Binding Modes Of Inhibitors Research Articles

Molecular dynamic simulations reveal structural insights into substrate and inhibitor binding modes and functionality of Ecto-Nucleoside Triphosphate Diphosphohydrolases

Ecto-nucleotidase enzymes catalyze the hydrolysis of extracellular nucleotides to their respective nucleosides. Herein, we place the focus on the elucidation of structural features of the cell surface located ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDase1-3 and 8). The physiological role of these isozymes is crucially important as they control purinergic signaling by modulating the extracellular availability of nucleotides. Since, crystal or NMR structure of the human isozymes are not available – structures have been obtained by homology modeling. Refinement of the homology models with poor stereo-chemical quality is of utmost importance in order to derive reliable structures for subsequent studies. Therefore, the resultant models obtained by homology modelling were refined by running molecular dynamic simulation. Binding mode analysis of standard substrates and of competitive inhibitor was conducted to highlight important regions of the active site involved in hydrolysis of the substrates and possible mechanism of inhibition.

Probing the binding mode and unbinding mechanism of LSD1 inhibitors by combined computational methods

Lysine specific demethylase 1 (LSD1) has emerged as a potential drug target in cancer therapy and a variety of inhibitors have been reported. We have recently reported the discovery of a series of triazole-dithiocarbamate based compounds, which were basically confirmed as cofactor flavin adenine dinucleotide (FAD)-competing inhibitors by experiments. However, the binding modes of the inhibitors to the binding site were undetermined. Here, we employed computational methods including molecular docking, classical molecular dynamics (MD) and steered molecular dynamics (SMD) simulations to investigate the potential binding modes of these inhibitors to LSD1. Based on the high correlation between the mean non-equilibrium pulling work W and experimental binding affinity, we identified the optimal binding modes of this class of compounds with LSD1. Using the optimal inhibitor binding conformation, we then performed SMD to study the ligand unbinding mechanism with a lower pulling velocity at 0.0005 nm ps-1. We found that residue Arg316 plays a crucial role in the binding/unbinding process. Furthermore, a gatekeeper residue Trp756 influences the ligand unbinding process by acting like a switch via steric hindrance but can enhance the hydrophobic interaction with the inhibitor. Hydrophobic interaction also dominated the interaction between LSD1 and the inhibitors. The pivotal residues and interactions between LSD1 and inhibitors determined from this study can be used to improve the inhibition activity of this series of inhibitors in development and to discover new scaffolds as FAD-competing inhibitors in compound screening.

Defining Dynamics of Membrane-Bound Pyrophosphatases by Experimental and Computational Single-Molecule FRET.

Membrane-bound pyrophosphatases couple the hydrolysis of inorganic pyrophosphate to the pumping of ions (sodium or protons) across a membrane in order to generate an electrochemical gradient. This class of membrane protein is widely conserved across plants, fungi, archaea, and bacteria, but absent in multicellular animals, making them a viable target for drug design against protozoan parasites such as Plasmodium falciparum. An excellent understanding of many of the catalytic states throughout the enzymatic cycle has already been afforded by crystallography. However, the dynamics and kinetics of the catalytic cycle between these static snapshots remain to be elucidated. Here, we employ single-molecule Förster resonance energy transfer (FRET) measurements to determine the dynamic range and frequency of conformations available to the enzyme in a lipid bilayer during the catalytic cycle. First, we explore issues related to the introduction of fluorescent dyes by cysteine mutagenesis; we discuss the importance of residue selection for dye attachment, and the balance between mutating areas of the protein that will provide useful dynamics while not altering highly conserved residues that could disrupt protein function. To complement and guide the experiments, we used all-atom molecular dynamics simulations and computational methods to estimate FRET efficiency distributions for dye pairs at different sites in different protein conformational states. We present preliminary single-molecule FRET data that points to insights about the binding modes of different membrane-bound pyrophosphatase substrates and inhibitors.

Analysis of the binding mode of a novel HIF1α inhibitor through molecular modelling.

Hypoxia inducible factor-1 (HIF1) is an important transcription factor related with tumor metastasis. As a subunit of HIF1, HIF1α plays an important role in regulation of the hypoxic response. HIF1α inhibitor could be a promising treatment for certain cancers. In the present study, we try to model the binding mode of the recent reported new series of HIF1α inhibitors with the purpose of further improving the performance of these inhibitors. Molecular docking was first employed to predict the binding modes of the protein-ligand complexes, followed by molecular dynamics simulations and MMGBSA free energy calculations. According to the predicted binding modes, these molecules form two important hydrogen bonds with HIF1α. Moreover, if the molecules could form a stable π-π interaction with HIF1α, the potency of the inhibitors can be greatly improved. Hydrophobic interactions between the molecules and HIF1α are also a key factor, especially the interactions between the hydrophobic groups (benzimidazole) of the molecules and the hydrophobic residues inside the binding cave. Molecular modeling could be a useful method in the future drug design. In this study, our calculation can help to design and develop high potent HIF1α inhibitors in future.

Similarity search combined with docking and molecular dynamics for novel hAChE inhibitor scaffolds.

The main purpose of this study was to address the performance of virtual screening methods based on ligands and the protein structure of acetylcholinesterase (AChE) in order to retrieve novel human AChE (hAChE) inhibitors. In addition, a protocol was developed to identify novel hit compounds and propose new promising AChE inhibitors from the ZINC database with 10 million commercially available compounds. In this sense, 3D similarity searches using rapid overlay of chemical structures and similarity analysis through comparison of electrostatic overlay of docked hits were used to retrieve AChE inhibitors from collected databases. Molecular dynamics simulation of 100ns was carried out to study the best docked compounds from similarity searches. Some key residues were identified as crucial for the dual binding mode of inhibitor with the interaction site. All results indicated the relevant use of EON and docking strategy for identifying novel hit compounds as promising potential anticholinesterase candidates, and seven new structures were selected as potential hAChE inhibitors. Graphical abstract Compound N01 in the 4M0E hAChE crystallography structure from docking results. Yellow dashed lines Hydrogen bonds, blue dashed lines π-stacking interactions, green dashed lines cation-π interactions.

A novel small molecule displays two different binding modes during inhibiting H1N1 influenza A virus neuraminidases.

Neuraminidase (NA) inhibitors can suppress NA activity to block the release of progeny virions and are effective against influenza viruses. As potential anti-flu drugs with unique functions, NA inhibitors are greatly concerned by the worldwide scientists. It has been reported recently that one of the novel quindoline derivatives named 7a, could inhibit both A/Puerto Rico/8/34 (H1N1) NA (NAPR) and A/California/04/09 (H1N1) NA (NACA). However, potential structure differences in the active site could be easily detected between the NAPR and NACA according to the flexibilities of their 150-loops located catalytic site. And no obvious 150-cavity could be observed in NACA crystal structure. In order to explore whether 7a could trigger the inhibition against these two NAs in the same way, a serial molecular dynamics simulation approach were applied in this study. The results indicated that 7a could be adopted under a relatively extended pose in the active center of NAPR. While in NACA-7a complex, the derivate preferred to be recognized and located on the side of active center. Interestingly, the potential of 7a was also found to be able to change the flexibility of the 150-loop in NACA that is absent of 150-cavity. Furthermore, a 150-cavity-like architecture could be induced in the active site of NACA. The results of this study revealed two kinds of binding modes of this novel small molecule inhibitor against NAs that might provide a theoretical basis for proposing novel inhibition mechanism and developing future influenza A virus inhibitors.

Three-Dimensional Analysis of the Interactions between hLDH5 and Its Inhibitors.

Inhibitors of human lactate dehydrogenase (hLDH5)—the enzyme responsible for the conversion of pyruvate to lactate coupled with oxidation of NADH to NAD+—are promising therapeutic agents against cancer because this enzyme is generally found to be overexpressed in most invasive cancer cells and is linked to their vitality especially under hypoxic conditions. Consequently, significant efforts have been made for the identification of small-molecule hLDH5 inhibitors displaying high inhibitory potencies. X-ray structure of hLDH5 complexes as well as molecular modeling studies contribute to identify and explain the main binding modes of hLDH5 inhibitors reported in literature. The purpose of this review is to analyze the main three-dimensional interactions between some of the most potent inhibitors and hLDH5, in order to provide useful suggestions for the design of new derivatives.

Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination of an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.

Modification of Bischler-Möhlau indole derivatives through palladium catalyzed Suzuki reaction as effective cholinesterase inhibitors, their kinetic and molecular docking studies

Due to the immense importance of aryl indole nucleus, herein we report the palladium-catalyzed arylation of N-substituted 2-aryl indole utilizing Suzuki-Miyaura cross coupling methodology. The biological screening for cholinesterase inhibition of the resulted biaryl indole moieties was carried out to evaluate their pharmacological potential, expecting to involve the development of new therapeutics for various inflammatory, cardiovascular, gastrointestinal and neurological diseases. This research work also involved the use of utilization of microwave-assisted organic synthesis (MAOS) for the synthesis of Bischler-Möhlau indole which is further biarylated via palladium-catalyzed cross coupling reaction. All the synthetic compounds (3a-n) were tested for cholinesterase inhibition and exhibited high level of AChE inhibitory activities. Interestingly, compounds 3m and 3n were found to be dual inhibitors, however, remaining compound exhibited no inhibitory activity against BChE. The biological potential of the resulted compounds was explained on the basis of molecular docking studies, performed against AChE and BChE, exploring the probable binding modes of most potent inhibitors.

Structural Insight into Inhibition of CsrA-RNA Interaction Revealed by Docking, Molecular Dynamics and Free Energy Calculations

The carbon storage regulator A (CsrA) and its homologs play an important role in coordinating the expression of bacterial virulence factors required for successful host infection. In addition, bacterial pathogens with deficiency of CsrA are typically attenuated for virulence. In 2016, the first series of small-molecule inhibitors of CsrA-RNA interaction were identified, which were found to achieve the CsrA-RNA inhibition by binding to the CsrA, without interfering with the RNA. However, the binding mechanism of these inhibitors of CsrA is not known. Herein, we applied molecular docking, molecular dynamics and binding free energy calculations to investigate the binding mode of inhibitors to CsrA. We found that the G11(RNA)-binding site is the most important binding site for CsrA inhibitors. An inhibitor with the proper size range can bind to that site and form a stable complex. We also found that inhibitors with larger size ranges bind to the entire CsrA-RNA interface, but have loose binding. However, this loose binding still resulted in inhibitory activity. The calculated binding free energy from MM/GBSA has a good correlation with the derived experimental binding energy, which might be used as a tool to further select CsrA inhibitors after a first-round of high-throughput virtual screening.

DINC 2.0: A New Protein-Peptide Docking Webserver Using an Incremental Approach.

Molecular docking is a standard computational approach to predict binding modes of protein-ligand complexes by exploring alternative orientations and conformations of the ligand (i.e., by exploring ligand flexibility). Docking tools are largely used for virtual screening of small drug-like molecules, but their accuracy and efficiency greatly decays for ligands with more than 10 flexible bonds. This prevents a broader use of these tools to dock larger ligands, such as peptides, which are molecules of growing interest in cancer research. To overcome this limitation, our group has previously proposed a meta-docking strategy, called DINC, to predict binding modes of large ligands. By incrementally docking overlapping fragments of a ligand, DINC allowed predicting binding modes of peptide-based inhibitors of transcription factors involved in cancer. Here, we describe DINC 2.0, a revamped version of the DINC webserver with enhanced capabilities and a more user-friendly interface. DINC 2.0 allows docking ligands that were previously too challenging for DINC, such as peptides with more than 25 flexible bonds. The webserver is freely accessible at http://dinc.kavrakilab.org, together with additional documentation and video tutorials. Our team will provide continuous support for this tool and is working on extending its applicability to other challenging fields, such as personalized immunotherapy against cancer. Cancer Res; 77(21); e55-57. ©2017 AACR.

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 50ns 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.

Design and synthesis of 2,6-di(substituted phenyl)thiazolo[3,2-b]-1,2,4-triazoles as α-glucosidase and α-amylase inhibitors, co-relative Pharmacokinetics and 3D QSAR and risk analysis

Ten fused heterocyclic derivatives bearing the 2,6-di(subsituted phenyl)thiazolo[3,2-b]-1,2,4-triazoles as central rings were synthesized and structures of the compounds were established by analytical and spectral data using FTIR, EI-MS, 1H NMR and 13C NMR techniques. In vitro inhibitory activities of synthesized compounds on α-amylase, α-glucosidase and α-burylcholinesterase (α-BuChE) were evaluated using a purified enzyme assays. Compound 5c demonstrated strong and selective α-amylase inhibitory activity (IC50=1.1μmol/g). 5g exhibited excellent inhibition against α-glucosidase (IC50=1.2μmol/g) when compared with acarbose (IC50=4.7μmol/g) as a positive reference. Compound 5i was found to be most potent derivative against α-BuChE with the IC50 of 1.5μmol/g which was comparable to the value obtained for (4.7μmol/g) positive control (i.e. galantamine hydrobromide). Molecular dockings of synthesized compounds into the binding sites of human pancreatic α-amylase, intestinal maltase-glucoamylase and neuronal α-butrylcholinesterase allowed to shed light on the affinity and binding mode of these novel inhibitors. Preliminary structure–activity relationship (SAR) studies were carried out to understand the relationship between molecular structural features and inhibition activities of synthesized derivatives. These data suggested that compounds 5c, 5g and 5i are promising candidates for hitto- lead follow-up in the drug-discovery process for the treatment of Alzheimer’s disease and hyperinsulinamia.

Therapeutic efficacy of a respiratory syncytial virus fusion inhibitor

Respiratory syncytial virus is a major cause of acute lower respiratory tract infection in young children, immunocompromised adults, and the elderly. Intervention with small-molecule antivirals specific for respiratory syncytial virus presents an important therapeutic opportunity, but no such compounds are approved today. Here we report the structure of JNJ-53718678 bound to respiratory syncytial virus fusion (F) protein in its prefusion conformation, and we show that the potent nanomolar activity of JNJ-53718678, as well as the preliminary structure–activity relationship and the pharmaceutical optimization strategy of the series, are consistent with the binding mode of JNJ-53718678 and other respiratory syncytial virus fusion inhibitors. Oral treatment of neonatal lambs with JNJ-53718678, or with an equally active close analog, efficiently inhibits established acute lower respiratory tract infection in the animals, even when treatment is delayed until external signs of respiratory syncytial virus illness have become visible. Together, these data suggest that JNJ-53718678 is a promising candidate for further development as a potential therapeutic in patients at risk to develop respiratory syncytial virus acute lower respiratory tract infection.

Recent Research Advances in Selective Matrix Metalloproteinase-13 Inhibitors as Anti-Osteoarthritis Agents.

Matrix metalloproteinase-13 (MMP-13) plays a key role in the degradation of type II collagen in cartilage and bone in osteoarthritis (OA). The subtle differences between the S1' loop of MMP-13 and that of other MMPs offer a structural base for the design of selective MMP-13 inhibitors to mitigate the unperceived risk associated with inhibiting other MMP isoforms. In this review, we summarize zinc-binding and non-zinc-binding selective MMP-13 inhibitors. The zinc-binding MMP-13 inhibitors contain a small set of zinc-binding groups (ZBGs), including hydroxamic acid, pyrimidinetrione, reversed hydroxamic acid and hydantoin, carboxylic acid, 1,2,4,-triazole, and 1,2,4,-triazolone. The non-zinc-binding MMP-13 inhibitors have different structural scaffolds, including diphenyl ethers, biaryls (aryltetrazoliums, arylfurans, pyrazole-indoles), pyrimidines, and aryl/cycloalkyl-fused pyrimidines. This review provides a systematic overview of recent developments in MMP-13 inhibitors for the treatment of OA, with emphasis on their enzyme inhibitory potency, selectivity, and biological activities, and highlights the various binding modes of typical inhibitors with MMP-13.

Synthesis of Alkynylated Dihydrofuran‐2(3H)‐ones as Potent and Selective Inhibitors of Tissue Non‐Specific Alkaline Phosphatase

AbstractThe synthesis of new derivatives of 3‐(3‐arylprop‐2‐ynyl)dihydrofuran‐2(3H)‐ones by Sonogashira reactions and their screening as potent and selective inhibitors of b‐TNAP is reported. Although a few derivatives exhibited non‐selective inhibition of c‐IAP, most of the derivatives were found to be potent and selective inhibitors of b‐TNAP. The selective and potent inhibitors of b‐TNAP might be useful for the treatment of vascular calcification. Among the derivatives studied, one compound was identified as the most potent inhibitor of b‐TNAP with a high inhibitory value, exhibiting a 6 fold more selective activity towards b‐TNAP as compared to c‐IAP. The higher inhibitory activity of this compound towards b‐TNAP is caused by the presence of two furan rings. Similarly, another derivative, having a furan ring, exhibited a lower inhibitory potential towards b‐TNAP, but this compound was found to be the most potent inhibitor of c‐IAP. Furthermore, molecular docking studies of these potent compounds were carried out to see the possible binding modes of potent inhibitors inside the active pocket of respective enzyme.

Abstract 4100: Mechanisms of resistance to type I and type II MET inhibitors in non-small cell lung cancer

Abstract Background: MET targeted therapies are clinically effective in MET amplified and MET exon 14 deletion mutant non-small cell lung cancers (NSCLC). At least 8 MET tyrosine kinase inhibitors (TKIs), including both type I and II, have been developed and are under clinical evaluation. We recently described a resistance mechanism in a patient with a unique MET secondary mutation, D1228V, refractory to all type I but sensitive to type II MET TKIs (Bahcall et al. Cancer Discovery 2016). We examined whether the sensitivity and resistance of other MET secondary mutations were similarly dependent on the mode of inhibitor binding, and sought to determine the optimal sequence of MET inhibitor use, so as to achieve the longest combined latency before the emergence of resistance. Methods: TPR-MET Ba/F3 cells were mutagenized with ENU and treated with each of the type I TKIs - crizotinib; savolitinib; capmatinib, or type II TKIs - cabozantinib; glesatinib; merestinib, until resistant clones emerged. Resistant mutations were identified by sequencing, constructed in the TPR-MET background, and expressed in Ba/F3 and NIH-3T3 cells. Cross-resistance to the 6 MET TKIs was evaluated. Next, low frequency (0.1%) of mutant Ba/F3 cells was spiked into parental TPR-MET Ba/F3 cells and sequentially exposed to different MET TKIs to identify the sequence associated with the longest combined time to resistance. Clonal evolution was assessed by droplet digital PCR (ddPCR). Results: Of 300 plated wells per drug used at either 0.1μM or 0.5μM, crizotinib yielded 210 and 2 clones; savolitinib 51 and 1 clone; capmatinib 9 and 3 clones; cabozantinib 38 and 3 clones; glesatinib 300 and 300 clones; merestinib 18 and 3 clones, respectively. Of the mutations, those at Y1230 were common to all 3 type I TKIs and the most frequently seen with savolitinib and capmatinib; D1228 was shared by savolitinib and capmatinib; V1155L, the most common mutation with crizotinib, was shared with savolitinib; M1211L emerged with capmatinib. Mutated F1200 residue was shared by and exclusive to all type II TKIs and was the single identified mutation for both glesatinib and merestinib. Cabozantinib gave rise to a broader array of unique mutants - with F1200 mutations being the most common - and shared G1163R with crizotinib. We show D1228 and Y1230 mutations being moderately resistant to crizotinib and strongly resistant to both savolitinib and capmatinib, while retaining sensitivity to all type II TKIs. G1163R and L1195V were slightly to moderately resistant to crizotinib, cabozantinib and glesatinib, but strongly sensitive to savolitinib and capmatinib. In this assay, merestinib had the broadest, while crizotinib most narrow activity against the tested mutants. Conclusions: Here we highlight key differences between and within the 2 types of MET inhibitors that define their activity against MET secondary mutants likely to emerge in patients, providing rationale for the specific use of these inhibitors in the clinic. Citation Format: Magda Bahcall, Yanan Kuang, Cloud P. Paweletz, Pasi A. Jänne. Mechanisms of resistance to type I and type II MET inhibitors in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4100. doi:10.1158/1538-7445.AM2017-4100

The Ligand Binding Landscape of Diacylglycerol Kinases

Diacylglycerol kinases (DGKs) are integral components of signal transduction cascades that regulate cell biology through ATP-dependent phosphorylation of the lipid messenger diacylglycerol. Methods for direct evaluation of DGK activity in native biological systems are lacking and needed to study isoform-specific functions of these multidomain lipid kinases.Here, we utilize ATP acyl phosphate activity-based probes and quantitative mass spectrometry to define, for the first time, ATP and small-molecule binding motifs of representative members from all five DGK subtypes. We use chemical proteomics to discover an unusual binding mode for the DGKα inhibitor, ritanserin, including interactions at the atypical C1 domain distinct from the ATP binding region.Unexpectedly, deconstruction of ritanserin yielded afragment compound that blocks DGKα activitythrough a conserved binding mode and enhanced selectivity against the kinome. Collectively, our studies illustrate the power of chemical proteomics to profile protein-small molecule interactions of lipid kinases for fragment-based lead discovery.

Leucine rich repeat kinase 2 (LRRK2) inhibitors based on indolinone scaffold: Potential pro-neurogenic agents

Leucine-rich repeat kinase 2 (LRRK2) is one of the most pursued targets for Parkinson's disease (PD) therapy. Moreover, it has recently described its role in regulating Wnt signaling and thus, it may be involved in adult neurogenesis. This new hypothesis could give rise to double disease-modifying agents firstly by the benefits of inhibiting LRRK2 and secondly by promoting adult neurogenesis. Herein we report, the design, synthesis, biological evaluation, SAR and potential binding mode of indoline-like LRRK2 inhibitors and their preliminary neurogenic effect in neural precursor cells isolated from adult mice ventricular-subventricular zone. These results open new therapeutic horizons for the use of LRRK2 inhibitors as neuroregenerative agents. Moreover, the indolinone derivatives here prepared, inhibitors of the kinase activity of LRRK2, may be considered as pharmacological probes to study the potential neuroregeneration of the damaged brain.

Insights into the binding mode of MEK type-III inhibitors. A step towards discovering and designing allosteric kinase inhibitors across the human kinome.

Protein kinases are critical drug targets for treating a large variety of human diseases. Type-III kinase inhibitors have attracted increasing attention as highly selective therapeutics. Thus, understanding the binding mechanism of existing type-III kinase inhibitors provides useful insights into designing new type-III kinase inhibitors. In this work, we have systematically studied the binding mode of MEK-targeted type-III inhibitors using structural systems pharmacology and molecular dynamics simulation. Our studies provide detailed sequence, structure, interaction-fingerprint, pharmacophore and binding-site information on the binding characteristics of MEK type-III kinase inhibitors. We hypothesize that the helix-folding activation loop is a hallmark allosteric binding site for type-III inhibitors. Subsequently, we screened and predicted allosteric binding sites across the human kinome, suggesting other kinases as potential targets suitable for type-III inhibitors.