Ulf Peters

K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions.

Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner.

Chemical genetic strategy for targeting protein kinases based on covalent complementarity

The conserved nature of the ATP-binding site of the > 500 human kinases renders the development of specific inhibitors a challenging task. A widely used chemical genetic strategy to overcome the specificity challenge exploits a large-to-small mutation of the gatekeeper residue (a conserved hydrophobic amino acid) and the use of a bulky inhibitor to achieve specificity via shape complementarity. However, in a number of cases, introduction of a glycine or alanine gatekeeper results in diminished kinase activity and ATP affinity. A new chemical genetic approach based on covalent complementarity between an engineered gatekeeper cysteine and an electrophilic inhibitor was developed to address these challenges. This strategy was evaluated with Src, a proto-oncogenic tyrosine kinase known to lose some enzymatic activity using the shape complementarity chemical genetic strategy. We found that Src with a cysteine gatekeeper recapitulates wild type activity and can be irreversibly inhibited both in vitro and in cells. A cocrystal structure of T338C c-Src with a vinylsulfonamide-derivatized pyrazolopyrimidine inhibitor was solved to elucidate the inhibitor binding mode. A panel of electrophilic inhibitors was analyzed against 307 kinases and MOK (MAPK/MAK/MRK overlapping kinase), one of only two human kinases known to have an endogenous cysteine gatekeeper. This analysis revealed remarkably few off-targets, making these compounds the most selective chemical genetic inhibitors reported to date. Protein engineering studies demonstrated that it is possible to increase inhibitor potency through secondary-site mutations. These results suggest that chemical genetic strategies based on covalent complementarity should be widely applicable to the study of protein kinases.

Staurosporine-derived inhibitors broaden the scope of analog-sensitive kinase technology.

Analog-sensitive (AS) kinase technology is a powerful approach for studying phospho-signaling pathways in diverse organisms and physiological processes. The key feature of this technique is that a kinase-of-interest can be mutated to sensitize it to inhibitor analogs that do not target wild-type (WT) kinases. In theory, this enables specific inhibition of any kinase in cells and in mouse models of human disease. Typically, these inhibitors are identified from a small library of molecules based on the pyrazolopyrimidine (PP) scaffold. However, we recently identified a subset of native human kinases, including the Ephrin A kinase family, that are sensitive to commonly used PP inhibitors. In an effort to develop a bioorthogonal AS-kinase inhibitor and to extend this technique to PP-sensitive kinases, we sought an alternative inhibitor scaffold. Here we report the structure-based design of synthetically tractable, potent, and extremely selective AS-kinase inhibitors based on the natural product staurosporine. We demonstrate that these molecules, termed staralogs, potently target AS kinases in cells, and we employ X-ray crystallography to elucidate their mechanism of efficacy. Finally, we demonstrate that staralogs target AS mutants of PP-sensitive kinases at concentrations where there is little to no inhibition of native human kinases. Thus, staralogs represent a new class of AS-kinase inhibitors and a core component of the chemical genetic tool kit for probing kinase-signaling pathways.

Abstract 5077: A novel small molecule menin-MLL inhibitor for potential treatment of MLL-rearranged leukemias

Patients with MLL-rearranged leukemia typically have a poor prognosis. With chemotherapy and stem cell transplantation as current standard of care, the 5-year survival rate is estimated to be only about 35%. As the leukemogenic activity of MLL fusion proteins has been shown to be dependent on their direct interaction with menin, development of small molecules that block the menin-MLL interaction is a promising therapeutic strategy for the treatment of this disease. Although small molecule menin-MLL inhibitors have been reported, previously published compounds exhibited modest cellular potency and/or poor pharmacokinetic properties. We now describe a novel, potent, and selective small molecule menin-MLL inhibitor that effectively treats MLL leukemias in in vivo models and demonstrate its potential clinical utility. The compound selectively inhibits the growth of a panel of MLL-rearranged cell lines relative to non MLL-rearranged cell lines, displays favorable pharmacokinetic properties, and induces tumor regression in an MV4;11 subcutaneous xenograft mouse model, with durable tumor suppression even after dosing is discontinued. In both the MV4;11 and MOLM13 disseminated leukemia models, the compound confers a prolonged survival benefit compared to an untreated group when dosed orally once daily at doses that are well-tolerated in the animals. We have used this potent and selective menin inhibitor to investigate the functional cellular response of MLL-rearranged cell lines to menin inhibition, assessing cell growth, cell cycle and gene expression responses over time, and compared that response to other epigenetic inhibitors and demonstrate significantly increased apoptosis which correlates with tumor regression measured both in early and long term readouts in xenograft and systemic leukemia models. We describe a menin-MLL inhibitor with optimized drug-like properties that demonstrates potential clinical utility in preclinical models of MLL leukemias. The compound is currently under further preclinical evaluation. Citation Format: Tao Wu, Linda Kessler, Shuangwei Li, Trupta Purohit, Shisheng Li, Hongzhi Miao, Brian Linhares, Rasmus Hansen, Jeff Kucharski, Yi Wang, Ke Yu, Katarzyna Kempinska, Tess Ely, Szymon Klossowski, Ata Zarieh, Ulf Peters, Jun Feng, Yvonne Yao, Yuan Liu, Bo Wen, Francis Burrows, Duxin Sun, Jingchuan Zhang, Levan Darjania, Dana Hu-Lowe, Patrick Zarrinkar, Liansheng Li, Tomasz Cierpicki, Jolanta Grembecka, Pingda Ren, Yi Liu. A novel small molecule menin-MLL inhibitor for potential treatment of MLL-rearranged leukemias [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 5077. doi:10.1158/1538-7445.AM2017-5077

Abstract PL04-03: Selective inhibition of K-Ras G12C through allosteric control of GTP affinity and effector interactions.

Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies1-3. Efforts to directly target this oncogene have faced difficulties due to its picomolar affinity for GTP/GDP4 and the absence of known allosteric regulatory sites.Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis5,6. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent upon relative nucleotide affinity and concentration. This gives GTP an advantage over GDP7 and increases the proportion of active GTP-bound Ras. Here, we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras G12C.These compounds rely on the mutant cysteine for binding and therefore do not affect the wild type protein (WT). Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras G12C disrupts both switch-I and switch-II, subverting the native nucleotide preference to favor GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a novel allosteric regulatory site on Ras that is targetable in a mutant specific manner. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PL04-03. Citation Format: Jonathan M. Ostrem, Ulf Peters, Martin L. Sos, James A. Wells, Kevan M. Shokat. Selective inhibition of K-Ras G12C through allosteric control of GTP affinity and effector interactions. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PL04-03.

Abstract LB-311: Development of mutant-specific small molecule inhibitors of K-Ras.

Activating mutations in K-Ras are among the most common lesions found in human cancer, and such mutations are generally associated with poor prognosis. Despite numerous efforts in academia and industry, small molecule inhibitors that directly target K-Ras remain elusive. Even more highly desired are molecules that selectively target mutant K-Ras while sparing the wild type protein. We have used a fragment-based screen to discover oncogenic mutant-specific inhibitors of K-Ras. Crystallographic studies with multiple inhibitors in complex with K-Ras reveal that the compounds bind in a novel hydrophobic pocket that is not apparent in any published crystal structure of Ras. These inhibitors disrupt the conformations of Switch I and Switch II, domains that are essential for the association and activation of downstream signaling partners. Our medicinal chemistry effort has greatly improved potency, showing that this pocket is particularly amenable to chemical optimization. In vitro biochemical characterization of these inhibitors confirms that they block Ras function. Our discovery of a new druggable pocket in K-Ras, and a set of inhibitors that bind to it in a mutant-specific fashion, provides a promising new avenue for the direct pharmacological inhibition of oncogenic Ras. Citation Format: Jonathan M. Ostrem, Ulf Peters, Pingda Ren, Yi Liu, James A. Wells, Kevan M. Shokat. Development of mutant-specific small molecule inhibitors of K-Ras. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-311. doi:10.1158/1538-7445.AM2013-LB-311