Bhavatarini Vangamudi

Discovery of a potent inhibitor of replication protein a protein-protein interactions using a fragment-linking approach.

Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA)-binding protein, is involved in nearly all cellular DNA transactions. The RPA N-terminal domain (RPA70N) is a recruitment site for proteins involved in DNA-damage response and repair. Selective inhibition of these protein-protein interactions has the potential to inhibit the DNA-damage response and to sensitize cancer cells to DNA-damaging agents without affecting other functions of RPA. To discover a potent, selective inhibitor of the RPA70N protein-protein interactions to test this hypothesis, we used NMR spectroscopy to identify fragment hits that bind to two adjacent sites in the basic cleft of RPA70N. High-resolution X-ray crystal structures of RPA70N-ligand complexes revealed how these fragments bind to RPA and guided the design of linked compounds that simultaneously occupy both sites. We have synthesized linked molecules that bind to RPA70N with submicromolar affinity and minimal disruption of RPAs interaction with ssDNA.

Discovery of a potent stapled helix peptide that binds to the 70N domain of replication protein A

Stapled helix peptides can serve as useful tools for inhibiting protein–protein interactions but can be difficult to optimize for affinity. Here we describe the discovery and optimization of a stapled helix peptide that binds to the N-terminal domain of the 70 kDa subunit of replication protein A (RPA70N). In addition to applying traditional optimization strategies, we employed a novel approach for efficiently designing peptides containing unnatural amino acids. We discovered hot spots in the target protein using a fragment-based screen, identified the amino acid that binds to the hot spot, and selected an unnatural amino acid to incorporate, based on the structure–activity relationships of small molecules that bind to this site. The resulting stapled helix peptide potently and selectively binds to RPA70N, does not disrupt ssDNA binding, and penetrates cells. This peptide may serve as a probe to explore the therapeutic potential of RPA70N inhibition in cancer.

Surface Reengineering of RPA70N Enables Cocrystallization with an Inhibitor of the Replication Protein A Interaction Motif of ATR Interacting Protein.

Replication protein A (RPA) is the primary single-stranded DNA (ssDNA) binding protein in eukaryotes. The N-terminal domain of the RPA70 subunit (RPA70N) interacts via a basic cleft with a wide range of DNA processing proteins, including several that regulate DNA damage response and repair. Small molecule inhibitors that disrupt these protein-protein interactions are therefore of interest as chemical probes of these critical DNA processing pathways and as inhibitors to counter the upregulation of DNA damage response and repair associated with treatment of cancer patients with radiation or DNA-damaging agents. Determination of three-dimensional structures of protein-ligand complexes is an important step for elaboration of small molecule inhibitors. However, although crystal structures of free RPA70N and an RPA70N-peptide fusion construct have been reported, RPA70N-inhibitor complexes have been recalcitrant to crystallization. Analysis of the P61 lattice of RPA70N crystals led us to hypothesize that the ligand-binding surface was occluded. Surface reengineering to alter key crystal lattice contacts led to the design of RPA70N E7R, E100R, and E7R/E100R mutants. These mutants crystallized in a P212121 lattice that clearly had significant solvent channels open to the critical basic cleft. Analysis of X-ray crystal structures, target peptide binding affinities, and (15)N-(1)H heteronuclear single-quantum coherence nuclear magnetic resonance spectra showed that the mutations do not result in perturbations of the RPA70N ligand-binding surface. The success of the design was demonstrated by determining the structure of RPA70N E7R soaked with a ligand discovered in a previously reported molecular fragment screen. A fluorescence anisotropy competition binding assay revealed this compound can inhibit the interaction of RPA70N with the peptide binding motif from the DNA damage response protein ATRIP. The implications of the results are discussed in the context of ongoing efforts to design RPA70N inhibitors.

Diphenylpyrazoles as replication protein a inhibitors.

Replication Protein A is the primary eukaryotic ssDNA binding protein that has a central role in initiating the cellular response to DNA damage. RPA recruits multiple proteins to sites of DNA damage via the N-terminal domain of the 70 kDa subunit (RPA70N). Here we describe the optimization of a diphenylpyrazole carboxylic acid series of inhibitors of these RPA-protein interactions. We evaluated substituents on the aromatic rings as well as the type and geometry of the linkers used to combine fragments, ultimately leading to submicromolar inhibitors of RPA70N protein-protein interactions.

Identification and Optimization of Anthranilic Acid Based Inhibitors of Replication Protein A.

Replication protein A (RPA) is an essential single‐stranded DNA (ssDNA)‐binding protein that initiates the DNA damage response pathway through protein–protein interactions (PPIs) mediated by its 70N domain. The identification and use of chemical probes that can specifically disrupt these interactions is important for validating RPA as a cancer target. A high‐throughput screen (HTS) to identify new chemical entities was conducted, and 90 hit compounds were identified. From these initial hits, an anthranilic acid based series was optimized by using a structure‐guided iterative medicinal chemistry approach to yield a cell‐penetrant compound that binds to RPA70N with an affinity of 812 nm. This compound, 2‐(3‐ (N‐(3,4‐dichlorophenyl)sulfamoyl)‐4‐methylbenzamido)benzoic acid (20 c), is capable of inhibiting PPIs mediated by this domain.

Abstract 3016: Identification of protein arginine methyltransferase 1 as novel epigenetic vulnerability in KRAS/p53 mutant PDAC primary patient models

Pancreatic ductal adenocarcinoma (PDAC) is a rapidly progressing disease associated with less than 10% 5-year survival rate. Various treatment regimens failed to improve survival of PDAC patients, thus a critical need exists to identify druggable targets essential for tumor maintenance. We developed a powerful in vivo platform that enables the identification of new molecular drivers in the PDAC context where activating mutation of KRAS gene and loss of p53 dominate the genetic landscape. Through an in vivo loss of function screen performed in KRAS/p53 mutant PDAC primary patient models, we identify protein arginine methyltransferase 1 (PRMT1) as top scoring hit. This novel dependency in PDAC was subsequently validated in multiple PDAC models using both shRNA mediated as well as CRISPR base genetic inhibition and we demonstrated that PRMT1 knockdown induces a significant growth inhibition in vitro. Methylation of arginine 3 on histone H4 (H4R3me2a) as well as global arginine methylation was also evaluated and showed a dramatic reduction upon PRMT1 knockdown, correlating observed phenotype with target engagement. To further confirm a role for PRMT1 in tumor maintenance, we developed inducible PRMT1 knockdown in a primary patient model and showed a dramatic tumor growth inhibition (TGI) in vivo upon PRMT1 knockdown. PRMT1 is the primary enzyme responsible for arginine asymmetric demethylation, however other members of the Type I family are also involved in this process and we evaluated the role of protein arginine methyltransferase 4 (PRMT4) and 6 (PRMT6) in our workhorse model. Surprisingly, no significant phenotypic response was observed upon genetic inhibition of PRMT4 or PRMT6 suggesting no redundancy between different PRMT type I and a unique dependency on PRMT1. To strengthen and complement the genetic validation, we leveraged a PRMT Type I inhibitor and confirmed in vitro results as well as in vivo efficacy at tolerated doses (xenograft vs allograft). Key models have been prioritized in order to inform on PRMT1 dependency and to refine responder population. Our research has identified and validated for the first time an arginine methyltransferase as a novel genetic vulnerability in PDAC and strongly suggest PRMT1 as a new therapeutic opportunity in PDAC cancers. Citation Format: Virginia Giuliani, Bhavatarini Vangamudi, Erika Suzuki, Meredith Miller, Chiu-Yi Liu, Alessandro Carugo, Christopher Bristow, Guang Gao, Jing Han, Yuting Sun, Ningping Feng, Edward Chang, Joseph Marszalek, Jeffrey Kovacs, Maria Emilia Di Francesco, Carlo Toniatti, Timothy Heffernan, Philip Jones, Giulio Draetta. Identification of protein arginine methyltransferase 1 as novel epigenetic vulnerability in KRAS/p53 mutant PDAC primary patient models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3016. doi:10.1158/1538-7445.AM2017-3016

Abstract 3340: Stapled helix peptides as probes to evaluate targeted disruption of protein-protein interactions mediated by RPA70N.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Replication Protein A (RPA) is a major regulator of checkpoint activation and enhanced DNA repair in cancer cells. In response to genotoxic stress, the RPA complex binds to and protects ssDNA while serving as a scaffold to recruit critical checkpoint and DNA-damage response proteins through the N-terminal region of the 70 kDa subunit of RPA (RPA70N). Specific disruption of the RPA protein-protein interactions mediated by the RPA70N domain has the potential to produce selective killing of cancer cells without the risk of cytotoxicity due to interference in the ssDNA-binding function. Stapled helix peptides are an emerging technology for the inhibition of protein-protein interactions. Incorporation of a hydrocarbon “staple” has the potential to increase the potency, stability, and cell permeability of peptides. Here, we report the development of a potent stapled helix peptide probe, derived from the endogenous RPA binding partner ATRIP (ATR-interacting protein), that binds to and inhibits the RPA70N protein-protein interaction surface. An initial alanine scan of the native ATRIP-derived sequence identified residues critical for peptide binding to RPA70N. In addition, the scan revealed residue positions that were dispensable and therefore suitable as sites for incorporation of a staple. Introduction of a conserved WFA motif derived from analysis of the p53-binding sequence produced a 10-fold gain in potency over the native ATRIP peptide. To facilitate entry into cells, negatively charged residues were replaced by alanines or by neutral, polar residues. In most instances, residues that improved the net charge had a deleterious effect on binding affinity. The resulting peptide, chosen for stapling, represented a balance between net charge and potency and was intended to offer the best chance of cell penetration while still maintaining affinity. Based on the alanine scan data, two positions were chosen for incorporation of a hydrocarbon staple; however, only one of these stapled peptides maintained binding affinity for RPA70N. The optimized peptide was cell penetrant, able to enter the nucleus, and co-localized with RPA in the nucleus at sites of DNA damage. In this study, we further examine the functional consequences of RPA70N disruption by ATRIP-derived hydrocarbon stapled peptides and discuss the use of them as tools to probe the therapeutic relevance of RPA inhibition in breast and other cancers. Citation Format: Bhavatarini Vangamudi, Andreas O. Frank, Elaine M. Souza-Fagundes, Michael D. Feldkamp, Edward T. Olejniczak, Olivia W. Rossanese, Stephen W. Fesik. Stapled helix peptides as probes to evaluate targeted disruption of protein-protein interactions mediated by RPA70N. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3340. doi:10.1158/1538-7445.AM2013-3340