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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Protein recognition by short peptide reversible inhibitors of the chromatin-modifying LSD1/CoREST lysine demethylase.

The combinatorial assembly of protein complexes is at the heart of chromatin biology. Lysine demethylase LSD1(KDM1A)/CoREST beautifully exemplifies this concept. The active site of the enzyme tightly associates to the N-terminal domain of transcription factors of the SNAIL1 family, which therefore can competitively inhibit the binding of the N-terminal tail of the histone substrate. Our enzymatic, crystallographic, spectroscopic, and computational studies reveal that LSD1/CoREST can bind to a hexapeptide derived from the SNAIL sequence through recognition of a positively charged α-helical turn that forms upon binding to the enzyme. Variations in sequence and length of this six amino acid ligand modulate affinities enabling the same binding site to differentially interact with proteins that exert distinct biological functions. The discovered short peptide inhibitors exhibit antiproliferative activities and lay the foundation for the development of peptidomimetic small molecule inhibitors of LSD1.

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.

An improved replica-exchange sampling method: temperature intervals with global energy reassignment.

In a molecular dynamics (MD) simulation, representative sampling over the entire phase space is desired to obtain an accurate canonical distribution at a given temperature. For large molecules, such as proteins, this is problematic because systems tend to become trapped in local energy minima. The extensively used replica-exchange molecular dynamics (REMD) simulation technique overcomes this kinetic-trapping problem by allowing Boltzmann-weighted configuration exchange processes to occur between numerous thermally adjacent and compositionally identical simulations that are thermostated at sequentially higher temperatures. While the REMD method provides much better sampling than conventional MD, there are two substantial difficulties that are inherent in its application: (1) the large number of replicas that must be used to span a designated temperature range and (2) the subsequent long time required for configurations sampled at high temperatures to exchange down for potential inclusion within the low-temperature ensemble of interest. In this work, a new method based on temperature intervals with global energy reassignment (TIGER) is presented that overcomes both of these problems. A TIGER simulation is conducted as a series of short heating-sampling-quenching cycles. At the end of each cycle, the potential energies of all replicas are simultaneously compared at the same temperature using a Metropolis sampling method and then globally reassigned to the designated temperature levels. TIGER is compared with regular MD and REMD methods for the alanine dipeptide in water. The results indicate that TIGER increases sampling efficiency while substantially reducing the number of central processing units required for a comparable conventional REMD simulation.