Probing the interaction of HTI-286 with tubulin using a stilbene analogue

HTI-286 is a synthetic analogue of the natural product hemiasterlin. HTI-286 is a potent antitumor agent that induces tubulin oligomerization. To investigate the binding stoichiometry and the binding site during this ligand-induced tubulin association, we synthesized an analogue of HTI-286 containing the chromophore stilbene. Using the distinct absorbance of the stilbene analogue, we determined the amounts of inhibitors bound to different tubulin oligomers by analytical ultracentrifugation. Herein we describe our findings based on these experiments. At the ratio of inhibitor to protein equal to or greater than 1, the stilbene analogue induces oligomerization of tubulin to a ring structure. The binding stoichiometry in the ring is one inhibitor per tubulin monomer (defined as an alpha/beta-heterodimer). At the ratio of inhibitor to protein less than 1, tubulin forms multiple intermediates, with the binding stoichiometry less than one inhibitor per tubulin monomer for all intermediates. The stable complex between the inhibitor and tubulin monomer was not detected under our experimental conditions. The binding site of the stilbene analogue does not overlap with the classic tubulin-binding agent, colchicine.     

Structural basis for selective inhibition of Src family kinases by PP1.

BACKGROUND: Small-molecule inhibitors that can target individual kinases are powerful tools for use in signal transduction research. It is difficult to find such compounds because of the enormous number of protein kinases and the highly conserved nature of their catalytic domains. Recently, a novel, potent, Src family selective tyrosine kinase inhibitor was reported (PP1). Here, we study the structural basis for this inhibitor's specificity for Src family kinases. RESULTS: A single residue corresponding to Ile338 (v-Src numbering; Thr338 in c-Src) in Src family tyrosine kinases largely controls PP1's ability to inhibit protein kinases. Mutation of Ile338 to a larger residue such as methionine or phenylalanine in v-Src makes this inhibitor less potent. Conversely, mutation of Ile338 to alanine or glycine increases PP1's potency. PP1 can inhibit Ser/Thr kinases if the residue corresponding to Ile338 in v-Src is mutated to glycine. We have accurately predicted several non-Src family kinases that are moderately (IC(50) approximately 1 microM) inhibited by PP1, including c-Abl and the MAP kinase p38. CONCLUSIONS: Our mutagenesis studies of the ATP-binding site in both tyrosine kinases and Ser/Thr kinases explain why PP1 is a specific inhibitor of Src family tyrosine kinases. Determination of the structural basis of inhibitor specificity will aid in the design of more potent and more selective protein kinase inhibitors. The ability to desensitize a particular kinase to PP1 inhibition of residue 338 or conversely to sensitize a kinase to PP1 inhibition by mutation should provide a useful basis for chemical genetic studies of kinase signal transduction.     

Identification of the quinolinedione inhibitor binding site in Cdc25 phosphatase B through docking and molecular dynamics simulations

Cdc25 phosphatase B, a potential target for cancer therapy, is inhibited by a series of quinones. The binding site and mode of quinone inhibitors to Cdc25B remains unclear, whereas this information is important for structure-based drug design. We investigated the potential binding site of NSC663284 [DA3003-1 or 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5, 8-dione] through docking and molecular dynamics simulations. Of the two main binding sites suggested by docking, the molecular dynamics simulations only support one site for stable binding of the inhibitor. Binding sites in and near the Cdc25B catalytic site that have been suggested previously do not lead to stable binding in 50 ns molecular dynamics (MD) simulations. In contrast, a shallow pocket between the C-terminal helix and the catalytic site provides a favourable binding site that shows high stability. Two similar binding modes featuring protein-inhibitor interactions involving Tyr428, Arg482, Thr547 and Ser549 are identified by clustering analysis of all stable MD trajectories. The relatively flexible C-terminal region of Cdc25B contributes to inhibitor binding. The binding mode of NSC663284, identified through MD simulation, likely prevents the binding of protein substrates to Cdc25B. The present results provide useful information for the design of quinone inhibitors and their mechanism of inhibition.     

Inhibitor docking screened by the modified SAFE_p scoring function: application to cyclic urea HIV-1 PR inhibitors

Our laboratory has in the past developed a method for the prediction of ligand binding free energies to proteins, referred to as SAFE_p (Solvent free energy predictor). Previously, we have applied this protocol for the prediction of the binding free energy of peptidic and cyclic urea HIV-1 PR inhibitors, whose X-ray structures bound to enzyme are known. In this work, we present the first account of a docking simulation, where the ligand conformations were screened and inhibitor ranking was predicted on the basis of a modified SAFE_p approach, for a set of cyclic urea-HIV-1 PR complexes whose structures are not known. We show that the optimal dielectric constant for docking is rather high, in line with the values needed to reproduce some protein residue properties, like pKa's. Our protocol is able to reproduce most of the observed binding ranking, even in the case that the components of the equation are not fitted to experimental data. Partition of the binding free energy into pocket and residue contributions sheds light into the importance of the inhibitor's fragments and on the prediction of "hot spots" for resistance mutations.     

Photo-leucine incorporation reveals the target of a cyclodepsipeptide inhibitor of cotranslational translocation

Photoaffinity labeling is a powerful tool to identify protein targets of biologically active small molecules, yet is often limited by the size, chemical properties, and availability of photoreactive groups. We report an improved synthesis of photo-leucine, a diazirine-based photoreactive analogue of leucine, and demonstrate its incorporation into a cyclodepsipeptide inhibitor of cotranslational translocation. Photoaffinity labeling in a crude membrane fraction, followed by "click chemistry" with a rhodamine-azide reporter, enabled the identification of Sec61alpha, the structural core of the Sec61 translocation channel, as the inhibitor's target.     

A Protein‐Based Pentavalent Inhibitor of the Cholera Toxin B‐Subunit

Protein toxins produced by bacteria are the cause of many life‐threatening diarrheal diseases. Many of these toxins, including cholera toxin (CT), enter the cell by first binding to glycolipids in the cell membrane. Inhibiting these multivalent protein/carbohydrate interactions would prevent the toxin from entering cells and causing diarrhea. Here we demonstrate that the site‐specific modification of a protein scaffold, which is perfectly matched in both size and valency to the target toxin, provides a convenient route to an effective multivalent inhibitor. The resulting pentavalent neoglycoprotein displays an inhibition potency (IC₅₀) of 104 pM for the CT B‐subunit (CTB), which is the most potent pentavalent inhibitor for this target reported thus far. Complexation of the inhibitor and CTB resulted in a protein heterodimer. This inhibition strategy can potentially be applied to many multivalent receptors and also opens up new possibilities for protein assembly strategies.     

Rapid and accurate prediction of binding free energies for saquinavir-bound HIV-1 proteases

To explain drug resistance by computer simulations at the molecular level, we first have to assess the accuracy of theoretical predictions. Herein we report an application of the molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) technique to the ranking of binding affinities of the inhibitor saquinavir with the wild type (WT) and three resistant mutants of HIV-1 protease: L90M, G48V, and G48V/L90M. For each ligand-protein complex we report 10 ns of fully unrestrained molecular dynamics (MD) simulations with explicit solvent. We investigate convergence, internal consistency, and model dependency of MM/PBSA ligand binding energies. Converged enthalpy and entropy estimates produce ligand binding affinities within 1.5 kcal/mol of experimental values, with a remarkable level of correlation to the experimentally observed ranking of resistance levels. A detailed analysis of the enthalpic/entropic balance of drug-protease interactions explains resistance in L90M in terms of a higher vibrational entropy than in the WT complex, while G48V disrupts critical hydrogen bonds at the inhibitor's binding site and produces an altered, more unfavorable balance of Coulomb and polar desolvation energies.     

Fragment-based lead generation: identification of seed fragments by a highly efficient fragment screening technology

For the detection of the precise and unambiguous binding of fragments to a specific binding site on the target protein, we have developed a novel reporter displacement binding assay technology. The application of this technology for the fragment screening as well as the fragment evolution process with a specific modelling based design strategy is demonstrated for inhibitors of the protein kinase p38alpha. In a fragment screening approach seed fragments were identified which were then used to build compounds from the deep-pocket towards the hinge binding area of the protein kinase p38alpha based on a modelling approach. BIRB796 was used as a blueprint for the alignment of the fragments. The fragment evolution of these deep-pocket binding fragments towards the fully optimized inhibitor BIRB796 included the modulation of the residence time as well as the affinity. The goal of our study was to evaluate the robustness and efficiency of our novel fragment screening technology at high fragment concentrations, compare the screening data with biochemical activity data and to demonstrate the evolution of the hit fragments with fast kinetics, into slow kinetic inhibitors in an in silico approach.     

A general framework for inhibitor resistance in protein kinases

Protein kinases control virtually every aspect of normal and pathological cell physiology and are considered ideal targets for drug discovery. Most kinase inhibitors target the ATP binding site and interact with residue of a hinge loop connecting the small and large lobes of the kinase scaffold. Resistance to kinase inhibitors emerges during clinical treatment or as a result of in vitro selection approaches. Mutations conferring resistance to ATP site inhibitors often affect residues that line the ATP binding site and therefore contribute to selective inhibitor binding. Here, we show that mutations at two specific positions in the hinge loop, distinct from the previously characterized "gatekeeper," have general adverse effects on inhibitor sensitivity in six distantly related kinases, usually without consequences on kinase activity. Our results uncover a unifying mechanism of inhibitor resistance of protein kinases that might have widespread significance for drug target validation and clinical practice.     

Molecular interactions of nitrogen-containing bisphosphonates within farnesyl diphosphate synthase

Bisphosphonates, known for their effectiveness in the treatment of osteoporosis, inhibit bone resorption via mechanisms that involve binding to bone mineral and cellular effects on osteoclasts. The major molecular target of nitrogen-containing bisphosphonates (N-BPs) in osteoclasts is farnesyl diphosphate synthase (FPPS). N-BPs likely inhibit this enzyme by mimicking one or more of the natural isoprenoid lipid substrates (GPP/DMAPP and IPP) but the mode of inhibition is not established. The active site of FPPS comprises a subsite for each substrate. Kinetic studies with recombinant human FPPS indicate that both potent (risedronate) and weak (NE-58051) enzyme inhibitors compete with GPP for binding to FPPS, however, binding to this site does not completely explain the difference in potency of the two inhibitors, suggesting that a second binding site may also be a target of bisphosphonate inhibition. Using the docking software suite Autodock, we explored a dual inhibitor binding mode for recombinant human FPPS. Experimental support for dual binding is suggested by Dixon plots for the inhibitors. N-BPs may inhibit by binding to both the GPP and a second site with differences in potency at least partly arising from inhibition at the second site.     

Development of a potent Bcl-x(L) antagonist based on alpha-helix mimicry

The rational design of low-molecular weight ligands that disrupt protein-protein interactions is still a challenging goal in medicinal chemistry. Our approach to this problem involves the design of molecular scaffolds that mimic the surface functionality projected along one face of an alpha-helix. Using a terphenyl scaffold, which in a staggered conformation closely reproduces the projection of functionality on the surface of an alpha-helix, we designed mimics of the pro-apoptotic alpha-helical Bak-peptide as inhibitors of the Bak/Bcl-xL interaction. This led to the development of a potent Bcl-xL antagonist (KD = 114 nM), whose binding affinity for Bcl-xL was assessed by a fluorescence polarization assay. To determine the binding site of the developed inhibitor we used docking studies and an HSQC-NMR experiment with 15N-labeled Bcl-xL protein. These studies suggest that the inhibitor is binding in the same hydrophobic cleft as the Bak- and Bad-peptides.     

Oriented surface immobilization of antibodies at the conserved nucleotide binding site for enhanced antigen detection

The conserved nucleotide binding site (NBS), found on the Fab variable domain of all antibody isotypes, remains a not-so-widely known and unutilized site. Here, we describe a UV photo-cross-linking method (UV-NBS) that utilizes the NBS for oriented immobilization of antibodies onto surfaces, such that the antigen binding activity remains unaffected. Indole-3-butyric acid (IBA) has an affinity for the NBS with a K(d) ranging from 1 to 8 μM for different antibody isotypes and can be covalently photo-cross-linked to the antibody at the NBS upon exposure to UV light. Using the UV-NBS method, antibody was successfully immobilized on synthetic surfaces displaying IBA via UV photo-cross-linking at the NBS. An optimal UV exposure of 2 J/cm(2) yielded significant antibody immobilization on the surface with maximal relative antibody activity per immobilized antibody without any detectable damage to antigen binding activity. Comparison of the UV-NBS method with two other commonly used methods, ε-NH(3)(+) conjugation and physical adsorption, demonstrated that the UV-NBS method yields surfaces with significantly enhanced antigen detection efficiency, higher relative antibody activity, and improved antigen detection sensitivity. Taken together, the UV-NBS method provides a practical, site-specific surface immobilization method, with significant implications in the development of a large array of platforms with diverse sensor and diagnostic applications.     

Anti-cancer activity of novel dibenzo[b,f]azepine tethered isoxazoline derivatives

Background  

Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association.     

Evidence that monastrol is an allosteric inhibitor of the mitotic kinesin Eg5

Monastrol, a cell-permeable inhibitor of the kinesin Eg5, has been used to probe the dynamic organization of the mitotic spindle. The mechanism by which monastrol inhibits Eg5 function is unknown. We found that monastrol inhibits both the basal and the microtubule-stimulated ATPase activity of the Eg5 motor domain. Unlike many ATPase inhibitors, monastrol does not compete with ATP binding to Eg5. Monastrol appears to inhibit microtubule-stimulated ADP release from Eg5 but does not compete with microtubule binding, suggesting that monastrol binds a novel allosteric site in the motor domain. Finally, we established that (S)-monastrol, as compared to the (R)-enantiomer, is a more potent inhibitor of Eg5 activity in vitro and in vivo. Future structural studies should help in designing more potent Eg5 inhibitors for possible use as anticancer drugs and cell biological reagents.     

Exhaustive search of the configurational space of heat-shock protein 90 with its inhibitor by multicanonical molecular dynamics based dynamic docking

Multicanonical molecular dynamics based dynamic docking was used to exhaustively search the configurational space of an inhibitor binding to the N-terminal domain of heat-shock protein 90 (Hsp90). The obtained structures at 300 K cover a wide structural ensemble, with the top two clusters ranked by their free energy coinciding with the native binding site. The representative structure of the most stable cluster reproduced the experimental binding configuration, but an interesting conformational change in Hsp90 could be observed. The combined effects of solvation and ligand binding shift the equilibrium from a preferred loop-in conformation in the unbound state to an α-helical one in the bound state for the flexible lid region of Hsp90. Thus, our dynamic docking method is effective at predicting the native binding site while exhaustively sampling a wide configurational space, modulating the protein structure upon binding.     

Crystal structures of Candida albicans N-myristoyltransferase with two distinct inhibitors

Myristoyl-CoA:protein N-myristoyltransferase (Nmt) is a monomeric enzyme that catalyzes the transfer of the fatty acid myristate from myristoyl-CoA to the N-terminal glycine residue of a variety of eukaryotic and viral proteins. Genetic and biochemical studies have established that Nmt is an attractive target for antifungal drugs. We present here crystal structures of C. albicans Nmt complexed with two classes of inhibitor competitive for peptide substrates. One is a peptidic inhibitor designed from the peptide substrate; the other is a nonpeptidic inhibitor having a benzofuran core. Both inhibitors are bound into the same binding groove, generated by some structural rearrangements of the enzyme, with the peptidic inhibitor showing a substrate-like binding mode and the nonpeptidic inhibitor binding differently. Further, site-directed mutagenesis for C. albicans Nmt has been utilized in order to define explicitly which amino acids are critical for inhibitor binding. The results suggest that the enzyme has some degree of flexibility for substrate binding and provide valuable information for inhibitor design.     

Exploiting Atropisomerism to Increase the Target Selectivity of Kinase Inhibitors

Many biologically active molecules exist as rapidly interconverting atropisomeric mixtures. Whereas one atropisomer inhibits the desired target, the other can lead to off‐target effects. Herein, we study atropisomerism as a possibility to improve the selectivities of kinase inhibitors through the synthesis of conformationally stable pyrrolopyrimidines. Each atropisomer was isolated by HPLC on a chiral stationary phase and subjected to inhibitor profiling across a panel of 18 tyrosine kinases. Notably different selectivity patterns between atropisomers were observed, as well as improved selectivity compared to a rapidly interconverting parent molecule. Computational docking studies then provided insights into the structure‐based origins of these effects. This study is one of the first examples of the intentional preorganization of a promiscuous scaffold along an atropisomeric axis to increase target selectivity, and provides fundamental insights that may be applied to other atropisomeric target scaffolds.     

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。     

Molecular docking and competitive binding study discovered different binding modes of microsomal prostaglandin E synthase-1 inhibitors

Microsomal prostaglandin E synthase-1 (mPGES-1) is a newly recognized therapeutic target for the treatment of inflammation, pain, cancer, atherosclerosis, and stroke. Many mPGES-1 inhibitors have been discovered. However, as the structure of the binding site is not well-characterized, none of these inhibitors was designed based on the mPGES-1 structure, and their inhibition mechanism remains to be fully disclosed. Recently, we built a new structural model of mPGES-1 which was well supported by experimental data. Based on this model, molecular docking and competition experiments were used to investigate the binding modes of four representive mPGES-1 inhibitors. As the inhibitor binding sites predicted by docking overlapped with both the substrate and the cofactor binding sites, mPGES-1 inhibitors might act as dual-site inhibitors. This inhibitory mechanism was further verified by inhibitor-cofactor and inhibitor-substrate competition experiments. To investigate the potency-binding site relationships of mPGES-1 inhibitors, we also carried out molecular docking studies for another series of compounds. The docking results correlated well with the different inhibitory effects observed experimentally. Our data revealed that mPGES-1 inhibitors could bind to the substrate and the cofactor binding sites simultaneously, and this dual-site binding mode improved their potency. Future rational design and optimization of mPGES-1 inhibitors can be carried out based on this binding mechanism.     

Pharmacophore anchor models of ATAT1 to discover potential inhibitors and lead optimization

Post-translation modification of microtubules is associated with many diseases like cancer. Alpha Tubulin Acetyltransferase 1 (ATAT1) is a major enzyme that acetylates ‘Lys-40’ in alpha-tubulin on the luminal side of microtubules and is a drug target that lacks inhibitors. Here, we developed pharmacophore anchor models of ATAT1 which were constructed statistically using thousands of docked compounds, for drug design and investigating binding mechanisms. Our models infer the compound moiety preferences with the physico-chemical properties for the ATAT1 binding site. The results from the pharmacophore anchor models show the three main sub-pockets, including S1 acetyl site, S2 adenine site, and S3 diphosphate site with anchors, where conserved moieties interact with respective sub-pocket residues in each site and help in guiding inhibitor discovery. We validated these key anchors by analyzing 162 homologous protein sequences (>99 species) and over 10 structures with various bound ligands and mutations. Our results were consistent with previous works also providing new interesting insights. Our models applied in virtual screening predicted several ATAT1 potential inhibitors. We believe that our model is useful for future inhibitor discovery and for guiding lead optimization.