KRAS G12C fragment screening renders new binding pockets.

KRAS genes belong to the most frequently mutated family of oncogenes in cancer. The G12C mutation, found in a third of lung, half of colorectal and pancreatic cancer cases, is believed to be responsible for a substantial number of cancer deaths. For 30 years, KRAS has been the subject of extensive drug-targeting efforts aimed at targeting KRAS protein itself, but also its post-translational modifications, membrane localization, protein-protein interactions and downstream signalling pathways. So far, most KRAS targeting strategies have failed, and there are no KRAS-specific drugs available. However, clinical candidates targeting the KRAS G12C protein have recently been developed. MRTX849 and recently approved Sotorasib are covalent binders targeting the mutated cysteine 12, occupying Switch II pocket.Herein, we describe two fragment screening drug discovery campaigns that led to the identification of binding pockets on the KRAS G12C surface that have not previously been described. One screen focused on non-covalent binders to KRAS G12C, the other on covalent binders.

Automated harvesting and processing of protein crystals through laser photoablation.

Currently, macromolecular crystallography projects often require the use of highly automated facilities for crystallization and X-ray data collection. However, crystal harvesting and processing largely depend on manual operations. Here, a series of new methods are presented based on the use of a low X-ray-background film as a crystallization support and a photoablation laser that enable the automation of major operations required for the preparation of crystals for X-ray diffraction experiments. In this approach, the controlled removal of the mother liquor before crystal mounting simplifies the cryocooling process, in many cases eliminating the use of cryoprotectant agents, while crystal-soaking experiments are performed through diffusion, precluding the need for repeated sample-recovery and transfer operations. Moreover, the high-precision laser enables new mounting strategies that are not accessible through other methods. This approach bridges an important gap in automation and can contribute to expanding the capabilities of modern macromolecular crystallography facilities.

Discovery of (2S)-8-[(3R)-3-methylmorpholin-4-yl]-1-(3-methyl-2-oxobutyl)-2-(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one: a novel potent and selective inhibitor of Vps34 for the treatment of solid tumors.

Vps34 (the human class III phosphoinositide 3-kinase) is a lipid kinase involved in vesicle trafficking and autophagy and therefore constitutes an interesting target for cancer treatment. Because of the lack of specific Vps34 kinase inhibitors, we aimed to identify such compounds to further validate the role of this lipid kinase in cancer maintenance and progression. Herein, we report the discovery of a series of tetrahydropyrimidopyrimidinone derivatives. Starting with hit compound 1a, medicinal chemistry optimization led to compound 31. This molecule displays potent activity, an exquisite selectivity for Vps34 with excellent properties. The X-ray crystal structure of compound 31 in human Vps34 illustrates how the unique molecular features of the morpholine synthon bestows selectivity against class I PI3Ks. This molecule exhibits suitable in vivo mouse PK parameters and induces a sustained inhibition of Vps34 upon acute administration. Compound 31 constitutes an optimized Vps34 inhibitor that could be used to investigate human cancer biology.

Selective inhibition of mutant isocitrate dehydrogenase 1 (IDH1) via disruption of a metal binding network by an allosteric small molecule.

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg(2+). A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme.

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 PI3Kß inhibitors for the treatment of phosphatase and tensin homologue (PTEN)-deficient cancers.

Compelling molecular biology publications have reported the implication of phosphoinositide kinase PI3Kß in PTEN-deficient cell line growth and proliferation. These findings supported a scientific rationale for the development of PI3Kß-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 PI3Kß 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 p110ß 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.

Discovery and optimization of new benzimidazole- and benzoxazole-pyrimidone selective PI3Kß inhibitors for the treatment of phosphatase and TENsin homologue (PTEN)-deficient cancers.

Most of the phosphoinositide-3 kinase (PI3K) kinase inhibitors currently in clinical trials for cancer treatment exhibit pan PI3K isoform profiles. Single PI3K isoforms differentially control tumorigenesis, and PI3Kß has emerged as the isoform involved in the tumorigenicity of PTEN-deficient tumors. Herein we describe the discovery and optimization of a new series of benzimidazole- and benzoxazole-pyrimidones as small molecular mass PI3Kß-selective inhibitors. Starting with compound 5 obtained from a one-pot reaction via a novel intermediate 1, medicinal chemistry optimization led to the discovery of compound 8, which showed a significant activity and selectivity for PI3Kß and adequate in vitro pharmacokinetic properties. The X-ray costructure of compound 8 in PI3Kδ showed key interactions and structural features supporting the observed PI3Kß isoform selectivity. Compound 8 achieved sustained target modulation and tumor growth delay at well tolerated doses when administered orally to SCID mice implanted with PTEN-deficient human tumor xenografts.

Sulfonylthiadiazoles with an unusual binding mode as partial dual peroxisome proliferator-activated receptor (PPAR) γ/δ agonists with high potency and in†vivo efficacy.

Compounds that simultaneously activate the peroxisome proliferator-activated receptor (PPAR) subtypes PPARγ and PPARδ have the potential to effectively target dyslipidemia and type II diabetes in a single pharmaceutically active molecule. The frequently observed side effects of selective PPARγ agonists, such as edema and weight gain, are expected to be overcome by using partial instead of full agonists for this nuclear receptor family. Herein we report the discovery, synthesis, and optimization of a novel series of sulfonylthiadiazoles that are active as partial agonists. The initial compound 6 was discovered by high-throughput screening as a moderate partial PPARδ agonist; its optimization was based on the X-ray crystal structure in complex with PPARδ. In contrast to other PPARδ agonists, this ligand does not interact directly with residues from the activation helix AF-2, which might be linked to its partial agonistic effect. Interestingly, the thiadiazole moiety fills a novel subpocket, which becomes accessible after moderate conformational rearrangement. The optimization was focused on introducing conformational constraints and replacing intramolecular hydrogen bonding interactions. Highly potent molecules with activity as dual partial PPARγ/δ agonists in the low nanomolar range were then identified. One of the most active members, compound 20 a, displayed EC50 values of 1.6 and 336 nM for PPARδ and γ, respectively. The X-ray crystal structure of its complex with PPARδ confirms our design hypothesis. Compound 20 a clearly displayed in vivo activity in two chronic mice studies. Lipids were modified in a beneficial way in normolipidemic mice, and the development of overt diabetes could be prevented in pre-diabetic db/db mice. However, body weight gain was similar to that observed with the PPARγ agonist rosiglitazone. Hence, active compounds from this series can be considered as valuable tools to elucidate the complex roles of dual PPARγ/δ agonists for potential treatment of metabolic syndrome.

Requirements for specific binding of low affinity inhibitor fragments to the SH2 domain of (pp60)Src are identical to those for high affinity binding of full length inhibitors.

Results from a novel approach which uses protein crystallography for the screening of a low affinity inhibitor fragment library are analyzed by comparing the X-ray structures with bound fragments to the structures with the corresponding full length inhibitors. The screen for new phospho-tyrosine mimics binding to the SH2 domain of (pp60)src was initiated because of the limited cell penetration of phosphates. Fragments in our library typically had between 6 and 30 atoms and included compounds which had either millimolar activity in a Biacore assay or were suggested by the ab initio design program LUDI but had no measurable affinity. All identified fragments were located in the phospho-tyrosine pocket. The most promising fragments were successfully used to replace the phospho-tyrosine and resulted in novel nonpeptidic high affinity inhibitors. The significant diversity of successful fragments is reflected in the high flexibility of the phospho-tyrosine pocket. Comparison of the X-ray structures shows that the presence of the H-bond acceptors and not their relative position within the pharmacophore are essential for fragment binding and/or high affinity binding of full length inhibitors. The X-ray data show that the fragments are recognized by forming a complex H-bond network within the phospho-tyrosine pocket of SH2. No fragment structure was found in which this H-bond network was incomplete, and any uncompensated H-bond within the H-bond network leads to a significant decrease in the affinity of full length inhibitors. No correlation between affinity and fragment binding was found for these polar fragments and hence affinity-based screening would have overlooked some interesting starting points for inhibitor design. In contrast, we were unable to identify electron density for hydrophobic fragments, confirming that hydrophobic interactions are important for inhibitor affinity but of minor importance for ligand recognition. Our results suggest that a screening approach using protein crystallography is particularly useful to identify universal fragments for the conserved hydrophilic recognition sites found in target families such as SH2 domains, phosphatases, kinases, proteases, and esterases.

Principles governing the binding of a class of non-peptidic inhibitors to the SH2 domain of src studied by X-ray analysis.

A total of 11 structures of the (pp60)src SH2 domain with non-peptidic inhibitors based on the same two closely related inhibitor scaffolds were determined using X-ray crystallography. Surprisingly, the inhibitors that have an IC(50) value between 4 and 2700 nM bind in three different binding modes. Structure comparisons show that the inhibitors aim to maximize the interaction between the hydrophobic substituent and the hydrophobic pY+3 pocket. This is achieved either by an alternative binding mode of the phenyl phosphate or by including water molecules that mediate the interaction between the inhibitor scaffold and a rigid surface of the SH2 domain. The combination of the rigid pY+3 pocket and the rigid protein surface to which the scaffolds bind results in severe distance and angular restraints for putative scaffolds and their substituents. The X-ray data suggest that these restraints seem to be compensated in our system by including water molecules, thereby increasing the flexibility of the system.

SAR and X-ray. A new approach combining fragment-based screening and rational drug design: application to the discovery of nanomolar inhibitors of Src SH2.

(pp60)Src is a protein involved in signal transduction and is mainly expressed in neurones, platelets, and osteoclasts. Its precise biological role was recently discovered with the KO experiments by Soriano that gave rise to no other apparent phenotype than osteopetrosis, a disease resulting in excedent bone formation. The SH2 domain of the Src family specifically recognizes a sequence of tetrapeptide featuring a phosphotyrosine and a lipophilic aminoacid at the +1 and +3 positions. Recently we engaged in the search for SH2 ligands via modular peptidomimicry of this tetrapetide. This gave rise to several families of nanomolar inhibitors; the best one incorporated a caprolactam scaffold, a biphenyl moiety, and a phosphotyrosine. However, these inhibitors still incorporated the phosphate group that confers good binding affinity to the protein. Phosphates have undesirable features for drug candidates, namely, high rate of hydrolysis of the phosphate group by phosphatases and high charge content precluding cell penetration. Therefore, while searching for optimal non-peptide ligands for Src SH2, we looked for phosphate replacements. For this, we have designed an SAR by fragment crystallography approach. The start of this work resulted from two experimental observations. First, the fact that phenyl phosphate itself displayed detectable binding affinity for Src SH2 permitted us to perform a screening of small aromatic compounds as phenyl phosphate surrogates. Second, the obtention of large Src SH2 crystals displaying a channel large enough for soaking purposes allowed structure determination of over 40 of these small aromatic compounds bound in the phosphotyrosine binding pocket. This search and the way it gave rise to low nanomolar range Src SH2 inhibitors devoid of phosphate groups will be the subject of the present paper.