SOS-mediated Nucleotide Exchange Research Articles

KRAS Inhibitor that Simultaneously Inhibits Nucleotide Exchange Activity and Effector Engagement.

We describe a small molecule ligand ACA-14 (2-hydroxy-5-{[(2-phenylcyclopropyl) carbonyl] amino} benzoic acid) as an initial lead for the development of direct inhibitors of KRAS, a notoriously difficult anticancer drug target. We show that the compound binds to KRAS near the switch regions with affinities in the low micromolar range and exerts different effects on KRAS interactions with binding partners. Specifically, ACA-14 impedes the interaction of KRAS with its effector Raf and reduces both intrinsic and SOS-mediated nucleotide exchange rates. Likely as a result of these effects, ACA-14 inhibits signal transduction through the MAPK pathway in cells expressing mutant KRAS and inhibits the growth of pancreatic and colon cancer cells harboring mutant KRAS. We thus propose compound ACA-14 as a useful initial lead for the development of broad-acting inhibitors that target multiple KRAS mutants and simultaneously deplete the fraction of GTP-loaded KRAS while abrogating the effector-binding ability of the already GTP-loaded fraction.

Abstract B27: A DARPin-based toolbox to understand and treat RAS-addicted cancers

Abstract The repeating failure of small molecules as specific inhibitors of KRAS has drawn the attention to macromolecular structures, which can recognize their target with high affinity and specificity. Designed Ankyrin Repeat Proteins (DARPins) are multipurpose alternative affinity reagents that have proved to recognize targets with exceptional specificities and selectivity that often surpass those of antibodies. Due to their additional outstanding ability to act intracellularly within living cells and their recognition of structural rather than linear epitopes, DARPins have enabled a multitude of more advanced projects. In-house selections against GTP- or GDP-loaded KRAS were performed, and several hundred different DARPins could be identified. In subsequent validations, we not only analyzed their affinities, but also focused on the essential features of cross-reactivities, the recognition of various epitopes and their biologic functionalities. A number of candidates directly interfere with the RAS-Raf interaction and SOS-mediated nucleotide exchange by binding to the identical epitope on RAS as proved by crystal structures. One of these lead candidates with an affinity of 10 nM was used to elucidate whether anti-KRAS DARPins can mediate a biologic effect in model systems of human cancers. For this purpose, we chose cell lines categorized as KRAS dependent to generate stable cell lines expressing a highly active anti-KRAS DARPin under an inducible promotor, and it revealed potent antitumor activity, reducing the proliferation, colony formation and anchorage-independent growth. We could furthermore show that the observed effect resulted from reduced signaling of KRAS through its downstream MEK-ERK and PI3K-AKT pathways and the induction of apoptosis. Importantly, this anti-KRAS DARPin was shown to have no effects in the immortalized cell line HEK293T. In addition to traditional knockdown approaches, this model could be used to assess RAS dependency of human cancers. Combined with various suitable intracellular delivery techniques currently under development in our laboratory, the potential of this model will be further investigated for its potential in the treatment of solid tumors in a mouse model. In our broad effort many additional DARPins were identified that recognize different, nonoverlapping epitopes and most likely will interfere with other essential functions, such as the nanoclustering of RAS. This toolbox also allows the development of various biosensors in living cells. This large set of binders has the potential not just to help the community to gain detailed insights into the various functions and of RAS, but also might highlight novel vulnerabilities and innovative ways to finally make these key players druggable. Citation Format: Jonas N. Kapp, Jonas V. Schaefer, Wouter Verdurmen, Gabriela Nagy, Ralph Degen, Patrick Ernst, Claudia Scholl, Andreas Plückthun. A DARPin-based toolbox to understand and treat RAS-addicted cancers [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B27.

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.

Novel K-Ras G12C Switch-II Covalent Binders Destabilize Ras and Accelerate Nucleotide Exchange.

The success of targeted covalent inhibitors in the global pharmaceutical industry has led to a resurgence of covalent drug discovery. However, covalent inhibitor design for flexible binding sites remains a difficult task due to a lack of methodological development. Here, we compared covalent docking to empirical electrophile screening against the highly dynamic target K-RasG12C. While the overall hit rate of both methods was comparable, we were able to rapidly progress a docking hit to a potent irreversible covalent binder that modifies the inactive, GDP-bound state of K-RasG12C. Hydrogen-deuterium exchange mass spectrometry was used to probe the protein dynamics of compound binding to the switch-II pocket and subsequent destabilization of the nucleotide-binding region. SOS-mediated nucleotide exchange assays showed that, contrary to prior switch-II pocket inhibitors, these new compounds appear to accelerate nucleotide exchange. This study highlights the efficiency of covalent docking as a tool for the discovery of chemically novel hits against challenging targets.

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

Abstract 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

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 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.

Bisphenol A Binds to Ras Proteins and Competes with Guanine Nucleotide Exchange: Implications for GTPase-Selective Antagonists

We show for the first time that bisphenol A (10) has the capacity to interact directly with K-Ras and that Rheb weakly binds to bisphenol A (10) and 4,4'-biphenol derivatives. We have characterized these interactions at atomic resolution suggesting that these compounds sterically interfere with the Sos-mediated nucleotide exchange in H- and K-Ras. We show that 4,4'-biphenol (5) selectively inhibits Rheb signaling and induces cell death suggesting that this compound might be a novel candidate for treatment of tuberous sclerosis-mediated tumor growth. Our results propose a new mode of action for bisphenol A (10) that advocates a reduced exposure to this compound in our environment. Our data may lay the foundation for the future design of GTPase-selective antagonists with higher affinity to benefit of the treatment of cancer because K-Ras inhibition is regarded to be a promising strategy with a potential therapeutic window for targeting Sos in Ras-driven tumors.

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

Abstract 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.

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

Abstract 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

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.

Abstract IA24: Drugging the undruggable: Small-molecule inhibition of Ras oncoprotein

Abstract 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 report here the identification and characterization of small-molecule inhibitors of the Ras oncoprotein. 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. 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. 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 Ras. Our findings provide new opportunities to target the “undruggable” Ras oncoprotein. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr IA24.