Binding Affinity Of Inhibitors Research Articles

Ligand and structure-based computational designing of multi-target molecules directing FFAR-1, FFAR-4 and PPAR-G as modulators of insulin receptor activity

Multi-agent therapies are an important treatment modality in many diseases based on the assumption that combining agents may result in increased therapeutic benefit by overcoming the mechanism of resistance and providing superior efficiency. Extensively validated 3D pharmacophore models for free fatty acid receptor-1 (FFAR-1), free fatty acid receptor-4 (FFAR-4), and peroxisome proliferator-activated receptor-G (PPAR-G) was developed. The pharmacophore model for FFAR-1 (r2 = 0.98, q 2 = 0.90) and PPAR-G (r2 = 0.89, q 2 = 0.88) suggested that one hydrogen bond acceptor, one hydrogen bond donor, three aromatic rings, and two hydrophobic groups arranged in 3D space are essential for the binding affinity of FFAR-1 and PPAR-G inhibitors. Similarly, the pharmacophore model for FFAR-4 (r2 = 0.92, q 2 = 0.87) suggested that the presence of a hydrogen bond acceptor, one negative atom, two aromatic rings, and three hydrophobic groups plays a vital role in the binding of an inhibitor of FFAR-4. These pharmacophore models allowed searches for novel FFAR-1, PPAR-G, and FFAR-4 triple inhibitors from multi-conformer 3D databases (Asinex). Finally, the twenty-five best hits were selected for molecular docking, to study the interaction of their complexes with all the proteins and final binding orientations of these molecules. After molecular docking, ten hits have been predicted to possess good binding affinity as per the Molecular Mechanics Generalized Born Surface Area (MM-GBSA) calculation for FFAR-1, FFAR-4, and PPAR-G which can be further investigated for its experimental in-vitro/in-vivo anti-diabetic activities. Communicated by Ramaswamy H. Sarma

Comparative Study of Interactions between Human cGAS and Inhibitors: Insights from Molecular Dynamics and MM/PBSA Studies.

Recent studies have identified cyclic GMP-AMP synthase (cGAS) as an important target for treating autoimmune diseases, and several inhibitors of human cGAS (hcGAS) and their structures in complexation with hcGAS have been reported. However, the mechanisms via which these inhibitors interact with hcGAS are not completely understood. Here, we aimed to assess the performance of molecular mechanics/Poisson–Boltzmann solvent-accessible surface area (MM/PBSA) in evaluating the binding affinity of various hcGAS inhibitors and to elucidate their detailed interactions with hcGAS from an energetic viewpoint. Using molecular dynamics (MD) simulation and MM/PBSA approaches, the estimated free energies were in good agreement with the experimental ones, with a Pearson’s correlation coefficient and Spearman’s rank coefficient of 0.67 and 0.46, respectively. In per-residue energy decomposition analysis, four residues, K362, R376, Y436, and K439 in hcGAS were found to contribute significantly to the binding with inhibitors via hydrogen bonding, salt bridges, and various π interactions, such as π· · ·π stacking, cation· · ·π, hydroxyl· · ·π, and alkyl· · ·π interactions. In addition, we discussed other key interactions between specific residues and ligands, in particular, between H363 and JUJ, F379 and 9BY, and H437 and 8ZM. The sandwiched structures of the inhibitor bound to the guanidinium group of R376 and the phenyl ring of Y436 were also consistent with the experimental data. The results indicated that MM/PBSA in combination with other virtual screening methods, could be a reliable approach to discover new hcGAS inhibitors and thus is valuable for potential treatments of cGAS-dependent inflammatory diseases.

Molecular basis for diaryldiamine selectivity and competition with tRNA in a type 2 methionyl-tRNA synthetase from a Gram-negative bacterium

Gram-negative bacteria are responsible for a variety of human, animal, and plant diseases. The spread of multidrug-resistant Gram-negative bacteria poses a challenge to disease control and highlights the need for novel antimicrobials. Owing to their critical role in protein synthesis, aminoacyl-tRNA synthetases, including the methionyl-tRNA synthetases MetRS1 and MetRS2, are attractive drug targets. MetRS1 has long been exploited as a drug target in Gram-positive bacteria and protozoan parasites. However, MetRS1 inhibitors have limited action upon Gram-negative pathogens or on Gram-positive bacteria that produce MetRS2 enzymes. The underlying mechanism by which MetRS2 enzymes are insensitive to MetRS1 inhibitors is presently unknown. Herein, we report the first structures of MetRS2 from a multidrug-resistant Gram-negative bacterium in its ligand-free state and bound to its substrate or MetRS1 inhibitors. The structures reveal the binding mode of two diaryldiamine MetRS1 inhibitors that occupy the amino acid–binding site and a surrounding auxiliary pocket implicated in tRNA acceptor arm binding. The structural features associated with amino acid polymorphisms found in the methionine and auxiliary pockets reveal the molecular basis for diaryldiamine binding and selectivity between MetRS1 and MetRS2 enzymes. Moreover, we show that mutations in key polymorphic residues in the methionine and auxiliary pockets not only altered inhibitor binding affinity but also significantly reduced enzyme function. Our findings thus reinforce the tRNA acceptor arm binding site as a druggable pocket in class I aminoacyl-tRNA synthetases and provide a structural basis for optimization of MetRS2 inhibitors for the development of new antimicrobials against Gram-negative pathogens.

Design, synthesis and bioevaluation of inhibitors targeting HSP90-CDC37 protein-protein interaction based on a hydrophobic core

HSP90-CDC37 protein-protein interaction (PPI) works as a kinase specific-molecular chaperone system to regulate the maturation of kinases. Currently, selectively disrupting HSP90-CDC37 PPI, rather than the direct inhibition of the ATPase function of HSP90, is emerging as a promising strategy for cancer therapy by specifically blocking the maturation of kinases. However, due to the limited understanding of HSP90-CDC37 binding interface, design of small molecule inhibitors targeting HSP90-CDC37 PPI is challenging. In this work, based on the binding mode of compound 11 (previously reported by our group), we discovered a hydrophobic pocket centered on Phe213, which was previously unknown, contributing to the binding affinity of HSP90-CDC37 PPI inhibitors. A series of hydrophobic substituted inhibitors were utilized to confirm the importance of Phe213 hydrophobic core. Finally, we obtained an optimum compound DDO-5994 (exhibited an ideal binding pattern on hydrophobic core) with improved binding affinity (KD=5.52μM) and antiproliferative activity (IC50=6.34μM). Both invitro and invivo assays confirmed DDO-5994 as a promising inhibitor to exhibit ideal antitumor efficacy through blocking HSP90-CDC37 PPI.

Structural Comparison of Diverse HIV-1 Subtypes using Molecular Modelling and Docking Analyses of Integrase Inhibitors.

The process of viral integration into the host genome is an essential step of the HIV-1 life cycle. The viral integrase (IN) enzyme catalyzes integration. IN is an ideal therapeutic enzyme targeted by several drugs; raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG), and bictegravir (BIC) having been approved by the USA Food and Drug Administration (FDA). Due to high HIV-1 diversity, it is not well understood how specific naturally occurring polymorphisms (NOPs) in IN may affect the structure/function and binding affinity of integrase strand transfer inhibitors (INSTIs). We applied computational methods of molecular modelling and docking to analyze the effect of NOPs on the full-length IN structure and INSTI binding. We identified 13 NOPs within the Cameroonian-derived CRF02_AG IN sequences and further identified 17 NOPs within HIV-1C South African sequences. The NOPs in the IN structures did not show any differences in INSTI binding affinity. However, linear regression analysis revealed a positive correlation between the Ki and EC50 values for DTG and BIC as strong inhibitors of HIV-1 IN subtypes. All INSTIs are clinically effective against diverse HIV-1 strains from INSTI treatment-naïve populations. This study supports the use of second-generation INSTIs such as DTG and BIC as part of first-line combination antiretroviral therapy (cART) regimens, due to a stronger genetic barrier to the emergence of drug resistance.

DFG-1 Residue Controls Inhibitor Binding Mode and Affinity, Providing a Basis for Rational Design of Kinase Inhibitor Selectivity.

Selectivity remains a challenge for ATP-mimetic kinase inhibitors, an issue that may be overcome by targeting unique residues or binding pockets. However, to date only few strategies have been developed. Here we identify that bulky residues located N-terminal to the DFG motif (DFG-1) represent an opportunity for designing highly selective inhibitors with unexpected binding modes. We demonstrate that several diverse inhibitors exerted selective, noncanonical binding modes that exclusively target large hydrophobic DFG-1 residues present in many kinases including PIM, CK1, DAPK, and CLK. By use of the CLK family as a model, structural and biochemical data revealed that the DFG-1 valine controlled a noncanonical binding mode in CLK1, providing a rationale for selectivity over the closely related CLK3 which harbors a smaller DFG-1 alanine. Our data suggest that targeting the restricted back pocket in the small fraction of kinases that harbor bulky DFG-1 residues offers a versatile selectivity filter for inhibitor design.

Identification of key residues determining the binding specificity of human 4-hydroxyphenylpyruvate dioxygenase

4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) is the second enzyme of the tyrosine catabolic pathway. Its physiological function is to catalyze the conversion of 4-hydroxyphenylpyruvic acid to homogentisic acid, which displays different physiological effects in mammals and plants. Insights on the selective inhibition of human HPPD (hHPPD) by triketone inhibitors were furnished by the integrated application of molecular simulation and biological testing. The binding free energy of hHPPD and inhibitors was obtained through molecular dynamics (MD) simulations, and the result was in agreement with the inhibition experiment in vitro. The binding free energy contribution demonstrated that the formation of hHPPD-inhibitor complexes was mainly driven by van der Waals energy. Ser226, Asn241, Gln265, Phe336, Phe359 and Phe364 made great contributions to binding affinities of all the systems. Among the residues involved in the interaction between nitisinone (NTBC) and hHPPD, Tyr221 and Leu224, whose mutation into Ala caused significant decrease of NTBC binding ability, were two key residues in determining the selective binding affinity of inhibitor and hHPPD. This work provides valuable theoretical basis for rational design of highly selective inhibitors targeting hHPPD.

Estimation of the ligand-binding free energy of checkpoint kinase 1 via non-equilibrium MD simulations

Checkpoint kinase 1 (CHK1) is a serine/threonine-protein kinase that is involved in cell cycle regulation in eukaryotes. Inhibition of CHK1 is thus considered as a promising approach in cancer therapy. In this study, the fast pulling of ligand (FPL) process was applied to predict the relative binding affinities of CHK1 inhibitors using non-equilibrium molecular dynamics (MD) simulations. The work of external harmonic forces to pull the ligand out of the binding cavity strongly correlated with the experimental binding affinity of CHK1 inhibitors with the correlation coefficient of R = −0.88 and an overall root mean square error (RMSE) of 0.99 kcal/mol. The data indicate that the FPL method is highly accurate in predicting the relative binding free energies of CHK1 inhibitors with an affordable CPU time. A new set of molecules were designed based on the molecular modeling of interactions between the known inhibitor and CHK1 as inhibitory candidates. Molecular docking and FPL results exhibited that the binding affinities of developed ligands were similar to the known inhibitor in interaction with the catalytic site of CHK1, producing very potential CHK1 inhibitors of that the inhibitory activities should be further evaluated in vitro.

A Library of Thiazolidin‐4‐one Derivatives as Protein Tyrosine Phosphatase 1B (PTP1B) Inhibitors: An Attempt To Discover Novel Antidiabetic Agents

Protein tyrosine phosphatase 1B (PTP1B) is an important target for the treatment of diabetes. A series of thiazolidin-4-one derivatives 8-22 was designed, synthesized and investigated as PTP1B inhibitors. The new molecules inhibited PTP1B with IC50 values in the micromolar range. 5-(Furan-2-ylmethylene)-2-(4-nitrophenylimino)thiazolidin-4-one (17) exhibited potency with a competitive type of enzyme inhibition. structure-activity relationship studies revealed various structural facets important for the potency of these analogues. The findings revealed a requirement for a nitro group-including hydrophobic heteroaryl ring for PTP1B inhibition. Molecular docking studies afforded good correlation with experimental results. H-bonding and π-π interactions were responsible for optimal binding and effective stabilization of virtual protein-ligand complexes. Furthermore, in-silico pharmacokinetic properties of test compounds predicted their drug-like characteristics for potential oral use as antidiabetic agents.Additionally, a binding site model demonstrating crucial pharmacophoric characteristics influencing potency and binding affinity of inhibitors has been proposed, which can be employed in the design of future potential PTP1B inhibitors.

A comparison of steroid and lipid binding cytochrome P450s from Mycobacterium marinum and Mycobacterium tuberculosis

The steroid lipid binding cytochrome P450 (CYP) enzymes of Mycobacterium tuberculosis are essential for organism survival through metabolism of cholesterol and its derivatives. The counterparts to these enzymes from Mycobacterium marinum were studied to determine the degree of functional conservation between them. Spectroscopic analyses of substrate and inhibitor binding for the four M. marinum enzymes CYP125A6, CYP125A7, CYP142A3 and CYP124A1 were performed and compared to the equivalent enzymes of M. tuberculosis. The sequence of CYP125A7 from M. marinum was more similar to CYP125A1 from M. tuberculosis than CYP125A6 but both showed differences in the resting heme spin state and in the binding modes and affinities of certain azole inhibitors. CYP125A7 did not show a significant Type II inhibitor-like shift with any of the azoles tested. CYP142A3 bound a similar range of steroids and inhibitors to CYP142A1. However, there were some differences in the extent of the Type I shifts to the high-spin form with steroids and a higher affinity for the azole inhibitors compared to CYP142A1. The two CYP124 enzymes had similar substrate binding properties. M. marinum CYP124 was characterised by X-ray crystallography and displayed strong conservation of active site residues, except near the region where the carboxylate terminus of the phytanic acid substrate would be bound. As these enzymes in M. tuberculosis have been identified as candidates for inhibition the data here demonstrates that alternative strategies for inhibitor design may be required to target CYP family members from distinct pathogenic Mycobacterium species or other bacteria.

Evaluation of a sesquiterpene as possible drug lead against gelatinases via molecular dynamics simulations

Malignant tumors can be targeted by accounting for their metastatic capabilities. Matrix metalloproteinases (MMPs) are the key players in tumor metastasis facilitating through their proteolytic activities of angiogenesis and extracellular matrix components (ECM) degradation. MMP-2 and MMP-9 being the members of a distinguished class of MMPs more commonly known as gelatinases are the prominent enzymes which are involved in different cancer progression stages. Targeting these isoforms specifically has always been a challenging task due to highly similar structural and functional features among the other members of MMPs with well preserve active sites containing catalytic zinc atom that was the only reason that none of the MMP inhibitor has been successfully marketed for the tumor pathology up till now. Therefore, non-competitive inhibitors with different structural attributed are needed to be evaluated at the molecular level for further experiments. The present study deals with the application of molecular dynamics simulation for the investigation of an alternative pathway for the inhibition of MMP-2 and MMP-9 by a sesquiterpene isolated from Polygonum barbatum which demonstrates the characteristics binding to the S1′ subsite of the enzymes followed by in vitro gene expression studies. The simulation results provide information on the possible binding profile producing inhibitory effects imposed by the inhibitor to these enzymes by acquiring different structural and dynamical features. Moreover, thermodynamic quantities based on the computationally intensive thermodynamic integration approach were also obtained in terms of inhibitor binding affinity computed for the inhibitor against MMP-2 and MMP-9 that completely augmented the experimental gene expression study. Communicated by Ramaswamy H. Sarma

Virtual screening to identify novel potential inhibitors for Glutamine synthetase of Mycobacterium tuberculosis

Glutamine synthetase (GS) of Mycobacterium tuberculosis (Mtb) is an essential enzyme which is involved in nitrogen metabolism and cell wall synthesis. It is involved in the inhibition of phagosome-lysosome fusion by preventing acidification. Targeting GS can be helpful to control the infection of Mtb. In order to identify potential inhibitors, we screened chemical libraries (56,400 compounds of ChEMBL anti-mycobacterial, 1596 FDA approved drugs, 419 Natural product and 916 phytochemical) against this target using the virtual screening approach. Screening by molecular docking identified ten top-ranked compounds as GSMtb inhibitors and they were compared with known inhibitors (as control). Since GS enzyme (GSHs) is also present in human. We have compared the protein sequence of GS from Mtb and human using the P-BLAST in NCBI. We found ∼27% identity in between these two sequences, so we also compared the binding affinity of inhibitor between Mtb and human. Finally, we identified top two compounds namely CHEMBL387509, CHEMBL226198 from ChEMBL anti-mycobacterial dataset, and Eriocitrin and Malvidin from phytochemical dataset which showed lees binding affinity towards GSHs whereas Pamidronate, and Phentermine from FDA approved drugs and (-)-Quinic Acid, Hexopyranuronic acid, Quebrachit, and Castanospermine from natural product showed protein-ligand interaction with Mtb protein while no interaction with GSHs. The top two docked complexes were subjected to molecular dynamic simulation to understand the stability of the molecule. Further, we calculated the binding free energy of the docked complex and analyzed hydrogen bond, salt bridge, pie stacking, and hydrophobic interaction in the docking region. These ligands exhibited very good binding affinity GSMtb enzymes. Therefore, these ligands are novel and drug-likeness compounds, and they may be potential inhibitors of M tuberculosis.Communicated by Ramaswamy H. Sarma

Removal of an atypical region from a staphylococcal cyclophilin affects its structure, function, stability, and shape

SaCyp, a cyclophilin having 197 amino acid residues, acts both as a protein-folding catalyst and a virulence factor in Staphylococcus aureus. Interestingly, a region, homologous to the SaCyp region carrying 121–148 amino acid residues, is present in many putative cyclophilins but absent in well-studied cyclophilins. To determine the exact roles of this unusual region in SaCyp and related proteins, we have investigated a deletion mutant (rCypΔ) of a recombinant SaCyp (rCyp) using various probes. The data reveal that rCypΔ has significantly less catalytic activity and possesses altered structure and hydrophobic surface compared to rCyp. Conversely, the deletion substantially increased inhibitor binding affinity and altered the shape of rCyp. However, both proteins were unfolded by a non-two-state mechanism in the presence of urea. Additionally, the stability of rCyp was significantly reduced due to the deletion of the residues 121–148. Our MD simulation study also indicated the considerable alteration in structure, shape, and fluctuations of SaCyp due to the removal of the region carrying 121–148 residues. Hence, the atypical region located in SaCyp might be vital for maintaining its unique structure, function, stability, and shape.

Screening and identification of potential tyrosinase inhibitors from Semen Oroxyli extract by ultrafiltration LC-MS and in silico molecular docking.

There is an increasing interest in screening and developing natural tyrosinase inhibitors widely applied in medicinal and cosmetic products, as well as in the food industry. In this study, an approach by ultrafiltration LC-MS and molecular docking was used to screen and identify tyrosinase inhibitors from Semen Oroxyli extract. The samples were first incubated with the tyrosinase to select the optimal binding conditions including tyrosinase concentration, incubation time and the molecular weight of ultrafiltration membrane. By comparison of the chromatographic profiles of the extracts after ultrafiltration with activated and inactivated tyrosinase, the potential inhibitors were obtained and then identified by LC-MS. The relative binding affinities of the potential inhibitors were also calculated based on the decrease of peak areas of those. As a result, seven compounds were fished out as tyrosinase inhibitors by this assay. Among them, oroxin A and baicalein showed higher tyrosinase inhibitory than resveratrol as positive drug, and their binding mode with enzyme was further verified via the molecular docking analysis. The test results showed that the proposed method was a simple, rapid, effective, and reliable method for the discovery of natural bioactive compounds, and it can be extended to screen other bioactive compounds from traditional Chinese medicines.

Identification of novel inhibitors of p-hydroxyphenylpyruvate dioxygenase using receptor-based virtual screening

Abstract p-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) is a potential target for herbicide discovery, which catalyzes the conversion of p-hydroxyphenylpyruvate to homogentisate. Triketone HPPD inhibitors have been widely applied to weed control for many years. To discover potential HPPD inhibitors with novel scaffold, a virtual screening procedure combining pharmacophore model, molecular docking and molecular dynamics (MD) simulation was carried out. The crystal complex (PDB: 6ISD ) with sulcotrione was selected to establish the receptor-ligand pharmacophore model (CBP). The verified CBP model with key features and interactions was employed to seek structures with potential inhibitory from ZINC database. The best-fit compounds from pharmacophore screening (2050 compounds) were subsequently subjected to molecular docking with CDOCKER method to identify the binding affinity of inhibitors with amino acid residues in active pocket. Six compounds were obtained as the final hits with the Fe(Ш) coordination and π-π interaction with Phe381 and Phe424. MD results demonstrated that Phe424 made great contributions to binding affinities of all the systems. This work provided powerful insights into the design and development of novel HPPD inhibitor in silico techniques. ZINC000255329531 was identified as the most promising candidate due to its high HPPD inhibition effect (ΔGbind = 25.66 kcal mol−1), in comparison with the commercial triketone HPPD herbicide sulcotrione (ΔGbind = 18.94 kcal mol−1).

Structure Dynamics Guides the Enhancement of Ligand Affinity for MdmX

Mdm2 and MdmX are highly homologous and Mdm2 inhibitors exhibit weak affinity for MdmX. The structural mechanism underlying how Mdm2/MdmX distinguish ligand binding affinity remains elusive. Guided by NMR study, we have identified three flexible regions on N-MdmX that affected inhibitor-binding affinity. Furthermore, using disulfide staple, we found that the Phe19-binding site on MdmX was one of the most flexible regions weakening nutlin-3a binding. On the other hand, we continued to pursue high-resolution structure of Mdm2 in complex with ntutin-3a. An X-ray crystallographic structure of Mdm2/nutlin-3a at 1.10 Å identified that His72 had two conformations, a bound state and an unbound state. Molecular dynamics simulation also revealed that His72 in MdmX existed two states. Thus, our work started to search fragments that could rigidify Phe19-binding site. We designed a fusion protein model that p5315-29 was fused with MdmX through a flexible linker, i.e., p5315-29-MdmX, for fragment screening. By use of the intrinsic fluorescence signal of p5315-29 to monitor high-throughput screening, we found that the imidazoindole was a bona fide scaffold for template-based design of MdmX inhibitors. Next the p5315-29-MdmX fusion protein was modified to leave Phe19-binding site unoccupied, i.e., p5323-29-MdmX. By screening a fragment library, a small fragment, CPD-01, could bind to Phe19-binding site in an allosteric mode that enhanced the binding of p5323-29 to MdmX. Furthermore, CPD-01 was conjugated with p5323-29 to synthesize p53 peptide analogues with different linkers. These peptide analogues exhibited enhanced binding affinity for MdmX, compared with p5323-29. Then, CPD-01 was integrated into nutlin-3a to replace Phe19-binding-site-pharmacophore. The new nutlin analogue exhibited high-affinity binding MdmX and Mdm2 as well. Taken together, our work provided a promising strategy to design inhibitors of MdmX/Mdm2. This strategy should be applicable for lead optimization in drug discovery.

Highly potent bacterial neuraminidase inhibitors, chromenone derivatives from Flemingia philippinensis.

The chromenone derivatives (1-4) from the root part of Flemingia philippinensis showed a significant inhibition against bacterial neuraminidase (NA) which plays a pivotal role in a cellular interaction including pathogenesis of bacterial infection and subsequent inflammation. The compounds 1 and 2 were the new compounds, philippin D (1) and philippin E (2). In particular, compounds (1-3) exhibited sub micromolar levels of IC50 values with 0.75, 0.54, and 0.07 μM. This is the first report that chromenone skeleton emerged as a lead structure of bacterial NA inhibition. In kinetic study, 8,8-diprenyl compounds displayed competitive inhibitory mode, whereas 4a,8-diprenyl ones showed noncompetitive behavior. It was manifested that all competitive inhibitors (1 and 2) were simple reversible slow-binding against bacterial NA. The binding affinities (KSV) of inhibitors to enzyme were agreement with their respective inhibitory potencies. Molecular docking data confirmed that the position of 3-methyl-2-butenyl substituent affects inhibitory mechanism against CpNanI. The tri-arginyl cluster of R266, R555, and R615 and D291 in NanI tightly interact with the competitive inhibitors.

The facile and visualizable identification of broad-spectrum inhibitors of MDM2/p53 using co-expressed protein complexes.

MDM2 is a well-known oncoprotein overexpressed in a variety of cancers, and the identification of inhibitors that disrupt the MDM2/p53 interaction is of great interest in anticancer drug development. Here we designed a platform for the facile and visualizable identification of inhibitors of MDM2 using co-expressed protein complexes of MDM2/p53. A hexahistidine-tag on MDM2 allows the binding of the protein complex to the Ni-NTA affinity resin, while the fluorescent protein fused to p53 enables the direct visualization of the interaction of p53 with MDM2. Hence, the inhibition of the MDM2/p53 interaction can be observed with the naked eye. The assay can be set up by directly loading cell lysate to the Ni-NTA affinity resin, and no chemical modification of proteins is needed. In addition to the qualitative analyses, the binding affinity of inhibitors to the MDM2 protein can be quantified by fluorescence titration. The applications of this system have been verified using small molecules and peptide inhibitors. As a proof of concept, we screened a small library using this platform. Interestingly, two types of novel inhibitors of MDM2, including cyclohexyl-triphenylamine derivatives and platinum complexes, were identified and their binding affinities were obtained. Quantitative measurements show that these new types of inhibitors demonstrate a high binding affinity (up to Kd = 51.9 nM) to MDM2.

Molecular insight into the T798M gatekeeper mutation-caused acquired resistance to tyrosine kinase inhibitors in ErbB2-positive breast cancer

Human epidermal growth factor receptor 2 (ErbB2) is an attractive therapeutic target for metastatic breast cancer. The kinase has been clinically observed to harbor a gatekeeper mutation T798M in its active site, which causes acquired resistance to the first-line targeted breast cancer therapy with small-molecule tyrosine kinase inhibitors. Previously, several theories have been proposed to explain the molecular mechanism of gatekeeper mutation-caused drug resistance, such as blocking of inhibitor binding and increasing of ATP affinity. In the current study, the direct binding of three wild type-selective inhibitors (Lapatinib, AEE788 and TAK-285) and two wild type-sparing inhibitors (Staurosporine and Bosutinib) to the wild-type ErbB2 and its T798M mutant are investigated in detail by using rigorous computational analysis and binding affinity assay. Substitution of the polar threonine with a bulky methionine at residue 798 can impair and improve the direct binding affinity of wild type-selective and wild type-sparing inhibitors, respectively. Hindrance effect is responsible for the affinity decrease of wild type-selective inhibitors, while additional nonbonded interactions contribute to the affinity increase of wild type-sparing inhibitors, thus conferring selectivity to the inhibitors for mutant over wild type. The binding affinity of Staurosporine and Bosutinib to ErbB2 kinase domain is improved by 11.9-fold and 2.1-fold upon T798M mutation, respectively. Structural analysis reveals that a nonbonded network of S–π contact interactions (for Staurosporine) or an S-involving halogen bond (for Bosutinib) forms with the sulfide group of mutant Met798 residue.

Molecular mechanism study of several inhibitors binding to BRD9 bromodomain based on molecular simulations

Bromodomain-containing protein 9 (BRD9) has been employed as a potential target for anticancer drugs in recent years. In this work, molecular docking, molecular dynamics (MD) simulations, binding free energy calculations, and per residue energy decomposition approaches were performed to elucidate the different binding modes between four pyridinone-like scaffold inhibitors and BRD9 bromodomain. Analysis results indicate that non-polar contribution mainly deriving from van der Waals energy is a critical impact on binding affinity of inhibitors against BRD9. Some key residues Phe44, Phe47, Val49, and Ile53 (at ZA loop) enhance the binding energy of inhibitors in BRD9 by means of providing hydrophobic interactions. Moreover, it is observed that BRD9 is anchored by the formation of a stable hydrogen bond between the carbonyl of the inhibitors and the residue Asn100 (at BC loop), and a strong π–π stacking interaction formed between the residue Tyr106 (at BC loop) and the inhibitors. The existence of dimethoxyphenyl structure and the aromatic ring merged to pyridinone scaffold are useful to enhance the BRD9 binding affinity. These findings should guide the rational design of more prospective inhibitors targeting BRD9.Communicated by Ramaswamy H. Sarma