Jason Phan

Discovery of Small Molecules that Bind to K-Ras and Inhibit Sos-Mediated Activation.

K-Ras is a small GTPase that functions as a molecular switch cycling between inactive (GDP-bound) and active (GTP-bound) states. The conversion of K-Ras-GDP to K-Ras-GTP is the rate-limiting step in the activation of K-Ras and is catalyzed by guanine nucleotide exchange factors such as the son of sevenless (Sos). Mutations in K-Ras fix the protein in the active state and endow cells with capabilities that represent the hallmarks of cancer.[1] These include the ability to proliferate, evade apoptosis, reprogram cell metabolism, induce angiogenesis, activate invasion and metastasis, and escape immune destruction.[2] Indeed, aberrant K-Ras signaling plays a role in 30% of all human cancers, with the highest incidence of activating mutations found in pancreatic (70-90%), colon (30-50%), and lung (20-30%) carcinomas.[3] Downregulation of activated Ras reverses the transformed phenotype of cells and results in the dramatic regression of tumors in murine xenograft models.[4] Thus, K-Ras inhibition represents an attractive therapeutic strategy for many cancers. However, Ras activation and signaling is accomplished primarily through protein-protein interactions. Such protein interfaces typically lack well-defined binding pockets and have been difficult to target with small molecules.[5]

Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange

Significance Ras is one of the most highly validated targets in cancer; however, the discovery of potent inhibitors of Ras has been difficult to achieve. We report the discovery of small molecules that bind to a pocket on the Ras:Son of Sevenless:Ras complex and alter Ras activity in biochemical and cell-based experiments. High-resolution cocrystal structures define the protein–ligand interactions, and the lead compounds provide a starting point for the discovery of potent inhibitors of Ras signaling. Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure–activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

Fragment-Based Screening of the Bromodomain of ATAD2.

Cellular and genetic evidence suggest that inhibition of ATAD2 could be a useful strategy to treat several types of cancer. To discover small-molecule inhibitors of the bromodomain of ATAD2, we used a fragment-based approach. Fragment hits were identified using NMR spectroscopy, and ATAD2 was crystallized with three of the hits identified in the fragment screen.

A method for the second-site screening of K-Ras in the presence of a covalently attached first-site ligand.

K-Ras is a well-validated cancer target but is considered to be “undruggable” due to the lack of suitable binding pockets. We previously discovered small molecules that bind weakly to K-Ras but wanted to improve their binding affinities by identifying ligands that bind near our initial hits that we could link together. Here we describe an approach for identifying second site ligands that uses a cysteine residue to covalently attach a compound for tight binding to the first site pocket followed by a fragment screen for binding to a second site. This approach could be very useful for targeting Ras and other challenging drug targets.

Discovery of Potent 2-Aryl-6,7-dihydro-5H-pyrrolo[1,2-a]imidazoles as WDR5-WIN-Site Inhibitors Using Fragment-Based Methods and Structure-Based Design

WDR5 is a chromatin-regulatory scaffold protein overexpressed in various cancers and a potential epigenetic drug target for the treatment of mixed-lineage leukemia. Here, we describe the discovery of potent and selective WDR5-WIN-site inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified several chemically distinct hit series that bind to the WIN site within WDR5. Members of a 6,7-dihydro-5 H-pyrrolo[1,2- a]imidazole fragment class were expanded using a structure-based design approach to arrive at lead compounds with dissociation constants <10 nM and micromolar cellular activity against an AML-leukemia cell line. These compounds represent starting points for the discovery of clinically useful WDR5 inhibitors for the treatment of cancer.

High-throughput screening identifies small molecules that bind to the RAS:SOS:RAS complex and perturb RAS signaling.

K-RAS is mutated in approximately 30% of human cancers, resulting in increased RAS signaling and tumor growth. Thus, RAS is a highly validated therapeutic target, especially in tumors of the pancreas, lung and colon. Although directly targeting RAS has proven to be challenging, it may be possible to target other proteins involved in RAS signaling, such as the guanine nucleotide exchange factor Son of Sevenless (SOS). We have previously reported on the discovery of small molecules that bind to SOS1, activate SOS-mediated nucleotide exchange on RAS, and paradoxically inhibit ERK phosphorylation (Burns et al., PNAS, 2014). Here, we describe the discovery of additional, structurally diverse small molecules that also bind to SOS1 in the same pocket and elicit similar biological effects. We tested >160,000 compounds in a fluorescence-based assay to assess their effects on SOS-mediated nucleotide exchange. X-Ray structures revealed that these small molecules bind to the CDC25 domain of SOS1. Compounds that elicited high levels of nucleotide exchange activity in vitro increased RAS-GTP levels in cells, and inhibited phospho ERK levels at higher treatment concentrations. The identification of structurally diverse SOS1 binding ligands may assist in the discovery of new molecules designed to target RAS-driven tumors.

Discovery of Quinazolines That Activate SOS1-Mediated Nucleotide Exchange on RAS

Proteins in the RAS family are important regulators of cellular signaling and, when mutated, can drive cancer pathogenesis. Despite considerable effort over the last 30 years, RAS proteins have proven to be recalcitrant therapeutic targets. One approach for modulating RAS signaling is to target proteins that interact with RAS, such as the guanine nucleotide exchange factor (GEF) son of sevenless homologue 1 (SOS1). Here, we report hit-to-lead studies on quinazoline-containing compounds that bind to SOS1 and activate nucleotide exchange on RAS. Using structure-based design, we refined the substituents attached to the quinazoline nucleus and built in additional interactions not present in the initial HTS hit. Optimized compounds activate nucleotide exchange at single-digit micromolar concentrations in vitro. In HeLa cells, these quinazolines increase the levels of RAS-GTP and cause signaling changes in the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway.

Abstract 4417: Targeting Ras with small molecules that activate SOS-mediated nucleotide exchange

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Aberrant activation of Ras, by mutation or constitutively active upstream kinases, occurs in nearly 30% of all human cancers, rendering Ras one of the most validated targets in cancer drug discovery. Historically, the direct inhibition of Ras with small molecules has proven extremely difficult. Here we report the discovery of compounds that bind to a unique pocket on the Ras:SOS:Ras complex, which increase the rate of SOScat-catalyzed nucleotide exchange in vitro and modulate Ras signaling pathways in cells. X-ray crystallography reveals that the molecules bind in a hydrophobic pocket in the CDC25 domain of SOS, adjacent to the Switch II region of Ras. Structure-based mutational analyses confirmed the functional relevance of this binding site and demonstrated that it is essential for compound activity. Consistent with their increased exchange activity in vitro, these molecules increase Ras-GTP levels in cells. However, the compounds inhibit ERK and AKT phosphorylation; the ERK inhibition at high concentrations is accompanied by an increase of p-ERK levels at lower compound concentrations. These molecules represent a unique tool to study the acute activation of Ras and highlight a novel pocket on SOS that may be exploited to modulate Ras signaling. Citation Format: Olivia W. Rossanese, Michael C. Burns, Qi Sun, R. Nathan Daniels, DeMarco V. Camper, J. Phillip Kennedy, Jason Phan, Edward T. Olejniczak, Taekyu Lee, Alex G. Waterson, Stephen W. Fesik. Targeting Ras with small molecules that activate SOS-mediated nucleotide exchange. [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 4417. doi:10.1158/1538-7445.AM2014-4417

Abstract PR01: Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange.

Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active receptor tyrosine kinases (RTKs) results in the deregulation of cellular signals governing growth and survival in cancer. The guanine nucleotide exchange factor Son of Sevenless (SOS) catalyzes the rate-limiting step in the activation of Ras by exchanging GDP for GTP. SOS is therefore a key control point for the propagation of RTK and Ras signaling. Here we report the discovery of small molecules that bind to a unique pocket on the Ras:SOS:Ras complex, increase SOScat-catalyzed nucleotide exchange, and perturb Ras signaling pathways in cells. X-ray crystallographic studies of Ras:SOS:Ras complexed with these small molecules reveal that they bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of the x-ray structures of multiple co-complexes. In addition, structure-based mutational analyses indicate that this newly identified pocket is essential for compound activity. As predicted, these molecules increase Ras-GTP levels in cells. However, they unexpectedly inhibit MAPK and PI3K signaling. Our studies suggest a novel way to target K-Ras and offer possible starting points for the discovery of compounds that could be used to treat Ras-driven tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR01. Citation Format: Michael Burns, Qi Sun, Richard Daniels, J. Phillip Kennedy, DeMarco Camper, Jason Phan, Edward Olejniczak, Taekyu Lee, Alex Waterson, Olivia Rossanese, Stephen Fesik. Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange. [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 PR01.