Marina Godes

A stapled p53 helix overcomes HDMX-mediated suppression of p53.

Cancer cells neutralize p53 by deletion, mutation, proteasomal degradation, or sequestration to achieve a pathologic survival advantage. Targeting the E3 ubiquitin ligase HDM2 can lead to a therapeutic surge in p53 levels. However, the efficacy of HDM2 inhibition can be compromised by overexpression of HDMX, an HDM2 homolog that binds and sequesters p53. Here, we report that a stapled p53 helix preferentially targets HDMX, blocks the formation of inhibitory p53-HDMX complexes, induces p53-dependent transcriptional upregulation, and thereby overcomes HDMX-mediated cancer resistance in vitro and in vivo. Importantly, our analysis of p53 interaction dynamics provides a blueprint for reactivating the p53 pathway in cancer by matching HDM2, HDMX, or dual inhibitors to the appropriate cellular context.

A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers

Cancer cells subvert the natural balance between cellular life and death, achieving immortality through pathologic enforcement of survival pathways and blockade of cell death mechanisms. Pro-apoptotic BCL-2 family proteins are frequently disarmed in relapsed and refractory cancer through genetic deletion or interaction-based neutralization by overexpressed antiapoptotic proteins, resulting in resistance to chemotherapy and radiation treatments. New pharmacologic strategies are urgently needed to overcome these formidable apoptotic blockades. We harnessed the natural killing activity of BCL-2-interacting mediator of cell death (BIM), which contains one of the most potent BH3 death domains of the BCL-2 protein family, to restore BH3-dependent cell death in resistant hematologic cancers. A hydrocarbon-stapled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity, blocked inhibitory antiapoptotic interactions, directly triggered proapoptotic activity, and induced dose-responsive and BH3 sequence-specific cell death of hematologic cancer cells. The therapeutic potential of stapled BIM BH3 was highlighted by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. Thus, we found that broad and multimodal targeting of the BCL-2 family pathway can overcome pathologic barriers to cell death.

Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices

Significance KRAS is one of the most prevalent and vicious oncogenic proteins, yet no drugs are available to inhibit its pathologic activity in patients. We report that KRAS-targeting stapled peptides, modeled after the native son of sevenless 1 (SOS1) helical domain, engage wild-type and clinically relevant KRAS mutant proteins with nanomolar affinity. To our knowledge, these compounds represent the highest affinity and broadest spectrum binders of KRAS mutants reported to date. The stapled peptides disrupt the SOS1/KRAS protein interaction and directly inhibit nucleotide association to wild-type and mutant KRAS proteins. We correlate functional binding activity with SAH-SOS1 cytotoxicity across a 13-member panel of KRAS-driven cancer cells and demonstrate sequence- and dose-dependent inhibition of the ERK-MAP kinase phosphosignaling cascade downstream of KRAS in vitro and in vivo. Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) underlie the pathogenesis and chemoresistance of ∼30% of all human tumors, yet the development of high-affinity inhibitors that target the broad range of KRAS mutants remains a formidable challenge. Here, we report the development and validation of stabilized alpha helices of son of sevenless 1 (SAH-SOS1) as prototype therapeutics that directly inhibit wild-type and mutant forms of KRAS. SAH-SOS1 peptides bound in a sequence-specific manner to KRAS and its mutants, and dose-responsively blocked nucleotide association. Importantly, this functional binding activity correlated with SAH-SOS1 cytotoxicity in cancer cells expressing wild-type or mutant forms of KRAS. The mechanism of action of SAH-SOS1 peptides was demonstrated by sequence-specific down-regulation of the ERK-MAP kinase phosphosignaling cascade in KRAS-driven cancer cells and in a Drosophila melanogaster model of Ras85DV12 activation. These studies provide evidence for the potential utility of SAH-SOS1 peptides in neutralizing oncogenic KRAS in human cancer.

Biophysical determinants for cellular uptake of hydrocarbon-stapled peptide helices

Hydrocarbon-stapled peptides are a class of bioactive alpha-helical ligands developed to dissect and target protein interactions. While there is consensus that stapled peptides can be effective chemical tools for investigating protein regulation, their broader utility for therapeutic modulation of intracellular interactions remains an active area of study. In particular, the design principles for generating cell-permeable stapled peptides are empiric, yet consistent intracellular access is essential to in vivo application. Here, we used an unbiased statistical approach to determine which biophysical parameters dictate the uptake of stapled peptide libraries. We found that staple placement at the amphipathic boundary combined with optimal hydrophobic and helical content are the key drivers of cellular uptake, whereas excess hydrophobicity and positive charge at isolated amino acid positions can trigger membrane lysis at elevated peptide dosing. Our results provide a design roadmap for maximizing the potential to generate cell-permeable stapled peptides with on-mechanism cellular activity.

Mechanistic validation of a clinical lead stapled peptide that reactivates p53 by dual HDM2 and HDMX targeting

Hydrocarbon-stapled peptides that display key residues of the p53 transactivation domain have emerged as bona fide clinical candidates for reactivating the tumor suppression function of p53 in cancer by dual targeting of the negative regulators HDM2 and HDMX. A recent study questioned the mechanistic specificity of such stapled peptides based on interrogating their capacity to disrupt p53/HDM2 and p53/HDMX complexes in living cells using a new recombinase enhanced bimolecular luciferase complementation platform (ReBiL). Here, we directly evaluate the cellular uptake, intracellular targeting selectivity and p53-dependent cytotoxicity of the clinical prototype ATSP-7041. We find that under standard serum-containing tissue culture conditions, ATSP-7041 achieves intracellular access without membrane disruption, dose-dependently dissociates both p53/HDM2 and p53/HDMX complexes but not an unrelated protein complex in long-term ReBiL experiments, and is selectively cytotoxic to cancer cells bearing wild-type p53 by inducing a surge in p53 protein level. These studies underscore the importance of a thorough stepwise approach, including consideration of the time-dependence of cellular uptake and intracellular distribution, in evaluating and advancing stapled peptides for clinical translation.

BAD Modulates Counterregulatory Responses to Hypoglycemia and Protective Glucoprivic Feeding

Hypoglycemia or glucoprivation triggers protective hormonal counterregulatory and feeding responses to aid the restoration of normoglycemia. Increasing evidence suggests pertinent roles for the brain in sensing glucoprivation and mediating counterregulation, however, the precise nature of the metabolic signals and molecular mediators linking central glucose sensing to effector functions are not fully understood. Here, we demonstrate that protective hormonal and feeding responses to hypoglycemia are regulated by BAD, a BCL-2 family protein with dual functions in apoptosis and metabolism. BAD-deficient mice display impaired glycemic and hormonal counterregulatory responses to systemic glucoprivation induced by 2-deoxy-D-glucose. BAD is also required for proper counterregulatory responses to insulin-induced hypoglycemia as evident from significantly higher glucose infusion rates and lower plasma epinephrine levels during hyperinsulinemic hypoglycemic clamps. Importantly, RNA interference-mediated acute knockdown of Bad in the brain provided independent genetic evidence for its relevance in central glucose sensing and proper neurohumoral responses to glucoprivation. Moreover, BAD deficiency is associated with impaired glucoprivic feeding, suggesting that its role in adaptive responses to hypoglycemia extends beyond hormonal responses to regulation of feeding behavior. Together, these data indicate a previously unappreciated role for BAD in the control of central glucose sensing.

Iterative optimization yields Mcl-1-targeting stapled peptides with selective cytotoxicity to Mcl-1-dependent cancer cells

Significance Myeloid cell leukemia 1 (Mcl-1) is a key cancer survival protein that functions by binding to and blocking the activity of prodeath members of the Bcl-2 family. The prosurvival functionality of Mcl-1 can be inhibited by peptides that compete with the native prodeath factors for interaction with Mcl-1. However, unmodified peptide inhibitors of Mcl-1 are ineffective in cellular assays because they cannot access the cytoplasm. In this work, chemical modification and sequence optimization of Mcl-1 binding peptides generated compounds that have favorable biophysical properties, engage Mcl-1 in a distinctive binding mode, and can enter and selectively kill cancer cells dependent on Mcl-1 for survival. This detailed proof-of-principle study demonstrates how systematic optimization can transform a lead peptide into a drug prototype suitable for diagnostic and therapeutic development. Bcl-2 family proteins regulate apoptosis, and aberrant interactions of overexpressed antiapoptotic family members such as Mcl-1 promote cell transformation, cancer survival, and resistance to chemotherapy. Discovering potent and selective Mcl-1 inhibitors that can relieve apoptotic blockades is thus a high priority for cancer research. An attractive strategy for disabling Mcl-1 involves using designer peptides to competitively engage its binding groove, mimicking the structural mechanism of action of native sensitizer BH3-only proteins. We transformed Mcl-1–binding peptides into α-helical, cell-penetrating constructs that are selectively cytotoxic to Mcl-1–dependent cancer cells. Critical to the design of effective inhibitors was our introduction of an all-hydrocarbon cross-link or “staple” that stabilizes α-helical structure, increases target binding affinity, and independently confers binding specificity for Mcl-1 over related Bcl-2 family paralogs. Two crystal structures of complexes at 1.4 Å and 1.9 Å resolution demonstrate how the hydrophobic staple induces an unanticipated structural rearrangement in Mcl-1 upon binding. Systematic sampling of staple location and iterative optimization of peptide sequence in accordance with established design principles provided peptides that target intracellular Mcl-1. This work provides proof of concept for the development of potent, selective, and cell-permeable stapled peptides for therapeutic targeting of Mcl-1 in cancer, applying a design and validation workflow applicable to a host of challenging biomedical targets.

Abstract B41: Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices

Activating mutations in KRAS represent the most frequent oncogenic driving force among the RAS homologs K-, N- and H-RAS, and are associated with poor prognosis and chemoresistance. KRAS mutations are present in approximately 30% of tumors, and at even higher frequencies in cancers of the pancreas, lung, thyroid gland, colon, and liver. In pancreatic ductal adenocarcinomas (PDAC), which carries a 5-year survival rate of less than 5%, activating KRAS mutations are present in over 90% of tumors. Moreover, these mutations have been causally linked to the initiation and progression of PDAC. Despite the mechanistic insights into KRAS-mediated oncogenesis, development of high affinity targeted inhibitors remains a formidable challenge. Here, we report the synthesis and application of hydrocarbon-stapled peptides as prototype therapeutics for blocking wild type and mutant KRAS in vitro and in cancer cells. Stabilized Alpha-Helices of SOS1 (SAH-SOS1) were generated by inserting hydrocarbon staples into SOS1 peptide sequence to recapitulate the alpha-helical structure of the native KRAS-interaction domain. SAH-SOS1 peptides, but not negative control analogs, bound to a variety of KRAS constructs with nanomolar affinity, inhibiting nucleotide exchange. This sequence-specific biochemical activity correlated with impairment of KRAS-driven cancer cell viability and signal transduction. These studies provide proof-of-concept for the utility of SAH-SOS1 peptides in dissecting and targeting the oncogenic KRAS pathway in human cancer. Citation Format: Elizaveta S. Leshchiner, Joseph Bellairs, Gregory H. Bird, Kwadwo Opoku-Nsiah, Marina Godes, Loren D. Walensky. Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B41. doi: 10.1158/1557-3125.RASONC14-B41

Abstract 2597: Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Activating mutations in KRAS represent the most frequent oncogenic driving force among the RAS homologs K-, N- and H-RAS, and are associated with poor prognosis and chemoresistance. KRAS mutations are present in approximately 30% of tumors, and at even higher frequencies in cancers of the pancreas, lung, thyroid gland, colon, and liver. In pancreatic ductal adenocarcinomas (PDAC), which carries a 5-year survival rate of less than 5%, activating KRAS mutations are present in over 90% of tumors. Moreover, these mutations have been causally linked to the initiation and progression of PDAC. Despite the mechanistic insights into KRAS-mediated oncogenesis, development of high affinity targeted inhibitors remains a formidable challenge. Here, we report the synthesis and application of hydrocarbon-stapled peptides as prototype therapeutics for blocking wild type and mutant KRAS in vitro and in cancer cells. Stabilized Alpha-Helices of SOS1 (SAH-SOS1) were generated by inserting hydrocarbon staples into SOS1 peptide sequence to recapitulate the alpha-helical structure of the native KRAS-interaction domain. SAH-SOS1 peptides, but not negative control analogs, bound to a variety of KRAS constructs with nanomolar affinity, inhibiting nucleotide exchange. This sequence-specific biochemical activity correlated with impairment of KRAS-driven cancer cell viability and signal transduction. These studies provide proof-of-concept for the utility of SAH-SOS1 peptides in dissecting and targeting the oncogenic KRAS pathway in human cancer. Citation Format: Elizaveta Leshchiner, Joseph Bellairs, Gregory H. Bird, Kwadwo Opoku-Nsiah, Marina Godes, Lored D. Walensky. Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2597. doi:10.1158/1538-7445.AM2014-2597

Abstract 1775: Therapeutic reactivation of cell death in refractory hematologic cancers using a broad spectrum BIM BH3 death helix

BCL-2 family proteins are essential regulators of cellular life and death and, when deregulated, contribute to the development, maintenance, and chemoresistance of human cancer. Whereas multidomain anti-apoptotic proteins such as BCL-2 guard against apoptosis, the multidomain pro-apoptotic proteins BAX and BAK induce cell death through mitochondrial damage. The BH3-only members act as cellular antennae, poised to transmit signals of cellular injury to their multidomain counterparts, and deliver the death message via conserved alpha-helical BH3 domains. Interaction-based neutralization or genetic deletion of BCL-2 family death proteins is a common mechanism employed by cancer cells to mount an apoptotic blockade against chemotherapy and radiation treatments. The design of next-generation therapeutics based on the molecular architecture of the BIM BH3 helix offers the unique advantage of recapitulating BIM9s natural capacity to directly target the full complement of anti- and pro-apoptotic BCL-2 proteins. By inserting a hydrocarbon staple into BIM BH3, we developed a Stabilized Alpha-Helix of BCL-2 domain (SAHB) peptide capable of targeting the three modes of BCL-2 family-mediated apoptotic blockade, achieving (1) anti-apoptotic inhibition, (2) pro-apoptotic direct activation, and (3) BIM BH3 replacement. We demonstrate that BIM SAHB is helical and targets BCL-2 family proteins and their complexes with high affinity in vitro and in cells. BIM SAHB, but not a binding interface mutant, induces dose-responsive apoptosis of resistant hematologic cancer cells as measured by viability, annexin V binding, and caspase 3/7 activation assays. Importantly, we demonstrate by immunoprecipitation that reactivation of the death program correlates with MCL-1/BAK dissociation and BAX activation. Non-malignant mouse embryonic and human fibroblasts demonstrate relative resistance to BIM SAHB, indicative of a therapeutic window for treatment. To evaluate the therapeutic potential of BIM SAHB in vivo, we examined the effect of pharmacologic BIM BH3 replacement in the B-cell lymphoproliferative disease of Bim −/− bone marrow-reconstituted mice. Strikingly, animals treated with BIM SAHB displayed significant TUNEL positivity within the aberrant lymphoid infiltrates, whereas no effect was observed in mice treated with vehicle or the mutant control SAHB. In addition, the surrounding parenchymal tissue was unaffected by BIM SAHB treatment, highlighting the selectivity of action and therapeutic window in vivo. Thus, we find that broad and multimodal targeting of the BCL-2 family pathway by pharmacologic replacement of the BIM BH3 helix can overcome formidable pathologic barriers to cell death in vitro and in vivo, and represents a promising strategy for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1775. doi:10.1158/1538-7445.AM2011-1775