Cristina Lewis

Identification of GNE-477, a potent and efficacious dual PI3K/mTOR inhibitor

Efforts to identify potent small molecule inhibitors of PI3 kinase and mTOR led to the discovery of the exceptionally potent 6-aryl morpholino thienopyrimidine 6. In an effort to reduce the melting point in analogs of 6, the thienopyrimidine was modified by the addition of a methyl group to disrupt planarity. This modification resulted in a general improvement in in vivo clearance. This discovery led to the identification of GNE-477 (8), a potent and efficacious dual PI3K/mTOR inhibitor.

Potent and selective inhibitors of PI3Kδ: obtaining isoform selectivity from the affinity pocket and tryptophan shelf.

A potent inhibitor of PI3Kδ that is ≥ 200 fold selective for the remaining three Class I PI3K isoforms and additional kinases is described. The hypothesis for selectivity is illustrated through structure activity relationships and crystal structures of compounds bound to a K802T mutant of PI3Kγ. Pharmacokinetic data in rats and mice support the use of 3 as a useful tool compound to use for in vivo studies.

Structure-based optimization of pyrazolo-pyrimidine and -pyridine inhibitors of PI3-kinase.

Starting from HTS hit 1a, X-ray co-crystallization and molecular modeling were used to design potent and selective inhibitors of PI3-kinase. Bioavailablity in this series was improved through careful modulation of physicochemical properties. Compound 12 displayed in vivo knockdown of PI3K pharmacodynamic markers such as pAKT, pPRAS40, and pS6RP in a PC3 prostate cancer xenograft model.

Structure-based design of thienobenzoxepin inhibitors of PI3-kinase

Starting from thienobenzopyran HTS hit 1, co-crystallization, molecular modeling and metabolic analysis were used to design potent and metabolically stable inhibitors of PI3-kinase. Compound 15 demonstrated PI3K pathway suppression in a mouse MCF7 xenograft model.

Identification of GNE-293, a potent and selective PI3Kδ inhibitor: navigating in vitro genotoxicity while improving potency and selectivity.

In an effort to identify potent and isoform selective inhibitors of PI3Kδ, GNE-293 (34) was identified. Inhibitor 2 was found to induce micronuclei formation in both the MNT and HCA in vitro assays. Compounds testing negative for genotoxicity were successfully identified through modifications of the 2-benzimidazole substituent and the methylene moiety to disrupt planarity. A variety of heteroatom linkers were explored to examine their effect on potency and isoform selectivity by restricting torsional angles to favor ligand interactions with PI3Kδs Trp760. These modifications also resulted in an improved in vivo pharmacokinetic profile.

Discovery of thiazolobenzoxepin PI3-kinase inhibitors that spare the PI3-kinase β isoform.

A series of suitable five-membered heterocyclic alternatives to thiophenes within a thienobenzoxepin class of PI3-kinase (PI3K) inhibitors was discovered. Specific thiazolobenzoxepin 8-substitution was identified that increased selectivity over PI3Kβ. PI3Kβ-sparing compound 27 (PI3Kβ Ki,app/PI3Kα Ki,app=57) demonstrated dose-dependent knockdown of pAKT, pPRAS40 and pS6RP in vivo as well as differential effects in an in vitro proliferation cell line screen compared to pan PI3K inhibitor GDC-0941. A new structure-based hypothesis for reducing inhibition of the PI3K β isoform while maintaining activity against α, δ and γ isoforms is presented.

Case Studies of Minimizing Nonspecific Inhibitors in HTS Campaigns That Use Assay-Ready Plates

Identifying chemical lead matter by high-throughput screening (HTS) has been a common practice in early stage drug discovery. Evolution of small-molecule library composition to include more drug-like molecules with desirable physical chemical properties combined with improving assay technologies has vastly enhanced the capability of HTS. However, HTS campaigns can still be plagued by false positives arising from nonspecific inhibitors. The generation of assay-ready plates has permitted an incremental advancement to the speed and efficiency of HTS but has the potential to enhance the occurrence of nonspecific inhibitors. A subtle change in the order of reagent addition to the assay-ready plates can greatly alleviate false-positive inhibition. Our case studies with six different kinase and protease targets reveal that this type of inhibition affects targets regardless of enzyme class and is unpredictable based on protein construct or inhibitor chemical scaffold. These case studies support a model where a diversity set of compounds should be tested first for hit rates as a function of order of addition, carrier protein, and relevant mechanistic studies prior to launch of the HTS campaign.

Abstract DDT02-01: Discovery of GDC-0032: A beta-sparing PI3K inhibitor active against PIK3CA mutant tumors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Modifications of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway are frequent in cancer due to multiple mechanisms, including activating mutations of the alpha isoform of PI3K. The dysregulation of this pathway has been implicated in many processes involved in oncogenesis. Thus, PI3K is a promising therapeutic target for cancer. Previously we have disclosed GDC-0941, a class 1 selective PI3K inhibitor and our class 1 PI3K/mTOR kinase inhibitor, GDC-0980. In this presentation we describe the design and discovery of a new class of PI3K inhibitors, which selectively inhibit the activated PI3Kα isoform relative to the PI3Kβ isoform. A lead was identified from a high throughput screen (HTS) that resulted in a novel chemical series of kinase inhibitors. Through a structure-based approach, this lead was optimized to provide very potent inhibitors of PI3K. In addition, this chemical series allowed for designing molecules that have different selectivity patterns with respect to the class 1 PI3K isoforms. In particular, a series of inhibitors were designed that could preferentially inhibit PI3Kα relative to PI3Kβ (“beta-sparing”). Further modification of the physicochemical properties led to the discovery of GDC-0032. GDC-0032 is a potent inhibitor of PI3Kα (PIK3CA) isoform with a Ki =0.2 nM, and with reduced inhibitory activity against PI3Kβ. This selectivity profile allowed for greater efficacy in vivo at the maximum tolerated dose relative to a pan inhibitor in representative PI3Kα (PIK3CA) mutant xenografts. It is notable that GDC-0032 preferentially inhibited PI3Kα (PIK3CA) mutant cells relative to cells with wild-type PI3K. Taken together, GDC-0032 is a potent and effective beta-sparing PI3K inhibitor, which currently is in clinical trials. Citation Format: Alan G. Olivero, Timothy P. Heffron, Matthew Baumgardner, Marcia Belvin, Leanne Berry Ross, Nicole Blaquiere, Erin Bradley, Georgette Castanedo, Mika Derynck, Steven Do, Jennafer Dotson, Danette Dudley, Kyle Edgar, Adrian Folkes, Ross Francis, Tony Gianetti, Richard Goldsmith, Paul Goldsmith, Jane Guan, Trevor Harrison, Robert Heald, Jerry Hsu, Phillip Jackson, Graham Jones, Amy Kim, Aleks Kolesnikov, Mark Lackner, Leslie Lee, John Lesnick, Cristina Lewis, Michael Mamounas, Neville McLean, Jeremy Murray, Chudi Ndubaku, Jim Nonomiya, Jodie Pang, Neil Pegg, Wei Wei Prior, Laurent Salphati, Deepack Sampath, Stephen Sideris, Michael Siu, Steven Staben, Daniel Sutherlin, Mark Ultsch, Jeff Wallin, Lan Wang, Christian Wiesmann, Xiaolin Zhang, Lori S. Friedman. Discovery of GDC-0032: A beta-sparing PI3K inhibitor active against PIK3CA mutant tumors. [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 DDT02-01. doi:10.1158/1538-7445.AM2013-DDT02-01

Abstract 2787: Discovery of GDC-0980, a selective PI3K/mTOR inhibitor in clinical trials

PI3 Kinase and mTOR have been identified as promising targets for the treatment of cancer. These enzymes participate in related, but not redundant, signaling networks to transmit cellular growth and survival signals, which are hallmarks of tumor growth. An interest in targeting both of these two important points along this critical signaling pathway, and the ability to leverage the high degree of structural similarity in the active sites of PI3K and mTOR kinase, has resulted in the discovery of GDC-0980 as a Class I PI3K and mTOR kinase inhibitor for oncology indications. The structure, efficacy, and medicinal chemistry behind the discovery of this compound is described. Beginning with the morpholin-4-yl-thieno[3,2-d]pyrimidine core of the Class I PI3K inhibitor GDC-0941, structural substitutions were made external to the core that added mTOR potency, improved the metabolic stability in vitro and in vivo, and lowered the plasma protein binding of the scaffold. Homology models of mTOR using PI3Kγ structures with bound inhibitors provided hypotheses for increasing mTOR potency relative to previous compounds. The solubility of the modified compounds was improved through the addition of polar functionality in the solvent exposed region of the scaffold, resulting in GDC-0980. GDC-0980 is potent across Class I isoforms with IC509s of 5, 27, 7, and 14 nM for PI3Kα, β, Δ, and γ, and inhibits mTOR with a Ki of 17 nM. The compound is highly selective versus a large panel of kinases including others in the PIK family. Based on the excellent PK profile, linear increase in exposure, strong potency in a broad range of cancer cells, and high free fraction, GDC-0980 is efficacious in animal models of cancer when dosed orally at low doses. Furthermore, this compound is efficacious when dosed intermittently as well as on a daily schedule. These preclinical data provide compelling support for GDC-0980 as a clinical candidate, and early stage clinical trials are underway. 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 2787. doi:10.1158/1538-7445.AM2011-2787