Keith Pitts

Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity

The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.

A small molecule inhibitor of the Wnt antagonist secreted frizzled-related protein-1 stimulates bone formation

Canonical Wnt signaling has been demonstrated to increase bone formation, and Wnt pathway components are being pursued as potential drug targets for osteoporosis and other metabolic bone diseases. Deletion of the Wnt antagonist secreted frizzled-related protein (sFRP)-1 in mice activates canonical signaling in bone and increases trabecular bone formation in aged animals. We have developed small molecules that bind to and inhibit sFRP-1 in vitro and demonstrate robust anabolic activity in an ex vivo organ culture assay. A library of over 440,000 drug-like compounds was screened for inhibitors of human sFRP-1 using a cell-based functional assay that measured activation of canonical Wnt signaling with an optimized T-cell factor (TCF)-luciferase reporter gene assay. One of the hits in this screen, a diarylsulfone sulfonamide, bound to sFRP-1 with a K(D) of 0.35 microM in a tryptophan fluorescence quenching assay. This compound also selectively inhibited sFRP-1 with an EC(50) of 3.9 microM in the cell-based functional assay. Optimization of this high throughput screening hit for binding and functional potency as well as metabolic stability and other pharmaceutical properties led to improved lead compounds. One of these leads (WAY-316606) bound to sFRP-1 with a K(D) of 0.08 microM and inhibited it with an EC(50) of 0.65 microM. Moreover, this compound increased total bone area in a murine calvarial organ culture assay at concentrations as low as 0.0001 microM. This work demonstrates the feasibility of developing small molecules that inhibit sFRP-1 and stimulate canonical Wnt signaling to increase bone formation.

Identification of substituted 2-thio-6-oxo-1,6-dihydropyrimidines as inhibitors of human lactate dehydrogenase.

A novel 2-thio-6-oxo-1,6-dihydropyrimidine-containing inhibitor of human lactate dehydrogenase (LDH) was identified by high-throughput screening (IC50=8.1 μM). Biochemical, surface plasmon resonance, and saturation transfer difference NMR experiments indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of the screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.48 μM). A crystal structure of an optimized compound bound to human LDHA was obtained and explained many of the observed structure-activity relationships.

Modulation of Wnt signaling through inhibition of secreted frizzled-related protein I (sFRP-1) with N-substituted piperidinyl diphenylsulfonyl sulfonamides.

The diphenylsulfonyl sulfonamide scaffold represented by 1 (WAY-316606) are small molecule inhibitors of the secreted protein sFRP-1, an endogenous antagonist of the secreted glycoprotein Wnt. Modulators of the Wnt pathway have been proposed as anabolic agents for the treatment of osteoporosis or other bone-related disorders. Details of the structure-activity relationships and biological activity from the first structural class of this scaffold will be discussed.

Identification of 2-amino-5-aryl-pyrazines as inhibitors of human lactate dehydrogenase.

A 2-amino-5-aryl-pyrazine was identified as an inhibitor of human lactate dehydrogenase A (LDHA) via a biochemical screening campaign. Biochemical and biophysical experiments demonstrated that the compound specifically interacted with human LDHA. Structural variation of the screening hit resulted in improvements in LDHA biochemical inhibition and pharmacokinetic properties. A crystal structure of an improved compound bound to human LDHA was also obtained and it explained many of the observed structure-activity relationships.

Identification of substituted 3-hydroxy-2-mercaptocyclohex-2-enones as potent inhibitors of human lactate dehydrogenase.

A novel class of 3-hydroxy-2-mercaptocyclohex-2-enone-containing inhibitors of human lactate dehydrogenase (LDH) was identified through a high-throughput screening approach. Biochemical and surface plasmon resonance experiments performed with a screening hit (LDHA IC50=1.7 μM) indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of this screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.18 μM). Two crystal structures of optimized compounds bound to human LDHA were obtained and explained many of the observed structure-activity relationships. In addition, an optimized inhibitor exhibited good pharmacokinetic properties after oral administration to rats (F=45%).

Small molecule inhibitors of HIV RT Ribonuclease H.

Two classes of compounds, thiocarbamates 1 and triazoles 2, have been identified as HIV RT RNase H inhibitors using a novel FRET-based HTS assay. The potent analogs in each series exhibited selectivity and were active in cell-based assays. In addition, saturable, 1:1 stoichiometric binding to target was established and time of addition studies were consistent with inhibition of RT-mediated HIV replication.

Modulation of Wnt signaling through inhibition of secreted frizzled-related protein I (sFRP-1) with N-substituted piperidinyl diphenylsulfonyl sulfonamides: Part II

Piperidinyl diphenylsulfonyl sulfonamides are a novel class of molecules that have inhibitory binding affinity for sFRP-1. As a secreted protein sFRP-1 inhibits the function of the secreted Wnt glycoprotein. Therefore, as inhibitors of sFRP-1 these small molecules facilitate the Wnt/beta-catenin canonical signaling pathway. Details of the structure-activity relationships and biological activity of this structural class of compounds will be discussed.

CHAPTER 2:Getting the Most Value from Your Screens: Advances in Hardware, Software, and Methodologies to Enhance Surface Plasmon Resonance Based Fragment Screening and Hit-to-Lead Support

The development and adoption of fragment-based lead discovery is partly driven by the deployment and refinement of the technologies that enable this drug-discovery approach. Advances in the capabilities of the core discovery functions have historically impacted various elements of the discovery process. For example, early fragment screens produced more structure information earlier in a screening cascade through use of high-resolution protein-detected NMR or crystallography based screening approaches. These screens were rich in information but slow, expensive, and limited to small libraries. The wider spread adoption of ligand-detected NMR and surface plasmon resonance (SPR) binding assays, with their higher throughput and lower sample consumption, allowed fragment screening cascades to be reformulated to more closely resemble HTS. The lower cost, high-throughput, lower information single-dose experiments are performed early, followed by more information-rich, but slower and more costly structural experiments on the reduced set of characterized binders. Refinements and innovations in hardware, software, and practical methodologies have continued to advance the ease and scope of the implementation of SPR-based biosensors. In this chapter we describe a number of these recent advances that streamline the workflow and can give screening groups more options and flexibility to bring higher information content data to teams earlier. With these tools in hand, third-generation screening cascades can be proposed that can give fragment discovery efforts more traction and momentum in the early lead-discovery setting.

Abstract 4759: Drugging the undruggable: Small molecule inhibition of the Ras oncoprotein

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Ras is a nucleotide-dependent switch that converts from an inactive GDP-bound state to an active GTP-bound state when activated by guanine nucleotide exchange factors, such as SOS. Active RasGTP then binds to and activates downstream signaling effectors. Ras is the most frequently mutated oncogene and hyperactive mutant Ras constitutively signals to effectors to promote cell survival, proliferation and metastasis. Thus, Ras oncoprotein has been considered by the cancer community to be one of the most important oncology drug targets. Despite the enormous interest and extensive exploratory efforts in industry and academia, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. We describe in this abstract the identification and characterization of small-molecule inhibitors of the Ras oncoprotein. Materials and Methods: To explore a new means of directly targeting Ras, we used a fragment-based lead discovery approach via an NMR-based screen. Hits from the fragment screen were characterized for their interactions with Ras by NMR and X-ray crystallography and for their effects on Ras activation and signaling in reconstituted biochemical assays in vitro and in cellular assays in vivo. Results: From the fragment-based screen, we identified a group of small molecules that each bind to a common site adjacent to the switch I/II regions in the Ras protein. X-ray crystallography studies of three compound-Ras complexes indicate that the binding site can be expanded upon ligand binding. Nucleotide exchange factors, notably SOS, are required to convert inactive RasGDP to active RasGTP. We determined that the compound-binding site is located at the interface of Ras and SOS. A subset of our Ras-binding molecules indeed inhibited SOS-mediated nucleotide exchange. Further mechanistic studies revealed that through steric hindrance the compounds block the formation of the Ras-SOS complex, a key intermediate of the exchange reaction. At the cellular level, our compounds inhibit the formation of active RasGTP and prevent Ras signaling to downstream effectors. To define the potential clinic utility of these compounds, we performed biological characterization of Ras-driven tumors and identified a subset of Ras mutant tumors that depend on nucleotide exchange factors for the activation of Ras, suggesting a specific profile for the use of exchange inhibitors. Conclusions: We conclude that the compounds act as competitive inhibitors of nucleotide exchange to prevent the activation of Ras. The discovery of a binding pocket on Ras with functional significance represents a breakthrough finding that will offer a new direction for therapeutic intervention of the Ras oncoprotein. Our findings provide new opportunities to target the “undruggable” Ras oncoprotein. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4759. doi:1538-7445.AM2012-4759