Nadeem A. Vellore

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.

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 ROS1D2033N mutation in a patient with CD74-ROS1–rearranged lung adenocarcinoma and acquired resistance to crizotinib. Crizotinib resistance of CD74-ROS1D2033N was functionally evaluated using cell-based assays and structural modeling. Results: In biochemical and cell-based assays, the CD74-ROS1D2033N 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.

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.

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.

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

Significance Targeting oncogenic ROS1 fusion proteins with crizotinib has shown promising clinical outcomes in non-small cell lung cancer (NSCLC) patients, but emergence of resistance to therapy has been reported. By profiling the activity of clinically viable ROS1/anaplastic lymphoma kinase (ALK) inhibitors, we discovered that the Food and Drug Administration (FDA)-approved inhibitor cabozantinib potently inhibits native ROS1 and the crizotinib-resistant ROS1G2032R mutant, suggesting potential utility for treatment of ROS1-rearranged lung cancer. Notably, cabozantinib is ineffective against the closely related ALK kinase. Molecular modeling shows specific structural differences between the kinase domains of ROS1 and ALK that explain selective binding of cabozantinib to ROS1. These findings reveal limitations pertaining to the widely presumed inhibitory reciprocity of ROS1 and ALK inhibitors and may facilitate rational design of new ROS1-selective 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-ROS1G2032R 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.

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.

Radotinib is an effective inhibitor of native and kinase domain-mutant BCR-ABL1

Philadelphia chromosome positive (Ph+) leukemia is driven by the constitutive enzymatic activity of the BCR-ABL1 fusion kinase.1 Tyrosine kinase inhibitors (TKIs) that block the activity of BCR-ABL1 are successfully used clinically to treat chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). The Food and Drug Administration (FDA) granted regulatory approval for the first of these, imatinib, in 2001. Emergence of clinical imatinib resistance, chiefly due to BCR-ABL1 kinase domain mutations, motivated the development and regulatory approval of new TKIs, including nilotinib, dasatinib, bosutinib and ponatinib.1–3 One strategy, as exemplified by nilotinib (Fig. 1A), is rational design of imatinib derivatives with substantially higher binding affinity. Nilotinib is approved for first-line use in the U.S. and is an order of magnitude more potent than imatinib, which translates into improved inhibitory activity against many of the common BCR-ABL1 mutants.4 The most important mutational liability is BCR-ABL1T315I, which is completely insensitive to all approved TKIs except ponatinib.1, 5

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

BackgroundLysine 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.ResultsWe 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.ConclusionThe 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.

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.

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.