Molecular docking of cathepsin L inhibitors in the binding site of papain

The papain/CLIK-148 coordinate system was employed as a model to study the interactions of a nonpeptide thiocarbazate inhibitor of cathepsin L ( 1). This small molecule inhibitor, a thiol ester containing a diacyl hydrazine functionality and one stereogenic center, was most active as the S-enantiomer, with an IC 50 of 56 nM; the R-enantiomer ( 2) displayed only weak activity (33 microM). Correspondingly, molecular docking studies with Extra Precision Glide revealed a correlation between score and biological activity for the two thiocarbazate enantiomers when a structural water was preserved. The molecular interactions between 1 and papain were very similar to the interactions observed for CLIK-148 ( 3a and 3b) with papain, especially with regard to the hydrogen-bonding and lipophilic interactions of the ligands with conserved residues in the catalytic binding site. Subsequent docking of virtual compounds in the binding site led to the identification of a more potent inhibitor ( 5), with an IC 50 of 7.0 nM. These docking studies revealed that favorable energy scores and correspondingly favorable biological activities could be realized when the virtual compound design included occupation of the S2, S3, and S1' subsites by hydrophobic and aromatic functionalities of the ligand, and at least three hydrogen bonding contacts between the ligand and the conserved binding site residues of the protein.     

Interactions between procyanidin oligomers and the active form of matrix metalloproteinase-7: A theoretical insight

The objective of this study was to determine the sites and modes of interaction of the most common oligomers of B-type procyanidins (PCs) with the active form of the matrix metalloproteinase-7 (MMP-7) and some molecular properties of the PCs by using theoretical methods. These data may provide useful insights into the ability of PCs to act as selective MMP-7 inhibitors. Some stereoisomers that predominated in the analyzed PCs (PB2, PC1, tetramer) could act as selective inhibitors of MMP-7 due to their interaction with amino acids of the subsites S2 and/or S1′ in the active site, and with some specific amino acids of MMP-7 that bind to cholesterol sulfate to promote proteolysis of the cell membrane. Hydrogen bonding was the main interaction of PB2 and PC1 with MMP-7, while in the tetramer, the van der Waals interactions prevailed. The determination of molecular properties such as chemical reactivity and stability by the highest occupied molecular orbital and lowest unoccupied molecular orbital gap, revealed that oligomers of PCs acquired very stable poses in their docking with MMP-7. Polarizability seems to be an important factor for PCs with large molecular volume (PC1, tetramer) to establish contact with amino acids of narrow subsites in the active site (S2, S1′) and amino acids located outside the active site.     

Studying enzyme binding specificity in acetylcholinesterase using a combined molecular dynamics and multiple docking approach

A combined molecular dynamics simulation and multiple ligand docking approach is applied to study the binding specificity of acetylcholinesterase (AChE) with its natural substrate acetylcholine (ACh), a family of substrate analogues, and choline. Calculated docking energies are well correlated to experimental k(cat)/K(M) values, as well as to experimental binding affinities of a related series of TMTFA inhibitors. The "esteratic" and "anionic" subsites are found to act together to achieve substrate binding specificity. We find that the presence of ACh in the active site of AChE not only stabilizes the setup of the catalytic triad but also tightens both subsites to achieve better binding. The docking energy gained from this induced fit is 0.7 kcal/mol for ACh. For the binding of the substrate tailgroup to the anionic subsite, both the size and the positive charge of the tailgroup are important. The removal of the positive charge leads to a weaker binding of 1 kcal/mol loss in docking energy. Substituting each tail methyl group with hydrogen results in both an incremental loss in docking energy and also a decrease in the percentage of structures docked in the active site correctly set up for catalysis.     

Probing structural determinants distal to the site of hydrolysis that control substrate specificity of the 20S proteasome

The 20S proteasome is a large multicomponent protease complex. Relatively little is known about the mechanisms that control substrate specificity of its multiple active sites. We present here the crystal structure at 2.95 A resolution of a beta2-selective inhibitor (MB1) bound to the yeast 20S proteasome core particle (CP). This structure is compared to the structure of the CP bound to a general inhibitor (MB2) that covalently modified all three (beta1, beta2, beta5) catalytic subunits. These two inhibitors differ only in their P3 and P4 residues, thereby highlighting binding interactions distal to the active site threonine that control absolute substrate specificity of the complex. Comparisons of the CP-bound structures of MB1, MB2, and the natural products epoxomycin and TMC-95A also provide information regarding general binding modes for several classes of proteasome inhibitors.     

Vertebrate collagenase inhibitor. II. Tetrapeptidyl hydroxamic acids.

To develop a potent and specific collagenase inhibitor, a series of tetrapeptidyl hydroxamic acids were synthesized, based on the previous findings with tripeptidyl derivatives (Chem. Pharm. Bull., 38, 1007-1011, 1990). Among the series of tetrapeptidyl derivatives synthesized, R-Gly-Pro-Leu-Ala-NHOH and R-Gly-Pro-D-Leu-D-Ala-NHOH were found to be highly specific and potent inhibitors against vertebrate collagenase with an IC50 of 10(-6) M order, where R stands for Boc or acyl group. Analysis of their structure-activity relationships showed a characteristic feature of the substrate-binding site of collagenase as follows: 1) the S1 subsite forms a shallow hydrophobic pocket, although glycine residue corresponds to the subsite of the natural collagen substrate: 2) the S2 subsite constitutes a bulky pocket with less requirement for hydrophobicity: 3) the S3 subsite preferentially accommodates Pro residue: and 4) the accommodation of the P4-P1 subsites of peptidyl collagenase inhibitor to the S4-S1 subsites is required to form a tight binding of its hydroxamic acid moiety to the zinc ion at the catalytic site of the enzyme. The introduction of an enantiometric dipeptide unit, D-Leu-D-Ala, to the P2-P1 subsites demonstrated an increased binding capacity to the extended S4-S1 subsites of collagenase, thus providing proteinase-resistant inhibitor.     

Understanding binding affinity: a combined isothermal titration calorimetry/molecular dynamics study of the binding of a series of hydrophobically modified benzamidinium chloride inhibitors to trypsin

The binding of a series of p-alkylbenzamidinium chloride inhibitors to the serine proteinase trypsin over a range of temperatures has been studied using isothermal titration (micro)calorimetry and molecular dynamics simulation techniques. The inhibitors have small structural variations at the para position of the benzamidinium ion. They show small differences in relative binding affinity but large compensating differences in enthalpy and entropy. Binding affinity decreases with increased branching at the first carbon but increases with increasing the length of a linear alkyl substituent, suggesting that steric hindrance and hydrophobic interactions play dominant roles in binding. Structural analysis showed that the backbone of the enzyme was unaffected by the change of the para substituent. In addition, binding does not correlate strongly with octanol/water partition data. To further characterize this system, the change in the heat capacity on binding, the change in solvent-accessible surface area on binding, the effect of inhibitor binding on the hydration of the active site, the pK(a) of His57, and interactions within the catalytic triad have been investigated. Although the changes in inhibitor structure are small, it is demonstrated that simple concepts such as steric hindrance, hydrophobicity, and buried surface area are insufficient to explain the binding data. Other factors, such as access to the binding site and the cost of dehydration of the active site, are of equal or greater importance.     

Bcl-2蛋白抑制剂结合腔的性质分析

采用多重拷贝同时搜寻(MCSS)等方法对Bcl-2蛋白抑制剂结合腔进行分析. 结果显示, 结合腔可分成P1, L1, P2, P3和P4等5个区域, 其底部呈疏水性, 而P3部位不适合芳香性大基团的结合. 结合腔侧面和边缘处分布有可与配体形成除疏水以外作用的多个重要残基. MCSS计算得到的各种性质官能团在结合腔内的能量优势位置和取向能与已知结合模式的高活性抑制剂的重要基团位置吻合得较好.     

Hydroxamate类抑制剂与MMP-2的分子对接研究

通过分子动力学模拟研究了MMP-2和hydroxamate抑制剂之间的作用模式。在分子动力学模拟中,对于催化区的锌离子和其共价结合的配体(包括抑制剂和组氨酸)采用了键合的模型。从模拟的结果可以看到,R^1取代基和MMP-2的S1疏水口袋中的部分残基能形成很好的几何匹配,从而可以产生很强的范德华和疏水相互作用。模拟结果也表明,两个抑制剂和MMP-2之间分别能形成5个和8个氢键,抑制剂B比A活性更高的原因就是能够形成更加有利氢键作用模式。在整个模拟过程中,催化锌都能保持好的五配位形式,配位键的长度也处于稳定的状态,预测得到的MMP-2和其抑制剂的相互作用模式对于全新抑制剂的设计提供了非常重要的结构信息。     

Allostery Inhibition of BACE1 by Psychotic and Meroterpenoid Drugs in Alzheimer’s Disease Therapy

In over a century since its discovery, Alzheimer’s disease (AD) has continued to be a global health concern due to its incurable nature and overwhelming increase among older people. In this paper, we give an overview of the efforts of researchers towards identifying potent BACE1 exosite-binding antibodies and allosteric inhibitors. Herein, we apply computer-aided drug design (CADD) methods to unravel the interactions of some proposed psychotic and meroterpenoid BACE1 allosteric site inhibitors. This study is aimed at validating the allosteric potentials of these selected compounds targeted at BACE1 inhibition. Molecular docking, molecular dynamic (MD) simulations, and post-MD analyses are carried out on these selected compounds, which have been experimentally proven to exhibit allosteric inhibition on BACE1. The SwissDock software enabled us to identify more than five druggable pockets on the BACE1 structural surface using docking. Besides the active site region, a melatonin derivative (compound 1) previously proposed as a BACE1 allostery inhibitor showed appreciable stability at eight different subsites on BACE1. Refinement with molecular dynamic (MD) simulations shows that the identified non-catalytic sites are potential allostery sites for compound 1. The allostery and binding mechanism of the selected potent inhibitors show that the smaller the molecule, the easier the attachment to several enzyme regions. This finding hereby establishes that most of these selected compounds failed to exhibit strong allosteric binding with BACE1 except for compound 1. We hereby suggest that further studies and additional identification/validation of other BACE1 allosteric compounds be done. Furthermore, this additional allosteric site investigation will help in reducing the associated challenges with designing BACE1 inhibitors while exploring the opportunities in the design of allosteric BACE1 inhibitors.     

Computational study of the catalytic domain of human neutrophil collagenase. specific role of the S3 and S'3 subsites in the interaction with a phosphonate inhibitor

Human neutrophil collagenase (HNC, MMP-8) is one of the target enzymes for drug treatment of pathologic extracellular matrix degradation. Peptidomimetic inhibitors bind in the S-side of the enzyme active site occupying the S ₁primary specificity pocket by their large hydrophobic side-chains. The crystal structure of the complex between the catalytic domain of MMP-8 and Pro-Leu-l-Trp^P(OH)₂(PLTP) showed that this phosphonate inhibitor binds in the S side of the active site. This finding was unexpected since it represents the first example of accommodation of the bulky Trp indolyl chain in the S₁rather than in the S ₁subsite. Dynamical and structural factors favouring this uncommon mode of binding were therefore investigated.MD simulations performed on the uncomplexed enzyme show that its structure in aqueous solution is only slightly different from the crystal structure found in the complex with PLTP. ED analysis of the MD simulations, performed on PLTP alternatively interacting with the S- or S-side of the active site, shows that the enzyme fluctuation increases in both cases. The main contribution to the overall enzyme fluctuation is given by the loop 164–173. The fluctuation of this loop is spread over more degrees of freedom when PLTP interacts with the S-side. This dynamical factor can enhance the preference of PLTP for the S subsites of MMP-8. MD simulations also show that ligation of PLTP in the S subsites is further favoured by better zinc chelation, a cation- interaction at the S₃subsite and unstrained binding conformations. The role of the S₃, S ₃and S ₁subsites in determining the inhibitor binding is discussed.     

Physical nature of interactions within the active site of cytosine-5-methyltransferase

The physical nature of interactions within the active site of cytosine-5-methyltransferase (CMT) was studied using a variation-perturbation energy decomposition scheme defining a sequence of approximate intermolecular interaction energy models. These models have been used to analyze the catalytic activity of residues constituting cytosine-5-methyltransferase active site as well their role in the binding group of de novo designed inhibitors. Our results indicate that Glu119, Arg163, and Arg165 appear to play the dominant role in stabilizing the protonated transition state structure and their influence can be qualitatively approximated by electrostatic interactions alone. The stabilization of neutral structures of the alternative reaction pathway is small, which might suggest the protonated pathway as preferred by the enzyme. Exchange and delocalization terms are negligible in most cases, or they cancel each other to some extent. Interactions of inhibitors with the CMT active site are dominated by electrostatic multipole contributions in analogy with previously studied transition state analogue inhibitors of leucyl aminopeptidase.     

Prediction of the binding sites of huperzine A in acetylcholinesterase by docking studies

Summary We have performed docking studies with the SYSDOC program on acetylcholinesterase (AChE) to predict the binding sites in AChE of huperzine A (HA), which is a potent and selective, reversible inhibitor of AChE. The unique aspects of our docking studies include the following: (i) Molecular flexibility of the guest and the host is taken into account, which permits both to change their conformations upon binding. (ii) The binding energy is evaluated by a sum of energies of steric, electrostatic and hydrogen bonding interactions. In the energy calculation no grid approximation is used, and all hydrogen atoms of the system are treated explicitly. (iii) The energy of cation- interactions between the guest and the host, which is important in the binding of AChE, is included in the calculated binding energy. (iv) Docking is performed in all regions of the host's binding cavity. Based on our docking studies and the pharmacological results reported for HA and its analogs, we predict that HA binds to the bottom of the binding cavity of AChE (the gorge) with its ammonium group interacting with Trp⁸⁴, Phe³³⁰, Glu¹⁹⁹ and Asp⁷² (catalytic site). At the the opening of the gorge with its ammonium group partially interacting with Trp²⁷⁹ (peripheral site). At the catalytic site, three partially overlapping subsites of HA were identified which might provide a dynamic view of binding of HA to the catalytic site.     

Atypical protonation states in the active site of HIV-1 protease: a computational study

The HIV protease (HIVP) is a prominent example for successful structure-based drug design. Besides its pharmaceutical impact, it is a well-studied system for which, as experimentally evidenced, protonation changes in the active site occur upon ligand binding. Therefore, it serves as an ideal candidate for a case study using our newly developed partial charge model, which was optimized toward the application of Poisson-Boltzmann based pK(a) calculations. The charge model suggests reliably experimentally determined protonation states in the active site of HIVP. Furthermore, we perform pKa calculations for two HIVP complexes with novel types of inhibitors developed and synthesized in our group. For these complexes, no experimental knowledge about the protonation states is given. For one of the compounds, containing a central pyrrolidine ring, the calculations predict that both catalytic aspartates should be deprotonated upon ligand binding.     

Effects of slow substrate binding and release in redox enzymes: theory and application to periplasmic nitrate reductase

For redox enzymes, the technique called protein film voltammetry makes it possible to determine the entire profile of activity against driving force by having the enzyme exchanging directly electrons with the rotating-disc electrode onto which it is adsorbed. Both the potential location of the catalytic response and its detailed shape report on the sequence of catalytic events, electron transfers and chemical steps, but the models that have been used so far to decipher this signal lack generality. For example, it was often proposed that substrate binding to multiple redox states of the active site may explain that turnover is greater in a certain window of electrode potential, but no fully analytical treatment has been given. Here, we derive (i) the general current equation for the case of reversible substrate binding to any redox states of a two-electron active site (as exemplified by flavins and Mo cofactors), (ii) the quantitative conditions for an extremum in activity to occur, and (iii) the expressions from which the substrate-concentration dependence of the catalytic potential can be interpreted to learn about the kinetics of substrate binding and how this affects the reduction potential of the active site. Not only does slow substrate binding and release make the catalytic wave shape highly complex, but we also show that it can have important consequences which will escape detection in traditional experiments: the position of the wave (this is the driving force that is required to elicit catalysis) departs from the reduction potential of the active site even at the lowest substrate concentration, and this deviation may be large if substrate binding is irreversible. This occurs in the reductive half-cycle of periplasmic nitrate reductase where irreversibility lowers the driving force required to reduce the active site under turnover conditions and favors intramolecular electron transfer from the proximal [4Fe4S]+ cluster to the active site Mo(V).     

Macrocyclic Protease Inhibitors with Reduced Peptide Character

There is a real need for simple structures that define a β‐strand conformation, a secondary structure that is central to peptide–protein interactions. For example, protease substrates and inhibitors almost universally adopt this geometry on active site binding. A planar pyrrole is used to replace two amino acids of a peptide backbone to generate a simple macrocycle that retains the required geometry for active site binding. The resulting β‐strand templates have reduced peptide character and provide potent protease inhibitors with the attachment of an appropriate amino aldehyde to the C‐terminus. Picomolar inhibitors of cathepsin L and S are reported and the mode of binding of one example to the model protease chymotrypsin is defined by X‐ray crystallography.     

Ligand- and protein-based modeling studies of the inhibitors of human cytochrome P450 2D6 and a virtual screening for potential inhibitors from the Chinese herbal medicine, Scutellaria baicalensis (Huangqin,Baikal Skullcap)

We have previously examined the binding patterns of various substrates to human cytochrome P450 2D6 (CYP2D6) using a series of molecular modeling methods. In this study, we further explored the binding modes of various types of inhibitors to CYP2D6 using a combination of ligand- and protein-based modeling approaches. Firstly, we developed and validated a pharmacophore model for CYP2D6 inhibitors, which consisted of two hydrophobic features and one hydrogen bond acceptor feature. Secondly, we constructed and validated a quantitative structure-activity relationship (QSAR) model for CYP2D6 inhibitors which gave a poor to moderate prediction accuracy. Thirdly, a panel of CYP2D6 inhibitors were subject to molecular docking into the active site of wild-type and mutated CYP2D6 enzyme. We demonstrated that 8 residues in the active site (Leu213, Glu216, Ser217, Gln244, Asp301, Ser304, Ala305, and Phe483) played an important role in the binding to the inhibitors via hydrogen bond formation and/or π-π stacking interaction. Apparent changes in the binding modes of the inhibitors have been observed with Phe120Ile, Glu216Asp, Asp301Glu mutations in CYP2D6. Finally, we screened for potential binders/inhibitors from the Chinese herbal medicine Scutellaria baicalensis (Huangqin, Baikal Skullcap) using the established pharmacophore model for CYP2D6 inhibitors and molecular docking approach. Overall, 18 out of 40 compounds from S. baicalensis were mapped to the pharmacophore model of CYP2D6 inhibitors and most herbal compounds from S. baicalensis could be docked into the active site of CYP2D6. Our study has provided insights into the molecular mechanisms of interaction of synthetic and herbal compounds with human CYP2D6 and further benchmarking studies are needed to validate our modeling and virtual screening results.     

Structure‐Based Design of Nonpeptidic Thrombin Inhibitors: Exploring the D‐Pocket and the Oxyanion Hole

Structure‐activity relationships for new members of a class of nonpeptidic, low‐molecular‐weight inhibitors of thrombin, a key serine protease in the blood coagulation cascade, are described. These compounds, which originate from X‐ray‐structure‐based design, feature a conformationally rigid, bi‐ or tricyclic core from which side chains diverge into the four major binding pockets (distal D, proximal P, recognition or specificity S1, and oxyanion hole O) at the thrombin active site (Fig. 1). Phenylamidinium is the side chain of choice for the S1 pocket, while the most active inhibitors orient an i‐Pr group into the P‐pocket (Table 1). The key step in the synthesis of the inhibitors is the construction of the central bi‐ or tricyclic scaffold by 1,3‐dipolar cycloaddition of an in situ prepared azomethine ylide and an N‐substituted maleimide (Schemes 1–3, and 8–10). One series of compounds was designed to explore the binding features of the large hydrophobic D pocket. This pocket provides space for lipophilic residues as bulky as benzhydryl groups. A new strategy was developed, allowing introduction of these sterically demanding substituents very late in the synthesis (Schemes 5 and 6). Benzhydryl derivative (±)‐ 2 was found to be the most selective member (K_i (trypsin)/K_i (thrombin)=1200) of this class of nonpeptidic thrombin inhibitors, while the ‘dipiperonyl' analog (±)‐ 3 (K_i=9 nM , 7.60‐fold selectivity) displays the highest potency of all compounds prepared so far (Table 1). A second series of inhibitors features side chains designed to orient into the oxyanion hole and to undergo H‐bonding with the backbone NH groups lining the catalytic site of the enzyme. Unfortunately, neither activity nor selectivity could be substantially improved by introduction of these substituents (Table 2). Presumably, the high degree of pre‐organization and the rigidity of the tightly bound scaffolds prevents the new substituents from assuming a position that would allow favorable interactions in the oxyanion hole. However, the oxyanion hole and the S1′ pocket next to it were found to be capable of accommodating quite large groups, which leaves much room for further exploration.     

Structure-based drug design: the discovery of novel nonpeptide orally active inhibitors of human renin

BACKGROUND: The aspartic proteinase renin plays an important physiological role in the regulation of blood pressure. It catalyses the first step in the conversion of angiotensinogen to the hormone angiotensin II. In the past, potent peptide inhibitors of renin have been developed, but none of these compounds has made it to the end of clinical trials. Our primary aim was to develop novel nonpeptide inhibitors. Based on the available structural information concerning renin-substrate interactions, we synthesized inhibitors in which the peptide portion was replaced by lipophilic moieties that interact with the large hydrophobic S1/S3-binding pocket in renin. RESULTS: Crystal structure analysis of renin-inhibitor complexes combined with computational methods were employed in the medicinal-chemistry optimisation process. Structure analysis revealed that the newly designed inhibitors bind as predicted to the S1/S3 pocket. In addition, however, these compounds interact with a hitherto unrecognised large, distinct, sub-pocket of the enzyme that extends from the S3-binding site towards the hydrophobic core of the enzyme. Binding to this S3(sp) sub-pocket was essential for high binding affinity. This unprecedented binding mode guided the drug-design process in which the mostly hydrophobic interactions within subsite S3(sp) were optimised. CONCLUSIONS: Our design approach led to compounds with high in vitro affinity and specificity for renin, favourable bioavailability and excellent oral efficacy in lowering blood pressure in primates. These renin inhibitors are therefore potential therapeutic agents for the treatment of hypertension and related cardiovascular diseases.     

Binding structure of elastase inhibitor scyptolin A

Natural bioactive compounds are of general interest to pharmaceutical research because they may be used as leads in drug development campaigns. Among them, scyptolin A and B from Scytonema hofmanni PCC 7110 are known to inhibit porcine pancreatic elastase, which in turn resembles the attractive drug target neutrophil elastase. The crystal structure of scyptolin A as bound to pancreatic elastase was solved at 2.8 A resolution. The inhibitor occupies the most prominent subsites S1 through S4 of the elastase and prevents a hydrolytic attack by covering the active center with its rigid ring structure. The observed binding structure may help to design potent elastase inhibitors.