Magali Mathieu

A highly potent and selective Vps34 inhibitor alters vesicle trafficking and autophagy

Vps34 is a phosphoinositide 3-kinase (PI3K) class III isoform that has attracted major attention over the recent years because of its role in autophagy. Herein we describe the biological characterization of SAR405, which is a low-molecular-mass kinase inhibitor of Vps34 (KD 1.5 nM). This compound has an exquisite protein and lipid kinase selectivity profile that is explained by its unique binding mode and molecular interactions within the ATP binding cleft of human Vps34. To the best of our knowledge, this is the first potent and specific Vps34 inhibitor described so far. Our results demonstrate that inhibition of Vps34 kinase activity by SAR405 affects both late endosome-lysosome compartments and prevents autophagy. Moreover, we show that the concomitant inhibition of Vps34 and mTOR, with SAR405 and the US Food and Drug Administration-approved mTOR inhibitor everolimus, results in synergistic antiproliferative activity in renal tumor cell lines, indicating a potential clinical application in cancer.

Discovery and optimization of pyrimidone indoline amide PI3Kβ inhibitors for the treatment of phosphatase and tensin homologue (PTEN)-deficient cancers.

Compelling molecular biology publications have reported the implication of phosphoinositide kinase PI3Kβ in PTEN-deficient cell line growth and proliferation. These findings supported a scientific rationale for the development of PI3Kβ-specific inhibitors for the treatment of PTEN-deficient cancers. This paper describes the discovery of 2-[2-(2,3-dihydro-indol-1-yl)-2-oxo-ethyl]-6-morpholin-4-yl-3H-pyrimidin-4-one (7) and the optimization of this new series of active and selective pyrimidone indoline amide PI3Kβ inhibitors. 2-[2-(2-Methyl-2,3-dihydro-indol-1-yl)-2-oxo-ethyl]-6-morpholin-4-yl-3H-pyrimidin-4-one (28), identified following a carefully designed methyl scan, displayed improved physicochemical and in vitro pharmacokinetic properties. Structural biology efforts enabled the acquisition of the first X-ray cocrystal structure of p110β with the selective inhibitor compound 28 bound to the ATP site. The nonplanar binding mode described herein is consistent with observed structure-activity relationship for the series. Compound 28 demonstrated significant in vivo activity in a UACC-62 xenograft model in mice, warranting further preclinical investigation. Following successful development, compound 28 entered phase I/Ib clinical trial in patients with advanced cancer.

Differential Water Thermodynamics Determine PI3K-Beta/Delta Selectivity for Solvent-Exposed Ligand Modifications

Phosphoinositide 3-kinases (PI3Ks) are involved in important cellular functions and represent desirable targets for drug discovery efforts, especially related to oncology; however, the four PI3K subtypes (α, β, γ, and δ) have highly similar binding sites, making the design of selective inhibitors challenging. A series of inhibitors with selectivity toward the β subtype over δ resulted in compound 3(S), which has entered a phase I/Ib clinical trial for patients with advanced PTEN-deficient cancer. Interestingly, X-ray crystallography revealed that the modifications making inhibitor 3(S) and related compounds selective toward the β-isoform do not interact directly with either PI3Kβ or PI3Kδ, thereby confounding rationalization of the SAR. Here, we apply explicit solvent molecular dynamics and solvent thermodynamic analysis using WaterMap in an effort to understand the unusual affinity and selectivity trends. We find that differences in solvent energetics and water networks, which are modulated upon binding of different ligands, explain the experimental affinity and selectivity trends. This study highlights the critical role of water molecules in molecular recognition and the importance of considering water networks in drug discovery efforts to rationalize and improve selectivity.

Structures of a pan‐specific antagonist antibody complexed to different isoforms of TGFβ reveal structural plasticity of antibody–antigen interactions

Various important biological pathways are modulated by TGFβ isoforms; as such they are potential targets for therapeutic intervention. Fresolimumab, also known as GC1008, is a pan‐TGFβ neutralizing antibody that has been tested clinically for several indications including an ongoing trial for focal segmental glomerulosclerosis. The structure of the antigen‐binding fragment of fresolimumab (GC1008 Fab) in complex with TGFβ3 has been reported previously, but the structural capacity of fresolimumab to accommodate tight interactions with TGFβ1 and TGFβ2 was insufficiently understood. We report the crystal structure of the single‐chain variable fragment of fresolimumab (GC1008 scFv) in complex with target TGFβ1 to a resolution of 3.00 Å and the crystal structure of GC1008 Fab in complex with TGFβ2 to 2.83 Å. The structures provide further insight into the details of TGFβ recognition by fresolimumab, give a clear indication of the determinants of fresolimumab pan‐specificity and provide potential starting points for the development of isoform‐specific antibodies using a fresolimumab scaffold.

Abstract 4746: Selective inhibition of mutant IDH1 via small molecule binding to the dimer interface

Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitric acid to α-ketoglutaric acid (αKG). Point mutations in IDH1 and IDH2 confer a neomorphic enzymatic activity: the reduction of αKG to D-2-hydroxyglutaric acid (2HG), which acts as an oncometabolite by inducing hypermethylation of histones and DNA. IDH1 mutations (predominantly R132H) have been found in 50-80% of grade II gliomas. To find potential IDH1-targeted therapeutics, we conducted a full small-molecule HTS and hit validation campaign using recombinant R132H IDH1. This campaign identified Compound 1, which belongs to a phenol-azole series, as a potent and selective inhibitor of mutant IDH1. We investigated the mode of inhibition of Compound 1 and a published IDH1 mutant inhibitor with a different chemical scaffold (Compound 2). Steady-state kinetics and biophysical studies showed that both inhibitors reversibly interact with both free and substrate-bound enzyme at an allosteric site. A crystal structure of Compound 1 complexed with R132H demonstrates binding of the inhibitor at the dimer interface. A competitive binding study showed that Compound 2 competes with Compound 1, suggesting that Compound 2 also binds at the dimer interface. This is contrary to a previously published statement that Compound 2 is a competitive inhibitor with respect to αKG. In cancer cell lines engineered to produce high levels of 2HG, both inhibitors penetrate cells efficiently and inhibit 2HG production with minimal cell toxicity. This study indicates that the dimer interface pocket is a druggable binding site for IDH1 inhibitors, and suggests that Compound 1 is a promising new starting point for future structure-based drug discovery efforts. Citation Format: Gejing Deng, Stuart Licht, Junqing Shen, Ming Yin, Jessica McManus, Patricia Gee, Tim He, Giang Gao, Bailin Zhang, Magali Mathieu, Alexey Rak, Olivier Bedel, Chaomei Shi, Stefan Gross, Dietmar Hoffmann, Eamonn Rooney, Aurelie Vassort, Walter Englaro, Yi Li, Dmitri Wiederschain, Vinod Patel, Francisco Adrian, Hong Cheng. Selective inhibition of mutant IDH1 via small molecule binding to the dimer interface. [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 4746. doi:10.1158/1538-7445.AM2014-4746