Identification of small-molecule protein-protein interaction inhibitors for NKG2D.

NKG2D (natural-killer group 2, member D) is a homodimeric transmembrane receptor that plays an important role in NK, γδ+, and CD8+ T cell-mediated immune responses to environmental stressors such as viral or bacterial infections and oxidative stress. However, aberrant NKG2D signaling has also been associated with chronic inflammatory and autoimmune diseases, and as such NKG2D is thought to be an attractive target for immune intervention. Here, we describe a comprehensive small-molecule hit identification strategy and two distinct series of protein-protein interaction inhibitors of NKG2D. Although the hits are chemically distinct, they share a unique allosteric mechanism of disrupting ligand binding by accessing a cryptic pocket and causing the two monomers of the NKG2D dimer to open apart and twist relative to one another. Leveraging a suite of biochemical and cell-based assays coupled with structure-based drug design, we established tractable structure-activity relationships with one of the chemical series and successfully improved both the potency and physicochemical properties. Together, we demonstrate that it is possible, albeit challenging, to disrupt the interaction between NKG2D and multiple protein ligands with a single molecule through allosteric modulation of the NKG2D receptor dimer/ligand interface.

Structural insight into selectivity and resistance profiles of ROS1 tyrosine kinase inhibitors.

Oncogenic ROS1 fusion proteins are molecular drivers in multiple malignancies, including a subset of non-small cell lung cancer (NSCLC). The phylogenetic proximity of the ROS1 and anaplastic lymphoma kinase (ALK) catalytic domains led to the clinical repurposing of the Food and Drug Administration (FDA)-approved ALK inhibitor crizotinib as a ROS1 inhibitor. Despite the antitumor activity of crizotinib observed in both ROS1- and ALK-rearranged NSCLC patients, resistance due to acquisition of ROS1 or ALK kinase domain mutations has been observed clinically, spurring the development of second-generation inhibitors. Here, we profile the sensitivity and selectivity of seven ROS1 and/or ALK inhibitors at various levels of clinical development. In contrast to crizotinib's dual ROS1/ALK activity, cabozantinib (XL-184) and its structural analog foretinib (XL-880) demonstrate a striking selectivity for ROS1 over ALK. Molecular dynamics simulation studies reveal structural features that distinguish the ROS1 and ALK kinase domains and contribute to differences in binding site and kinase selectivity of the inhibitors tested. Cell-based resistance profiling studies demonstrate that the ROS1-selective inhibitors retain efficacy against the recently reported CD74-ROS1(G2032R) mutant whereas the dual ROS1/ALK inhibitors are ineffective. Taken together, inhibitor profiling and stringent characterization of the structure-function differences between the ROS1 and ALK kinase domains will facilitate future rational drug design for ROS1- and ALK-driven NSCLC and other malignancies.

LSD1/CoREST is an allosteric nanoscale clamp regulated by H3-histone-tail molecular recognition.

The complex of lysine-specific demethylase-1 (LSD1/KDM1A) with its corepressor protein CoREST is an exceptionally relevant target for epigenetic drugs. Here, we provide insight into the local and global changes of LSD1/CoREST conformational dynamics that occur upon H3 binding on the basis of a total cumulative time of one microsecond molecular dynamics simulation. The LSD1/CoREST complex functions as an allosteric nanoscale-binding clamp, which is regulated by substrate binding. In the unbound state, LSD1/CoREST reversibly visits clamp states that are more open or significantly more closed compared with the available X-ray crystal structures. The Lys triad of residues Lys355, Lys357, and Lys359 gates the entrance of the H3 pocket. H3 binding shifts the pocket breathing dynamics toward open, higher-volume states while reducing the overall flexibility of the LSD1/CoREST nanoscale clamp. We show that the H3 pocket is an allosteric site for the regulation of the rotation of the amino oxidase domain with respect to the Tower domain. The allosteric mechanism relies on the specific reduction of nanoscale domain rotation upon local H3-tail binding. Instead, clamp opening/closing motions that do not involve domain rotation only reduce in amplitude yet are dominant in the bound state. Overall, our data suggest that the H3 binding pocket is a central target site to (i) switch off LSD1 amino oxidase activity, thus H3-tail demethylation; (ii) block the competitive binding of transcription factors; and (iii) prevent chromatin anchoring to LSD1/CoREST. This study underscores the importance of receptor flexibility for future epigenetic drug discovery.

Expanding the druggable space of the LSD1/CoREST epigenetic target: new potential binding regions for drug-like molecules, peptides, protein partners, and chromatin.

Lysine specific demethylase-1 (LSD1/KDM1A) in complex with its corepressor protein CoREST is a promising target for epigenetic drugs. No therapeutic that targets LSD1/CoREST, however, has been reported to date. Recently, extended molecular dynamics (MD) simulations indicated that LSD1/CoREST nanoscale clamp dynamics is regulated by substrate binding and highlighted key hinge points of this large-scale motion as well as the relevance of local residue dynamics. Prompted by the urgent need for new molecular probes and inhibitors to understand LSD1/CoREST interactions with small-molecules, peptides, protein partners, and chromatin, we undertake here a configurational ensemble approach to expand LSD1/CoREST druggability. The independent algorithms FTMap and SiteMap and our newly developed Druggable Site Visualizer (DSV) software tool were used to predict and inspect favorable binding sites. We find that the hinge points revealed by MD simulations at the SANT2/Tower interface, at the SWIRM/AOD interface, and at the AOD/Tower interface are new targets for the discovery of molecular probes to block association of LSD1/CoREST with chromatin or protein partners. A fourth region was also predicted from simulated configurational ensembles and was experimentally validated to have strong binding propensity. The observation that this prediction would be prevented when using only the X-ray structures available (including the X-ray structure bound to the same peptide) underscores the relevance of protein dynamics in protein interactions. A fifth region was highlighted corresponding to a small pocket on the AOD domain. This study sets the basis for future virtual screening campaigns targeting the five novel regions reported herein and for the design of LSD1/CoREST mutants to probe LSD1/CoREST binding with chromatin and various protein partners.

Substrate selection influences molecular recognition in a screen for lymphoid tyrosine phosphatase inhibitors.

Assay design is an important variable that influences the outcome of an inhibitor screen. Here, we have investigated the hypothesis that protein tyrosine phosphatase inhibitors with improved biological activity could be identified from a screen by using a biologically relevant peptide substrate, rather than traditional phosphotyrosine mimetic substrates. A 2000-member library of drugs and drug-like compounds was screened for inhibitors of lymphoid tyrosine phosphatase (LYP) by using both a peptide substrate (Ac-ARLIEDNE-pCAP-TAREG-NH2, peptide 1) and a small-molecule phosphotyrosine mimetic substrate (difluoromethyl umbelliferyl phosphate, DiFMUP). The results demonstrate that compounds that inhibited enzyme activity on the peptide substrate had greater biological activity than compounds that only inhibited enzyme activity on DiFMUP. Finally, epigallocatechin-3,5-digallate was identified as the most potent inhibitor of lymphoid tyrosine phosphatase activity to date, with an IC50 of 50 nM and significant activity in T-cells. Molecular docking simulations provided a first model for binding of this potent inhibitor to LYP; this will constitute the platform for ongoing lead optimization efforts.

LSD1/CoREST reversible opening-closing dynamics: discovery of a nanoscale clamp for chromatin and protein binding.

LSD1 associated with its corepressor protein CoREST is an exceptionally relevant target for epigenetic drugs. Hypotheses for the role of LSD1/CoREST as a multidocking site for chromatin and protein binding would require significant molecular flexibility, and LSD1/CoREST large-amplitude conformational dynamics is currently unknown. Here, molecular dynamics simulation reveals that the LSD1/CoREST complex in solution functions as a reversible nanoscale binding clamp. We show that the H3 histone tail binding pocket is a potential allosteric site for regulation of the rotation of SWIRM/SANT2 domains around the Tower domain. Thus, targeting this site and including receptor flexibility are crucial strategies for future drug discovery.

Molecular docking of bisphenol A and its nitrated and chlorinated metabolites onto human estrogen-related receptor-gamma.

A xenoestrogen and known endocrine disruptor, bisphenol A (BPA) binds the human estrogen-related receptor-gamma (ERRγ) with high affinity (Kd ≈ 5.5 nM). It is likely that BPA undergoes oxidative biotransformation by hypochlorite/hypochlorous acid ((-)OCl/HOCl) and peroxynitrite (PN) and the products formed in these reactions may serve as secondary estrogens and contribute to the toxicodynamics of BPA. Therefore, in the present study we have examined the formation of chlorinated and nitrated BPA in reactions of BPA with (-)OCl/HOCl and PN(+CO(2)) performed around the neutral pH. We have identified four major products in these reactions and they include 3-chloro-BPA (CBPA), 3,3'-dichloro-BPA (DCBPA), 3-nitro-BPA (NBPA) and 3,3'-dinitro-BPA (DNBPA). Towards understanding the toxicodynamics and estrogenic activity of BPA in biological systems, we have performed molecular docking of BPA, CBPA, DCBPA, DNBPA and NBPA onto the ERRγ using AutoDock 4.2 software and compared the binding energies with those of estradiol, the natural ligand. Based on the genetic algorithm, the three best conformations were selected and averaged for each ligand and a detailed analysis of molecular interactions based on free energies of binding (kcal/mol) was computed. The results indicate the following rank order of binding to ERRγ: BPA (-8.78 ± 0.06) > CBPA (-8.53 ± 0.41) > NBPA (-7.36 ± 0.74) > DCBPA (-5.24 ± 0.17) > DNBPA (-4.95 ± 0.78) > estradiol (-4.94 ± 1.04). The docking studies revealed that the OH group of one of the phenyl rings forms a hydrogen bond with Glu275/Arg316, while the OH group of other phenyl ring was bound to Asp346. These results suggest that both BPA and its putative chlorinated and nitrated metabolites have strong binding affinity compared to estradiol.

Simulation of multiphase systems utilizing independent force fields to control intraphase and interphase behavior.

Fixed-charge empirical force fields have been developed and widely used over the past three decades for all-atom molecular simulations. Most simulation programs providing these methods enable only one set of force field parameters to be used for the entire system. Whereas this is generally suitable for single-phase systems, the molecular environment at the interface between two phases may be sufficiently different from the individual phases to require a different set of parameters to be used to accurately represent the system. Recently published simulations of peptide adsorption to material surfaces using the CHARMM force field have clearly demonstrated this issue by revealing that calculated values of adsorption free energy substantially differ from experimental results. Whereas nonbonded parameters could be adjusted to correct this problem, this cannot be done without also altering the conformational behavior of the peptide in solution, for which CHARMM has been carefully tuned. We have developed a dual-force-field approach (Dual-FF) to address this problem and implemented it in the CHARMM simulation package. This Dual-FF method provides the capability to use two separate sets of nonbonded force field parameters within the same simulation: one set to represent intraphase interactions and a separate set to represent interphase interactions. Using this approach, we show that interfacial parameters can be adjusted to correct errors in peptide adsorption free energy without altering peptide conformational behavior in solution. This program thus provides the capability to enable both intraphase and interphase molecular behavior to be accurately and efficiently modeled in the same simulation.

SIRT5 IS A DRUGGABLE METABOLIC VULNERABILITY IN ACUTE MYELOID LEUKEMIA.

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype-agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.

Assessment of the transferability of a protein force field for the simulation of peptide-surface interactions.

In order to evaluate the transferability of existing empirical force fields for all-atom molecular simulations of protein adsorption behavior, we have developed and applied a method to calculate the adsorption free energy (DeltaG(ads)) of model peptides on functionalized surfaces for comparison with available experimental data. Simulations were conducted using the CHARMM program and force field using a host-guest peptide with the sequence TGTG-X-GTGT (where G and T are glycine and threonine amino acid residues, respectively, with X representing valine, threonine, aspartic acid, phenylalanine or lysine) over nine different functionalized alkanethiol self-assembled monolayer (SAM) surfaces with explicitly represented solvent. DeltaG(ads) was calculated using biased-energy replica exchange molecular dynamics to adequately sample the conformational states of the system. The simulation results showed that the CHARMM force-field was able to represent DeltaG(ads) within 1 kcal/mol of the experimental values for most systems, while deviations as large as 4 kcal/mol were found for others. In particular, the simulations reveal that CHARMM underestimates the strength of adsorption on the hydrophobic and positively charged amine surfaces. These results clearly show that improvements in force field parameterization are needed in order to accurately represent interactions between amino acid residues and functional groups of a surface and they provide a means for force field evaluation and modification for the eventual development and validation of an interfacial force field for the accurate simulation of protein adsorption behavior.

BCR-ABL1 tyrosine kinase inhibitor K0706 exhibits preclinical activity in Philadelphia chromosome-positive leukemia.

BCR-ABL1 tyrosine kinase inhibitors (TKIs) are the cornerstone of treatment in chronic myeloid leukemia. Although there are now four TKIs approved for use in the front-line setting, acquired TKI resistance via secondary kinase domain mutations remains a problem for patients. K0706 is a novel BCR-ABL1 TKI currently under clinical investigation with structural elements similar to those of ponatinib and dasatinib. In this article, we functionally characterize the anti-leukemic activity of K0706 using cell proliferation assays in conjunction with drug resistance screening. We provide details from molecular modeling to support our in vitro findings and additionally describe our limited clinical experience with this drug in two patients treated on trial. We demonstrate that although K0706 retains efficacy against a large spectrum of clinically relevant mutations, it does not appear to have activity against BCR-ABL1T315I. Early trial experience suggests excellent tolerability, which may positively affect the place of K0706 within the ever-expanding chronic myeloid leukemia treatment paradigm.

Combining the Allosteric Inhibitor Asciminib with Ponatinib Suppresses Emergence of and Restores Efficacy against Highly Resistant BCR-ABL1 Mutants.

BCR-ABL1 point mutation-mediated resistance to tyrosine kinase inhibitor (TKI) therapy in Philadelphia chromosome-positive (Ph+) leukemia is effectively managed with several approved drugs, including ponatinib for BCR-ABL1T315I-mutant disease. However, therapy options are limited for patients with leukemic clones bearing multiple BCR-ABL1 mutations. Asciminib, an allosteric inhibitor targeting the myristoyl-binding pocket of BCR-ABL1, is active against most single mutants but ineffective against all tested compound mutants. We demonstrate that combining asciminib with ATP site TKIs enhances target inhibition and suppression of resistant outgrowth in Ph+ clinical isolates and cell lines. Inclusion of asciminib restores ponatinib's effectiveness against currently untreatable compound mutants at clinically achievable concentrations. Our findings support combining asciminib with ponatinib as a treatment strategy for this molecularly defined group of patients.

New Strategies in Myeloproliferative Neoplasms: The Evolving Genetic and Therapeutic Landscape.

The classical BCR-ABL1-negative myeloproliferative neoplasms (MPN) include essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF). Although these clonal disorders share certain clinical and genetic features, MF in particular is distinct for its complex mutational landscape, severe disease phenotype, and poor prognosis. The genetic complexity inherent to MF has made this disease extremely challenging to treat. Pharmacologic JAK inhibition has proven to be a transformative therapy in MPNs, alleviating symptom burden and improving survival, but has been hampered by off-target toxicities and, as monotherapy, has shown limited effects on mutant allele burden. In this review, we discuss the genetic heterogeneity contributing to the pathogenesis of MPNs, focusing on novel driver and epigenetic mutations and how they relate to combination therapeutic strategies. We discuss results from ongoing studies of new JAK inhibitors and report on new drugs and drug combinations that have demonstrated success in early preclinical and clinical trials, including type II JAK inhibitors, antifibrotic agents, and telomerase inhibitors.

Disarming an Electrophilic Warhead: Retaining Potency in Tyrosine Kinase Inhibitor (TKI)-Resistant CML Lines While Circumventing Pharmacokinetic Liabilities.

Pharmacologic blockade of the activation of signal transducer and activator of transcription 3 (STAT3) in tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia (CML) cell lines characterized by kinase-independent resistance was shown to re-sensitize CML cells to TKI therapy, suggesting that STAT3 inhibitors in combination with TKIs are an effective combinatorial therapeutic for the treatment of CML. Benzoic acid- and hydroxamic acid-based STAT3 inhibitors SH-4-054 and SH-5-007, developed previously in our laboratory, demonstrated promising activity against these resistant CML cell lines. However, pharmacokinetic studies in murine models (CD-1 mice) revealed that both SH-4-054 and SH-5-007 are susceptible to glutathione conjugation at the para position of the pentafluorophenyl group via nucleophilic aromatic substitution (SN Ar). To determine whether the electrophilicity of the pentafluorophenyl sulfonamide could be tempered, an in-depth structure-activity relationship (SAR) study of the SH-4-054 scaffold was conducted. These studies revealed that AM-1-124, possessing a 2,3,5,6-tetrafluorophenylsulfonamide group, retained STAT3 protein affinity (Ki =15 µm), as well as selectivity over STAT1 (Ki >250 µm). Moreover, in both hepatocytes and in in vivo pharmacokinetic studies (CD-1 mice), AM-1-124 was found to be dramatically more stable than SH-4-054 (t1/2 =1.42 h cf. 10 min, respectively). AM-1-124 is a promising STAT3-targeting inhibitor with demonstrated bioavailability, suitable for evaluation in preclinical cancer models.

A Novel Crizotinib-Resistant Solvent-Front Mutation Responsive to Cabozantinib Therapy in a Patient with ROS1-Rearranged Lung Cancer.

PURPOSE: Rearranged ROS1 is a crizotinib-sensitive oncogenic driver in lung cancer. The development of acquired resistance, however, poses a serious clinical challenge. Consequently, experimental and clinical validation of resistance mechanisms and potential second-line therapies is essential. EXPERIMENTAL DESIGN: We report the discovery of a novel, solvent-front ROS1(D2033N) mutation in a patient with CD74-ROS1-rearranged lung adenocarcinoma and acquired resistance to crizotinib. Crizotinib resistance of CD74-ROS1(D2033N) was functionally evaluated using cell-based assays and structural modeling. RESULTS: In biochemical and cell-based assays, the CD74-ROS1(D2033N) mutant demonstrated significantly decreased sensitivity to crizotinib. Molecular dynamics simulation revealed compromised crizotinib binding due to drastic changes in the electrostatic interaction between the D2033 residue and crizotinib and reorientation of neighboring residues. In contrast, cabozantinib binding was unaffected by the D2033N substitution, and inhibitory potency against the mutant was retained. Notably, cabozantinib treatment resulted in a rapid clinical and near-complete radiographic response in this patient. CONCLUSIONS: These results provide the first example of successful therapeutic intervention with targeted therapy to overcome crizotinib resistance in a ROS1-rearranged cancer. Clin Cancer Res; 22(10); 2351-8. ©2015 AACR.

A marine analgesic peptide, Contulakin-G, and neurotensin are distinct agonists for neurotensin receptors: uncovering structural determinants of desensitization properties.

Neurotensin receptors have been studied as molecular targets for the treatment of pain, schizophrenia, addiction, or cancer. Neurotensin (NT) and Contulakin-G, a glycopeptide isolated from a predatory cone snail Conus geographus, share a sequence similarity at the C-terminus, which is critical for activation of neurotensin receptors. Both peptides are potent analgesics, although affinity and agonist potency of Contulakin-G toward neurotensin receptors are significantly lower, as compared to those for NT. In this work, we show that the weaker agonist properties of Contulakin-G result in inducing significantly less desensitization of neurotensin receptors and preserving their cell-surface density. Structure-activity relationship (SAR) studies suggested that both glycosylation and charged amino acid residues in Contulakin-G or NT played important roles in desensitizing neurotensin receptors. Computational modeling studies of human neurotensin receptor NTS1 and Contulakin-G confirmed the role of glycosylation in weakening interactions with the receptors. Based on available SAR data, we designed, synthesized, and characterized an analog of Contulakin-G in which the glycosylated amino acid residue, Gal-GalNAc-Thr10, was replaced by memantine-Glu10 residue. This analog exhibited comparable agonist potency and weaker desensitization properties as compared to that of Contulakin-G, while producing analgesia in the animal model of acute pain following systemic administration. We discuss our study in the context of feasibility and safety of developing NT therapeutic agents with improved penetration across the blood-brain barrier. Our work supports engineering peptide-based agonists with diverse abilities to desensitize G-protein coupled receptors and further emphasizes opportunities for conotoxins as novel pharmacological tools and drug candidates.

Combined STAT3 and BCR-ABL1 inhibition induces synthetic lethality in therapy-resistant chronic myeloid leukemia.

Mutations in the BCR-ABL1 kinase domain are an established mechanism of tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive leukemia, but fail to explain many cases of clinical TKI failure. In contrast, it is largely unknown why some patients fail TKI therapy despite continued suppression of BCR-ABL1 kinase activity, a situation termed BCR-ABL1 kinase-independent TKI resistance. Here, we identified activation of signal transducer and activator of transcription 3 (STAT3) by extrinsic or intrinsic mechanisms as an essential feature of BCR-ABL1 kinase-independent TKI resistance. By combining synthetic chemistry, in vitro reporter assays, and molecular dynamics-guided rational inhibitor design and high-throughput screening, we discovered BP-5-087, a potent and selective STAT3 SH2 domain inhibitor that reduces STAT3 phosphorylation and nuclear transactivation. Computational simulations, fluorescence polarization assays and hydrogen-deuterium exchange assays establish direct engagement of STAT3 by BP-5-087 and provide a high-resolution view of the STAT3 SH2 domain/BP-5-087 interface. In primary cells from chronic myeloid leukemia (CML) patients with BCR-ABL1 kinase-independent TKI resistance, BP-5-087 (1.0 µM) restored TKI sensitivity to therapy-resistant CML progenitor cells, including leukemic stem cells. Our findings implicate STAT3 as a critical signaling node in BCR-ABL1 kinase-independent TKI resistance, and suggest that BP-5-087 has clinical utility for treating malignancies characterized by STAT3 activation.

Epigenetic molecular recognition: a biomolecular modeling perspective.

The abnormal regulation of epigenetic protein families is associated with the onset and progression of various human diseases. However, epigenetic processes remain relatively obscure at the molecular level, thus preventing the rational design of chemical therapeutics. An array of robust computational and modeling approaches can complement experiments to shed light on the complex mechanisms of epigenetic molecular recognition and can guide medicinal chemists in designing selective and potent drug molecules. Herein we present a review of studies focused on epigenetic molecular recognition from a biomolecular modeling viewpoint. Although the known epigenetic targets are numerous, this review focuses on the more limited protein families on which computational modeling has been successfully applied. Therefore, we review three main topics: 1) histone deacetylases, 2) histone demethylases, and 3) histone tail dynamics. A brief review of the biological background and biomedical relevance is presented for each topic, followed by a detailed discussion of the computational studies and their relevance.

BCR-ABL1 compound mutations combining key kinase domain positions confer clinical resistance to ponatinib in Ph chromosome-positive leukemia.

Ponatinib is the only currently approved tyrosine kinase inhibitor (TKI) that suppresses all BCR-ABL1 single mutants in Philadelphia chromosome-positive (Ph(+)) leukemia, including the recalcitrant BCR-ABL1(T315I) mutant. However, emergence of compound mutations in a BCR-ABL1 allele may confer ponatinib resistance. We found that clinically reported BCR-ABL1 compound mutants center on 12 key positions and confer varying resistance to imatinib, nilotinib, dasatinib, ponatinib, rebastinib, and bosutinib. T315I-inclusive compound mutants confer high-level resistance to TKIs, including ponatinib. In vitro resistance profiling was predictive of treatment outcomes in Ph(+) leukemia patients. Structural explanations for compound mutation-based resistance were obtained through molecular dynamics simulations. Our findings demonstrate that BCR-ABL1 compound mutants confer different levels of TKI resistance, necessitating rational treatment selection to optimize clinical outcome.

Molecular dynamics simulations indicate an induced-fit mechanism for LSD1/CoREST-H3-histone molecular recognition.

BACKGROUND: Lysine Specific Demethylase (LSD1 or KDM1A) in complex with its co-repressor protein CoREST catalyzes the demethylation of the H3 histone N-terminal tail and is currently one of the most promising epigenetic targets for drug discovery against cancer and neurodegenerative diseases. Models of non-covalent binding, such as lock and key, induced-fit, and conformational selection could help explaining the molecular mechanism of LSD1/CoREST-H3-histone association, thus guiding drug discovery and design efforts. Here, we quantify the extent to which LSD1/CoREST substrate binding is consistent with these hypothetical models using LSD1/CoREST conformational ensembles obtained through extensive explicit solvent molecular dynamics (MD) simulations. RESULTS: We find that an induced-fit model is the most representative of LSD1/CoREST-H3-histone non-covalent binding and accounts for the local conformational changes occurring in the H3-histone binding site. We also show that conformational selection - despite in principle not ruled out by this finding - is minimal, and only relevant when global properties are considered, e.g. the nanoscale motion of the LSD1/CoREST clamp. CONCLUSION: The induced-fit mechanism revealed by our MD simulation study will aid the inclusion of protein dynamics for the discovery and design of LSD1 inhibitors targeting the H3-histone binding region. On a general basis, our study indicates the importance of using multiple metrics or selection schemes when testing alternative hypothetical mechanistic models of non-covalent binding.