Charge optimization of the interface between protein kinases and their ligands

Examining the potential for electrostatic complementarity between a ligand and a receptor is a useful technique for rational drug design, and can demonstrate how a system prioritizes interactions when allowed to optimize its charge distribution. In this computational study, we implemented the previously developed, continuum solvent-based charge optimization theory with a simple, quadratic programming algorithm and the UHBD Poisson-Boltzmann solver. This method allows one to compute the best set of point charges for a ligand or ligand region based on the ligand and receptor shape, and the receptor partial charges, by optimizing the binding free energy obtained from a continuum-solvent model. We applied charge optimization to a fragment of the heat-stable protein kinase inhibitor (PKI) of protein kinase A (PKA), to three flavopiridol inhibitors of CDK2, and to cyclin A which interacts with CDK2 to regulate the cell cycle. We found that a combination of global (involving every charge) and local (involving only charges in a local region) optimization can give useful hints for designing better inhibitors. Although some parts of an inhibitor may already contribute significantly to binding, we found that they could still be the most important targets for modifications to obtain stronger binders. In studying the binding of flavopiridol inhibitors to CDK2, comparable binding affinity could be obtained regardless of whether the net charges of the inhibitors were constrained to -2, -1, 0, 1, or 2 during the optimization. This provides flexibility in inhibitor design when a certain net charge of the inhibitor is desired in addition to strong binding affinity. For the study of the PKA-PKI and CDK2-cyclin A interfaces, we identified residues whose charge distributions are already close to optimal and those whose charge distributions could be refined to further improve binding.     

Beta-lactones as specific inhibitors of ClpP attenuate the production of extracellular virulence factors of Staphylococcus aureus

To approach the daunting problem of multidrug resistant bacterial pathogens, a multidisciplinary chemical proteomic strategy was applied and functionalized beta-lactones were identified as potent, cell permeable inhibitors for specific and selective targeting of the key virulence regulator complex ClpP in S. aureus and methicillin resistant S. aureus (MRSA) strains. ClpP represents the central protease complex responsible for the activation of numerous virulence factors including many with devastating effects for human health such as hemolysins, proteases, lipases, and DNases. Although the crucial role of this enzyme was validated by genetic knockouts, no inhibitor has been reported to date. In fact, our most potent inhibitor was able to completely abolish hemolytic and proteolytic activities and showed a dramatic decrease in the activities of virulence associated lipases and DNases. These effects were also observed in a multiresistant strain emphasizing the potential value of such compounds. Targeting this virulence factor may therefore likely represent an attractive strategy for neutralizing the harmful effects of bacterial pathogens and help the host immune response to eliminate the disarmed bacteria. Since ClpP is not essential for viability and highly conserved in many pathogens, our strategy could represent a global approach for the treatment of infectious diseases without the pressing problem of antibiotic pressure and resistance development.     

Approaches to solving the rigid receptor problem by identifying a minimal set of flexible residues during ligand docking

BACKGROUND: Using fixed receptor sites derived from high-resolution crystal structures in structure-based drug design does not properly account for ligand-induced enzyme conformational change and imparts a bias into the discovery and design of novel ligands. We sought to facilitate the design of improved drug leads by defining residues most likely to change conformation, and then defining a minimal manifold of possible conformations of a target site for drug design based on a small number of identified flexible residues. RESULTS: The crystal structure of thymidylate synthase from an important pathogenic target Pneumocystis carinii (PcTS) bound to its substrate and the inhibitor, BW1843U89, is reported here and reveals a new conformation with respect to the structure of PcTS bound to substrate and the more conventional antifolate inhibitor, CB3717. We developed an algorithm for determining which residues provide 'soft spots' in the protein, regions where conformational adaptation suggests possible modifications for a drug lead that may yield higher affinity. Remodeling the active site of thymidylate synthase with new conformations for only three residues that were identified with this algorithm yields scores for ligands that are compatible with experimental kinetic data. CONCLUSIONS: Based on the examination of many protein/ligand complexes, we develop an algorithm (SOFTSPOTS) for identifying regions of a protein target that are more likely to accommodate plastically to regions of a drug molecule. Using these indicators we develop a second algorithm (PLASTIC) that provides a minimal manifold of possible conformations of a protein target for drug design, reducing the bias in structure-based drug design imparted by structures of enzymes co-crystallized with inhibitors.     

On-line immobilized acetylcholinesterase microreactor for screening of inhibitors from natural extracts by capillary electrophoresis

In this study we developed a simple capillary electrophoresis (CE) method with an on-line acetylcholinesterase (AChE) microreactor at the inlet of capillary for inhibitor screening. The fused-silica capillary surface was modified with a polycationic polyethylenimine coating. Solutions of the enzyme and chitosan were then injected to immobilize the enzyme in approximately 2.9?cm of the capillary inlet (total length of capillary 60.2?cm) by electrostatic interaction and the film overlay technique. Separation of enzyme reaction product (thiocholine, ThCh) and unreacted substrate (acetylthiocholine, AThCh) was achieved within 3.0?min. The conditions affecting the efficiency of reaction of the enzyme were optimized by measuring the peak area of ThCh. Under the optimum conditions, using Huperzine-A as model inhibitor, K (i) and IC (50) were 0.551?μmol?L(-1) and 1.52?μmol?L(-1), respectively, for immobilized AChE. Finally, screening of a small compound library containing two known AChE inhibitors and 30 natural extracts was conducted, and species with inhibition activity were directly identified. Compared with previous publications on screening for AChE inhibitors in natural products based on CE methods, the method developed in this work has the advantages of lower cost per analysis, less leakage, and better bioaffinity for the immobilized enzyme because of the unique properties of sodium alginate and chitosan.     

THE PRIMARY STRUCTURE OF ARROWHEAD PROTEINASE INHIBITOR

After the reduction and carboxymethylation of disulfide bonds, arrowhead proteinase inhibitors A and B were cleaved either by proteinases or by cyanogen bromide, the fractionated and purified peptides were then subjected to sequencing by a gas phase automatic sequencer, the primary structures were completed by the alignment of the peptides sequenced with overlapping peptides. Both inhibitors A and B consist of 150 amino acid residues with three pairs of disulfide bonds, share 90% homology in structure, and are markedly different from all other Ser proteinase inhibitors so far known. Hence, the arrowhead inhibitor may belong to a new inhibitor family. Based on their structure characteristics, it was deduced that both their two reactive sites might be located in the positions of Lys-Ser (45-46) and Arg-Tyr-Lys (77-79), respectively. Among 13 mutated residues in inhibitors A and B, the substitution of residue Arg in position 87 of inhibitor B for residue Leu in A might be the main cause of leading to diffe     

Understanding and modulating cyclin-dependent kinase inhibitor specificity: molecular modeling and biochemical evaluation of pyrazolopyrimidinones as CDK2/cyclin A and CDK4/cyclin D1 inhibitors

Summary Cyclin-dependent kinases (CDKs) play a key role in regulating the cell cycle. The cyclins, their activating agents, and endogenous CDK inhibitors are frequently mutated in human cancers, making CDKs interesting targets for cancer chemotherapy. Our aim is the discovery of selective CDK4/cyclin D1 inhibitors. An ATP-competitive pyrazolopyrimidinone CDK inhibitor was identified by HTS and docked into a CDK4 homology model. The resulting binding model was consistent with available SAR and was validated by a subsequent CDK2/inhibitor crystal structure. An iterative cycle of chemistry and modeling led to a 70-fold improvement in potency. Small substituent changes resulted in large CDK4/CDK2 selectivity changes. The modeling revealed that selectivity is largely due to hydrogen-bonded interactions with only two kinase residues. This demonstrates that small differences between enzymes can efficiently be exploited in the design of selective inhibitors.     

Small molecule inhibitors of the MDM2-p53 interaction discovered by ensemble-based receptor models

Five nonpeptide, small-molecule inhibitors of the human MDM2-p53 interaction are presented, and each inhibitor represents a new scaffold. The most potent compound exhibited a Ki of 110 +/- 30 nM. These compounds were identified using our multiple protein structure (MPS) method which incorporates protein flexibility into a receptor-based pharmacophore model that identifies appropriate hotspots of binding. Docking the inhibitors with an induced-fit docking protocol suggested that the inhibitors mimicked the three critical binding residues of p53 (Phe19, Trp23, and Leu26). Docking also predicted a new orientation of the scaffolds that more fully fills the binding cleft, enabling the inhibitors to take advantage of additional hydrogen-bonding possibilities not explored by other small molecule inhibitors. One inhibitor in particular was proposed to probe the hydrophobic core of the protein by taking advantage of the flexibility of the binding cleft floor. These results show that the MPS technique is a promising advance for structure-based drug discovery and that the method can truly explore broad chemical space efficiently in the quest to discover potent, small-molecule inhibitors of protein-protein interactions. Our MPS technique is one of very few ensemble-based techniques to be proven through experimental verification of the discovery of new inhibitors.     

Prospective CCR5 small molecule antagonist compound design using a combined mutagenesis/modeling approach

The viral resistance of marketed antiviral drugs including the emergence of new viral resistance of the only marketed CCR5 entry inhibitor, maraviroc, makes it necessary to develop new CCR5 allosteric inhibitors. A mutagenesis/modeling approach was used (a) to remove the potential hERG liability in an otherwise very promising series of compounds and (b) to design a new class of compounds with an unique mutant fingerprint profile depending on residues in the N-terminus and the extracellular loop 2. On the basis of residues, which were identified by mutagenesis as key interaction sites, binding modes of compounds were derived and utilized for compound design in a prospective manner. The compounds were then synthesized, and in vitro evaluation not only showed that they had good antiviral potency but also fulfilled the requirement of low hERG inhibition, a criterion necessary because a potential approved drug would be administered chronically. This work utilized an interdisciplinary approach including medicinal chemistry, molecular biology, and computational chemistry merging the structural requirements for potency with the requirements of an acceptable in vitro profile for allosteric CCR5 inhibitors. The obtained mutant fingerprint profiles of CCR5 inhibitors were used to translate the CCR5 allosteric binding site into a general pharmacophore, which can be used for discovering new inhibitors.     

Probing molecular mechanism of inhibitor bindings to bromodomain-containing protein 4 based on molecular dynamics simulations and principal component analysis

ABSTRACT

It is well known that bromodomain-containing protein 4 (BRD4) has been thought as a promising target utilized for treating various human diseases, such as inflammatory disorders, malignant tumours, acute myelogenous leukaemia (AML), bone diseases, etc. For this study, molecular dynamics (MD) simulations, binding free energy calculations, and principal component analysis (PCA) were integrated together to uncover binding modes of inhibitors 8P9, 8PU, and 8PX to BRD4(1). The results obtained from binding free energy calculations show that van der Waals interactions act as the main regulator in bindings of inhibitors to BRD4(1). The information stemming from PCA reveals that inhibitor associations extremely affect conformational changes, internal dynamics, and movement patterns of BRD4(1). Residue-based free energy decomposition method was wielded to unveil contributions of independent residues to inhibitor bindings and the data signify that hydrogen bonding interactions and hydrophobic interactions are decisive factors affecting bindings of inhibitors to BRD4(1). Meanwhile, eight residues Trp81, Pro82, Val87, Leu92, Leu94, Cys136, Asn140, and Ile146 are recognized as the common hot interaction spots of three inhibitors with BRD4(1). The results from this work are expected to provide a meaningfully theoretical guidance for design and development of effective inhibitors inhibiting of the activity of BRD4.     

白色念珠菌N-肉豆蔻酰基转移酶中药物结合位点的MCSS分析

采用多拷贝同时搜寻方法(MCSS)分析得到了CaNMT活性位点的疏水区域、氢键结合位点和负电性区域. MCSS计算结果显示, CaNMT活性位点有两个疏水性比较强的区域: 一个由Tyr107, Tyr109, Val108, Phe117, Phe123, Ala127, Phe176和Leu337等残基组成; 另一个由Phe115, Phe240和Phe339组成. CaNMT活性位点发现有两个氢键作用区域, 其中Tyr119, His227, Asn392和Leu451是与已有抑制剂的氢键结合位点, Tyr107, Asn175, Thr211和Asp412是新发现的氢键结合位点, 而且在NMT家族中高度稳定, 它们对设计新结构类型的CaNMT抑制剂具有重要作用. Leu451是负电性兼氢键作用位点, 是抑制剂设计时所必需考虑的位点.     

Squash inhibitors: from structural motifs to macrocyclic knottins

In this article, we will first introduce the squash inhibitor, a well established family of highly potent canonical serine proteinase inhibitors isolated from Cucurbitaceae. The squash inhibitors were among the first discovered proteins with the typical knottin fold shared by numerous peptides extracted from plants, animals and fungi. Knottins contain three knotted disulfide bridges, two of them arranged as a Cystine-Stabilized Beta-sheet motif. In contrast to cyclotides for which no natural linear homolog is known, most squash inhibitors are linear. However, Momordica cochinchinensis Trypsin Inhibitor-I and (MCoTI-I and -II), 34-residue squash inhibitors isolated from seeds of a common Cucurbitaceae from Vietnam, were recently shown to be macrocyclic. In these circular squash inhibitors, a short peptide linker connects residues that correspond to the N- and C-termini in homologous linear squash inhibitors. In this review we present the isolation, characterization, chemical synthesis, and activity of these macrocyclic knottins. The solution structure of MCoTI-II will be compared with topologically similar cyclotides, homologous linear squash inhibitors and other knottins, and potential applications of such scaffolds will be discussed.     

A high throughput scintillation proximity imaging assay for protein methyltransferases

Protein methyltransferases (PMTs) orchestrate epigenetic modifications through post-translational methylation of various protein substrates including histones. Since dysregulation of this process is widely implicated in many cancers, it is of pertinent interest to screen inhibitors of PMTs, as they offer novel target-based opportunities to discover small molecules with potential chemotherapeutic use. We have thus developed an enzymatic screening strategy, which can be adapted to scintillation proximity imaging assay (SPIA) format, to identify these inhibitors. We took advantage of S-adenosyl-L-[3H-methyl]-methionine availability and monitored the enzymatically catalyzed [3H]-methyl addition on lysine residues of biotinylated peptide substrates. The radiolabeled peptides were subsequently captured by streptavidin coated SPA imaging PS beads. We applied this strategy to four PMTs: SET7/9, SET8, SETD2, and EuHMTase1, and optimized assay conditions to achieve Z' values ranging from 0.48 to 0.91. The robust performance of this SPIA for the four PMTs was validated in a pilot screen of approximately 7,000 compounds. We identified 80 cumulative hits across the four targets. NF279, a suramin analogue, was found to specifically inhibit SET7/9 and SETD2 with IC50 values of 1.9 and 1.1 μM, respectively. Another identified compound, Merbromin, a topical antiseptic, was classified as a pan-active inhibitor of the four PMTs. These findings demonstrate that our proposed SPIA strategy is generic for multiple PMTs and can be successfully implemented to identify novel and specific inhibitors of PMTs. The specific PMT inhibitors may constitute a new class of anti-proliferative agents for potential therapeutic use.     

Selective Inhibitors of FKBP51 Employ Conformational Selection of Dynamic Invisible States

The recently discovered SAFit class of inhibitors against the Hsp90 co‐chaperone FKBP51 show greater than 10 000‐fold selectivity over its closely related paralogue FKBP52. However, the mechanism underlying this selectivity remained unknown. By combining NMR spectroscopy, biophysical and computational methods with mutational analysis, we show that the SAFit molecules bind to a transient pocket in FKBP51. This represents a weakly populated conformation resembling the inhibitor‐bound state of FKBP51, suggesting conformational selection rather than induced fit as the major binding mechanism. The inhibitor‐bound conformation of FKBP51 is stabilized by an allosteric network of residues located away from the inhibitor‐binding site. These residues stabilize the Phe67 side chain in a dynamic outward conformation and are distinct in FKBP52, thus rationalizing the basis for the selectivity of SAFit inhibitors. Our results represent a paradigm for the selective inhibition of transient binding pockets.     

Two-prong inhibitors for human carbonic anhydrase II

The enzyme inhibitors are usually designed by taking into consideration the overall dimensions of the enzyme's active site pockets. This conventional approach often fails to produce desirable affinities of inhibitors for their cognate enzymes. To circumvent such constraints, we contemplated enhancing the binding affinities of inhibitors by attaching tether groups, which would interact with the surface exposed amino acid residues. This strategy has been tested for the inhibition of human carbonic anhydrase II. Benzenesulfonamide serves as a weak inhibitor for the enzyme, but when it is conjugated to iminodiacetate-Cu2+ (which interacts with the surface-exposed His residues) via a spacer group, its binding affinity is enhanced by about 2 orders of magnitude. This "two-prong" approach is expected to serve as a general strategy for converting weak inhibitors of enzymes into tight-binding inhibitors.     

Solid-phase synthesis of a focused library of trypanothione reductase inhibitors

A focused library of inhibitors of the enzyme trypanothione reductase was prepared using solid-phase synthesis. The inhibitors were based on a previously identified, non-competitive, lead compound comprising of two Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl) side-chain protected, N-capped arginine residues linked by a spermidine bridge. In total six protecting groups and four capping groups were used to generate a 24-membered library. All compounds bearing the 5-methoxyindole-3-acetic acid capping group were found to have good activity. The most potent inhibitor was observed to contain the Mtr (4-methoxy-2,3,6-trimethylbenzenesulphonyl) protecting group on the arginine residue, terminated with tryptophan as the capping group.     

Novel inhibitors of trihydroxynaphthalene reductase with antifungal activity identified by ligand-based and structure-based virtual screening

Curvularia lunata is a dark pigmented fungus that is the causative agent of several diseases in plants and in both immunodeficient and immunocompetent patients. 1,8-Dihydroxynaphthalene-melanin is found in the cell wall of C. lunata and is believed to be the important virulence factor of dematiaceous fungi. Trihydroxynaphthalene reductase is an enzyme of the 1,8-dihydroxynaphthalene-melanin biosynthetic pathway, and it thus represents an emerging target for the development of novel fungicides and antimycotics. In the present study, we describe novel inhibitors of trihydroxynaphthalene reductase from C. lunata. These inhibitors were identified by ligand-based three-dimensional similarity searching and docking to a homology-built model and by subsequent biochemical and antifungal evaluation. Discovery of competitive inhibitors with K(i) values in low micromolar and even nanomolar concentration range proves the aplicability of homology-built model of 3HNR for hit finding by virtual screening methods.     

Constrained Peptides with Target‐Adapted Cross‐Links as Inhibitors of a Pathogenic Protein–Protein Interaction

Bioactive conformations of peptides can be stabilized by macrocyclization, resulting in increased target affinity and activity. Such macrocyclic peptides proved useful as modulators of biological functions, in particular as inhibitors of protein–protein interactions (PPI). However, most peptide‐derived PPI inhibitors involve stabilized α‐helices, leaving a large number of secondary structures unaddressed. Herein, we present a rational approach towards stabilization of an irregular peptide structure, using hydrophobic cross‐links that replace residues crucially involved in target binding. The molecular basis of this interaction was elucidated by X‐ray crystallography and isothermal titration calorimetry. The resulting cross‐linked peptides inhibit the interaction between human adaptor protein 14‐3‐3 and virulence factor exoenzyme S. Taking into consideration that irregular peptide structures participate widely in PPIs, this approach provides access to novel peptide‐derived inhibitors.     

Methylxanthine drugs are chitinase inhibitors: investigation of inhibition and binding modes

Family 18 chitinases play key roles in a range of pathogenic organisms and are overexpressed in the asthmatic lung. By screening a library of marketed drug molecules, we have identified methylxanthine derivatives as possible inhibitor leads. These derivatives, theophylline, caffeine, and pentoxifylline, are used therapeutically as antiinflammatory agents, with pleiotropic mechanisms of action. Here it is shown that they are also competitive inhibitors against a fungal family 18 chitinase, with pentoxifylline being the most potent (K(i) of 37 microM). Crystallographic analysis of chitinase-inhibitor complexes revealed specific interactions with the active site, mimicking the reaction intermediate analog, allosamidin. Mutagenesis identified the key active site residues, conserved in mammalian chitinases, which contribute to inhibitor affinity. Enzyme assays also revealed that these methylxanthines are active against human chitinases.     

Combating susceptibility to drug resistance: lessons from HIV-1 protease

Drug resistance is a major obstacle in modern medicine. However, resistance is rarely considered in drug development and may inadvertently be facilitated, as many designed inhibitors contact residues that can mutate to confer resistance, without significantly impairing function. Contemporary drug design often ignores the detailed atomic basis for function and primarily focuses on disrupting the target's activity, which is necessary but not sufficient for developing a robust drug. In this study, we examine the impact of drug-resistant mutations in HIV-1 protease on substrate recognition and demonstrate that most primary active site mutations do not extensively contact substrates, but are critical to inhibitor binding. We propose a general, structure-based strategy to reduce the probability of drug resistance by designing inhibitors that interact only with those residues that are essential for function.     

Structural analysis of charge discrimination in the binding of inhibitors to human carbonic anhydrases I and II

Despite the similarity in the active site pockets of carbonic anhydrase (CA) isozymes I and II, the binding affinities of benzenesulfonamide inhibitors are invariably higher with CA II as compared to CA I. To explore the structural basis of this molecular recognition phenomenon, we have designed and synthesized simple benzenesulfonamide inhibitors substituted at the para position with positively charged, negatively charged, and neutral functional groups, and we have determined the affinities and X-ray crystal structures of their enzyme complexes. The para-substituents are designed to bind in the midsection of the 15 A deep active site cleft, where interactions with enzyme residues and solvent molecules are possible. We find that a para-substituted positively charged amino group is more poorly tolerated in the active site of CA I compared with CA II. In contrast, a para-substituted negatively charged carboxylate substituent is tolerated equally well in the active sites of both CA isozymes. Notably, enzyme-inhibitor affinity increases upon neutralization of inhibitor charged groups by amidation or esterification. These results inform the design of short molecular linkers connecting the benzenesulfonamide group and a para-substituted tail group in "two-prong" CA inhibitors: an optimal linker segment will be electronically neutral, yet capable of engaging in at least some hydrogen bond interactions with protein residues and/or solvent. Microcalorimetric data reveal that inhibitor binding to CA I is enthalpically less favorable and entropically more favorable than inhibitor binding to CA II. This contrasting behavior may arise in part from differences in active site desolvation and the conformational entropy of inhibitor binding to each isozyme active site.