over multiple 100-ns conventional MD simulations in both the presence and absence of inhibitors. The S1, S2, and S3 pockets are stable as well. However, in two of eight simulations, the A132-G133 peptide bond in the binding pocket of S1' spontaneously flips to form a 310 -helix that corresponds to the inactive conformation of the oxyanion hole, and then maintains this conformation until the end of the 100-ns conventional MD simulations without inversion of the flip. This conformational change affects the S1' pocket in ZIKV NS2B/NS3 protease active site, which is important for small molecule binding. The simulation results provide evidence at the atomic level that the inactive conformation of the oxyanion hole is more favored energetically when no specific interactions are formed between substrate/inhibitor and oxyanion hole residues. Interestingly, however, transition between the active and inactive conformation of the oxyanion hole can be observed by boosting the valley potential in accelerated MD simulations. This supports a proposed induced-fit mechanism of ZIKV NS2B/NS3 protease from computational methods and provides useful direction to enhance inhibitor binding predictions in structure-based drug design.

Dissecting the role of mutations in chymase inhibition: Free energy and decomposition analysis

Chymase enzyme abundantly found in secretory granules of mast cells and catalyzes the hydrolysis of peptide bonds to generate angiotensin II via hydrolysis of angiotensin I and also activates transforming growth factor-b and MMP-9. MMP-9 and TGF-b have significant role in tissue inflammation and fibrosis. In present study, we investigated that Lys192Met mutation leads to a higher loss in binding energy of inhibitors than mutation Arg143Gln in chymase. The energy decomposition revealed that the contributing residues are almost same in all the forms with some change in energy value. All the results pointing that arginine and lysine residues of chymase play the most significant role in inhibitor binding revealed by energy decomposition. The Lys40, Arg90, Lys192 and Arg217 are found to be most prominent residues in two different inhibitor systems but the role of other lysine and arginine also important as they also have significant contribution in the total binding energy.

An integrated molecular modeling approach for in silico design of new tetracyclic derivatives as ALK inhibitors

Anaplastic lymphoma kinase (ALK), a promising therapeutic target for treatment of human cancers, is a receptor tyrosine kinase that instigates the activation of several signal transduction pathways. In the present study, in silico methods have been employed in order to explore the structural features and functionalities of a series of tetracyclic derivatives displaying potent inhibitory activity toward ALK. Initially docking was performed using GLIDE 5.6 to probe the bioactive conformation of all the compounds and to understand the binding modes of inhibitors. The docking results revealed that ligand interaction with Met 1199 plays a crucial role in binding of inhibitors to ALK. Further to establish a robust 3D-QSAR model using CoMFA and CoMSIA methods, the whole dataset was divided into three splits. Model obtained from Split 3 showed high accuracy ( of 0.700 and 0.682, of 0.971 and 0.974, of 0.673 and 0.811, respectively for CoMFA and CoMSIA). The key structural requirements for enhancing the inhibitory activity were derived from CoMFA and CoMSIA contours in combination with site map analysis. Substituting small electronegative groups at Position 8 by replacing either morpholine or piperidine rings and maintaining hydrophobic character at Position 9 in tetracyclic derivatives can enhance the inhibitory potential. Finally, we performed molecular dynamics simulations in order to investigate the stability of protein ligand interactions and MM/GBSA calculations to compare binding free energies of co-crystal ligand and newly designed molecule N1. Based on the coherence of outcome of various molecular modeling studies, a set of 11 new molecules having potential predicted inhibitory activity were designed.

Structural insights into binding of small molecule inhibitors to Enhancer of Zeste Homolog 2.

Enhancer of Zeste Homolog 2 (EZH2) is a SET domain protein lysine methyltransferase (PKMT) which has recently emerged as a chemically tractable and therapeutically promising epigenetic target, evidenced by the discovery and characterization of potent and highly selective EZH2 inhibitors. However, no experimental structures of the inhibitors co-crystallized to EZH2 have been resolved, and the structural basis for their activity and selectivity remains unknown. Considering the need to minimize cross-reactivity between prospective PKMT inhibitors, much can be learned from understanding the molecular basis for selective inhibition of EZH2. Thus, to elucidate the binding of small-molecule inhibitors to EZH2, we have developed a model of its fully-formed cofactor binding site and used it to carry out molecular dynamics simulations of protein-ligand complexes, followed by molecular mechanics/generalized born surface area calculations. The obtained results are in good agreement with biochemical inhibition data and reflect the structure-activity relationships of known ligands. Our findings suggest that the variable and flexible post-SET domain plays an important role in inhibitor binding, allowing possibly distinct binding modes of inhibitors with only small variations in their structure. Insights from this study present a good basis for design of novel and optimization of existing compounds targeting the cofactor binding site of EZH2.

Induced opening of influenza virus neuraminidase N2 150-loop suggests an important role in inhibitor binding

The recently discovered 150-cavity (formed by loop residues 147–152, N2 numbering) adjacent to the enzymatic active site of group 1 influenza A neuraminidase (NA) has introduced a novel target for the design of next-generation NA inhibitors. However, only group 1 NAs, with the exception of the 2009 pandemic H1N1 NA, possess a 150-cavity, and no 150-cavity has been observed in group 2 NAs. The role of the 150-cavity played in enzymatic activity and inhibitor binding is not well understood. Here, we demonstrate for the first time that oseltamivir carboxylate can induce opening of the rigid closed N2 150-loop and provide a novel mechanism for 150-loop movement using molecular dynamics simulations. Our results provide the structural and biophysical basis of the open form of 150-loop and illustrates that the inherent flexibility and the ligand induced flexibility of the 150-loop should be taken into consideration for future drug design.

Cysteine Residues in the Transmembrane (TM) 9 to TM11 Region of the Human Equilibrative Nucleoside Transporter Subtype 1 Play an Important Role in Inhibitor Binding and Translocation Function

Inhibitor and substrate interactions with equilibrative nucleoside transporter 1 (ENT1; SLC29A1) are known to be affected by cysteine-modifying reagents. A previous study from our laboratory established Cys222 in transmembrane (TM) 6 as the residue responsible for methyl methanethiosulfonate (a membrane-permeable sulfhydryl modifier)-mediated enhancement of the binding of the ENT1 inhibitor nitrobenzylmercaptopurine riboside (NBMPR) in intact cells. However, the capacity of charged sulfhydryl reagents to inhibit the binding of NBMPR in broken cell preparations (allowing cytoplasmic access) was not affected by mutation of any of the cysteines (Cys87, 193, 213, or 222) in the N-terminal half of the protein. We thus hypothesized that the inhibitory effects of the modifiers were due to the one or more of the six cysteine residues in the C-terminal half of ENT1, particularly one or both of those in the fifth intracellular loop (Cys414 and Cys416). Each of the cysteines were mutated to serine or alanine and expressed in nucleoside transport-deficient PK15 cells and probed with a series of methanethiosulfonate sulfhydryl-modifying reagents. Transporter function was assessed by the site-specific binding of [(3)H]NBMPR and the cellular uptake of [(3)H]2-chloroadenosine. These studies established that Cys378 is an extracellular-located residue modified by [2-(trimethylammonium)ethyl] methane-thiosulfonate (MTSET) to inhibit the binding of NBMPR to intact cells. Mutation of Cys414 led to an enhancement of the ability of MTSET to inhibit NBMPR binding, and this enhancement was eliminated by the comutation of Cys378, indicating that disruption of the fifth intracellular loop modifies the conformation of TM10 and its extracellular extension. Mutation of Cys416 led to the loss of the ability of the charged sulfhydryl reagents to inhibit NBMPR binding in isolated membranes and also led to the loss of transport function. This finding further supports an allosteric interaction between the fifth intracellular loop and the extracellular NBMPR binding domain and implicates this region in the translocation function of human ENT1.

The selectivity of tyrosine 280 of human 11β-hydroxysteroid dehydrogenase type 1 in inhibitor binding

11β-Hydroxysteroid dehydrogenase type 1 is a homodimer where the carboxyl terminus of one subunit covers the active site of the dimer partner. Based on the crystal structure with CHAPS, the carboxyl terminal tyrosine 280 (Y280) has been postulated to interact with the substrate/inhibitor at the binding pocket of the dimer partner. However, the co-crystal structure with carbenoxolone argues against this role. To clarify and reconcile these findings, here we report our mutagenesis data and demonstrate that Y280 is not involved in substrate binding but rather plays a selective role in inhibitor binding. The involvement of Y280 in inhibitor binding depends on the inhibitor chemical structure. While Y280 is not involved in the binding of carbenoxolone, it is critical for the binding of glycyrrhetinic acid.

The role of tyrosine 177 in human 11β-hydroxysteroid dehydrogenase type 1 in substrate and inhibitor binding: an unlikely hydrogen bond donor for the substrate

The catalytic motif (YSASK) at the active site of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is conserved across different species. The crystal structures of the human, guinea pig and mouse enzymes have been resolved to help identify the non-conserved residues at the active site. A tyrosine residue (Y177) upstream of the catalytic motif in human 11β-HSD1 represents the largest difference at the active sites between the human and the rodent enzyme where the corresponding residue is glutamine. Although Y177 was postulated as a potential hydrogen bond donor in substrate binding in crystal structure-based modeling, no experimental evidence is available to support this notion. Here, we report that Y177 is not a hydrogen bond donor in substrate binding because removal of the hydroxyl group from its side chain by mutagenesis (Y177F) did not significantly change the K m value for cortisone. However, removal of the hydrophobic side chain by changing tyrosine to alanine (Y177A) or substitution with a hydrophilic side chain by changing tyrosine to glutamine (Y177Q) increased K m values for cortisone. These data suggest that Y177 is involved in substrate binding through its hydrophobic side chain but not by hydrogen bonding. In addition, the three mutations had little effect on the binding of the rodent substrate 11-dehydrocorticosterone, suggesting that Y177 does not confer substrate specificity. However, the same mutations reduced the affinity of the licorice derived 11β-HSD1 inhibitor glycyrrhetinic acid by about 6- to 10-fold. Interestingly, the affinity of carbenoxolone, the hemisuccinate ester of glycyrrhetinic acid with a similar potency against the wildtype enzyme, was not drastically affected by the same mutations at Y177. These data suggest that Y177 has a unique role in inhibitor binding. Molecular modeling with glycyrrhetinic acid led to findings consistent with the experimental data and provided potential interaction mechanisms. Our data suggest that Y177 plays an important role in both substrate and inhibitor binding but it is unlikely a hydrogen bond donor for the substrate.

Residues Distant from the Active Site Influence Protein-tyrosine Phosphatase 1B Inhibitor Binding

Regions of protein-tyrosine phosphatase (PTP) 1B that are distant from the active site yet affect inhibitor binding were identified by a novel library screen. This screen was based on the observation that expression of v-Src in yeast leads to lethality, which can be rescued by the coexpression of PTP1B. However, this rescue is lost when yeast are grown in the presence of PTP1B inhibitors. To identify regions of PTP1B (amino acids 1-400, catalytic domain plus 80-amino acid C-terminal tail) that can affect the binding of the difluoromethyl phosphonate (DFMP) inhibitor 7-bromo-6-difluoromethylphosphonate 3-naphthalenenitrile, a library coexpressing PTP1B mutants and v-Src was generated, and the ability of yeast to grow in the presence of the inhibitor was evaluated. PTP1B inhibitor-resistant mutations were found to concentrate on helix alpha7 and its surrounding region, but not in the active site. No resistant amino acid substitutions were found to occur in the C-terminal tail, suggesting that this region has little effect on active-site inhibitor binding. An in-depth characterization of a resistant substitution localizing to region alpha7 (S295F) revealed that this change minimally affected enzyme catalytic activity, but significantly reduced the potency of a panel of structurally diverse DFMP PTP1B inhibitors. This loss of inhibitor potency was found to be due to the difluoro moiety of these inhibitors because only the difluoro inhibitors were shifted. For example, the inhibitor potency of a monofluorinated or non-fluorinated analog of one of these DFMP inhibitors was only minimally affected. Using this type of library screen, which can scan the nearly full-length PTP1B sequence (catalytic domain and C-terminal tail) for effects on inhibitor binding, we have been able to identify novel regions of PTP1B that specifically affect the binding of DFMP inhibitors.

Probing the ultra-high resolution structure of aldose reductase with molecular modelling and noncovalent mass spectrometry

Aldose reductase, the first and rate-limiting enzyme of the polyol pathway, is a target for drug design for the treatment of diabetes complications. The structures of aldose reductase in complex with the cyclic imide inhibitors Fidarestat and Minalrestat were recently determined at ultra-high resolution ( Proteins 2004, 55, 805). We have used the detailed structural information revealed at atomic resolution, including the assignment of protonation states for the inhibitors and active site residues, together with molecular modelling and noncovalent mass spectrometry to characterise the type and strength of the interactions between the enzyme and the inhibitors, and to attempt the design of novel potential inhibitors with enhanced binding energies of the complexes. The VC 50 values measured by mass spectrometry (accelerated voltage of ions needed to dissociate 50% of a noncovalent complex in the gas phase) for the aldose reductase inhibitors correlate with the IC 50 values (concentration of inhibitor giving 50% inhibition in solution) and with the electrostatic binding energies calculated between the active site residues Tyr48, His110 and Trp111 and the inhibitors, suggesting that electrostatic interactions play a major role in inhibitor binding. Our molecular modelling and design studies suggest that the replacement of the fluorine atom in Minalrestat's bromo-fluorobenzyl group with nitro, amide and carboxylate functional groups enhanced the predicted net binding energies of the complexes by 16%, 31% and 68%, respectively. When the carbamoyl group of Fidarestat was replaced with a nitro, 4-hydroxyl phenyl and carboxylate functional groups, the predicted net binding energies of the complexes were enhanced by 13%, 34% and 46%, respectively.

Structural Characterization of the GSK-3β Active Site Using Selective and Non-selective ATP-mimetic Inhibitors

GSK-3β is a regulatory serine/threonine kinase with a plethora of cellular targets. Consequently, selective small molecule inhibitors of GSK-3β may have a variety of therapeutic uses including the treatment of neurodegenerative diseases, type II diabetes and cancer. In order to characterize the active site of GSK-3β, we determined crystal structures of unphosphorylated GSK-3β in complex with selective and non-selective ATP-mimetic inhibitors. Analysis of the inhibitors' interactions with GSK-3β in the structures reveals how the enzyme can accommodate a number of diverse molecular scaffolds. In addition, a conserved water molecule near Thr138 is identified that can serve a functional role in inhibitor binding. Finally, a comparison of the interactions made by selective and non-selective inhibitors highlights residues on the edge of the ATP binding-site that can be used to obtain inhibitor selectivity. Information gained from these structures provides a promising route for the design of second-generation GSK-3β inhibitors.

Molecular basis for p38 protein kinase inhibitor specificity.

p38 is a member of the mitogen-activated protein (MAP) kinase family and is a critical enzyme in the proinflammatory cytokine pathway. Other MAP kinase group members that share both structural and functional homology to p38 include the c-Jun NH2-terminal kinases (JNKs or SAPKs) and the extracellular-regulated protein kinases (ERKs). In this study, we determined the molecular basis for p38alpha inhibitor specificity exhibited by five compounds in the diarylimidazole, triarylimidazole, and triarylpyrrole classes of protein kinase inhibitors. These compounds are significantly more potent inhibitors of p38 compared to the JNKs and ERKs. Three active site ATP-binding domain residues in p38, T106, M109, and A157, selected based on primary sequence alignment, molecular modeling, and X-ray crystal structure data, were mutated to assess their role in inhibitor binding and enzymatic catalysis. All mutants, with the exception of T106M, had kinase activity within 3-fold of wild-type p38. Mutation of T106 to glutamine, the residue present at the corresponding position in ERK-2, or methionine, the corresponding residue in p38gamma, p38delta, and the JNKs, rendered all five inhibitors ineffective. The diarylimidazoles had approximately a 6-fold decrease in potency toward M109A p38. For the mutant A157V, all diarylimidazoles and triarylimidazoles tested were 5-10-fold more potent compared with wild-type p38. In contrast, two triarylpyrroles were 15-40-fold less potent versus A157V p38. These results showed that the molecular basis for the specificity of the p38 inhibitors was attributed largely to threonine 106 in p38 and that methionine 109 contributes to increased binding affinity for imidazole based inhibitors.

Thermodynamics of a transition state analogue inhibitor binding to Escherichia coli chorismate mutase: probing the charge state of an active site residue and its role in inhibitor binding and catalysis.

Electrostatic interactions play important roles in the catalysis of chorismate to prephenate by chorismate mutase. Mutation of Gln88 to glutamate in the monofunctional chorismate mutase from Escherichia coli results in an enzyme with a pH profile of activity significantly different from that of the wild type protein. To investigate whether the mutation alters the substrate binding process or the catalysis, we have directly determined the thermodynamic parameters of a transition state analogue inhibitor binding to the wild-type chorismate mutase and its Q88E mutant using isothermal titration calorimetry. The results demonstrate that solvent reorganization and hydrophobic interactions contribute the predominant free energy to inhibitor binding. The charge state of Glu88 in the Q88E mutant was experimentally determined and was shown to be protonated at pH 4.5 and ionized at pH 7.8, consistent with earlier hypotheses. Most surprisingly, inhibitor binding energetics do not exhibit significant pH dependency for both enzymes. Our findings indicate that the charge state of Glu88 has a small impact on inhibitor binding but plays an important role in the catalytic process.

In vitro selection of HIV-1 resistance of ddI is not as efficient as for other antiretroviral drugs and yields virus which shows reduced cytophatic activity

The locations of HIV-1 RT nucleoside and non-nucleoside inhibitor-binding sites and inhibitor-resistance mutations are analyzed in the context of the three-dimensional structure of the enzyme and implications for mechanisms of drug inhibition and resistance are discussed. In order to help identify residues that may play a role in inhibitor binding, solvent accessibilities of amino acids that comprise the inhibitor-binding sites in the structure of HIV-1 RT complexed with a dsDNA template-primer are analyzed. While some mutations that cause resistance to nucleoside analogs, such as AZT, ddI, and ddC, are located near enough to the dNTP-binding site to direct interfere with binding of nucleoside analogs many are located away from the DNTP-binding site and more likely confer resistance by other mechanisms. Many of the latter mutations are located on the surface of the DNA-binding cleft and may lead to altered template-primer positioning or conformation, causing a distortion of the geometry of the polymerase active site and consequent discrimination between normal and altered DNTP substrates. Other nucleoside analog-resistance mutations located on the periphery of the DNTP-binding site may exert their effects via altered interactions with DNTP-binding site residues. The structure of the hydrophobic region in HIV-1 RT that binds non-nucleoside inhibitors, for example, nevirapine and TIBO, has been analyzed in the absence of bound ligand. The pocket that is present when non-nucleoside inhibitors are bond is not observed in the inhibitor-free structure of HIV-1 RT with dsdna. In particular it is filled by Tyr181 and Tyr188, suggesting that the pocket is formed primarily by rotation of these large aromatic side-chains. Existing biochemical data, taken together with the three-dimensional structure of HIV-1 RT, makes it possible to propose potential mechanisms of inhibition by non-nucleoside inhibitors. One such mechanism is local distortion of HIV-1 RT structural elements thought to participate in catalysis: the β9-β10 hairpin (which contains polymerase active site residues) and the β12-β13 hairpin ("primer grip"). An alternative possibility is restricted mobility of the p66 thumb subdomain, which is supported by the observation that structural elements of the non-nucleoside inhibitor-binding pocket may act as a "hinge" for the thumb. Mutations that have been shown to confer resistance to non-nucleoside inhibitors are observed to cluster around the pocket, suggesting that most of these resistance mutations lead to direct alteration of inhibitor binding. A comparison of residues in the non-nucleoside inhibitor-binding pocket of HIV-1 RT and the corresponding residues in HIV-2 RT can explain the low activity of non-nucleoside compounds against HIV-2 RT.

Vinylogous Sulfonylureas: A New Class of Acetohydroxyacid Synthase Inhibitors Incorporating a Large Bridging Moiety

Several acrylic acid derivatives incorporating elements of sulfonylurea structure around a central vinylogous core were synthesized and found to be moderate inhibitors of the enzyme, acetohydroxyacid synthase (AHAS). Some com pounds showed post-emergence herbicidal activity against mustard. Biological activity was found to be markedly influenced by the nature of the 2-arylcarbamoyl group. When this group contained a phenyl ring the compounds were inactive. A 4,6-dim ethoxypyrimidin-2-ylcarbamoyl function resulted in the best activity but this activity was highly dependent on the nature of the 4,6-substituents on the pyrimidine ring, with 4,6-dim ethylpyrimidines showing very little activity. The structures of the most active compound, ethyl 3-(2-chlorophenyl)sulfonylamino-3-m ethylthio-2-[(4,6-dimethoxypyrimidin- 2-yl)carbam oyl]acrylate, and an inactive analogue, 4,6-dimethylpyrimidin-2-yl-3-(2-chlorophenyl) sulfonylamino-3-methylthio-2-cyanoacrylate, were determined by X -ray crystallography. The resulting structures showed several conformational differences which may play an important role in inhibitor binding to AHAS and the resultant herbicidal activity.