Michael D. Feldkamp

Calmodulin Mutations Associated With Recurrent Cardiac Arrest in Infants

Background— Life-threatening disorders of heart rhythm may arise during infancy and can result in the sudden and tragic death of a child. We performed exome sequencing on 2 unrelated infants presenting with recurrent cardiac arrest to discover a genetic cause. Methods and Results— We ascertained 2 unrelated infants (probands) with recurrent cardiac arrest and dramatically prolonged QTc interval who were both born to healthy parents. The 2 parent-child trios were investigated with the use of exome sequencing to search for de novo genetic variants. We then performed follow-up candidate gene screening on an independent cohort of 82 subjects with congenital long-QT syndrome without an identified genetic cause. Biochemical studies were performed to determine the functional consequences of mutations discovered in 2 genes encoding calmodulin. We discovered 3 heterozygous de novo mutations in either CALM1 or CALM2, 2 of the 3 human genes encoding calmodulin, in the 2 probands and in 2 additional subjects with recurrent cardiac arrest. All mutation carriers were infants who exhibited life-threatening ventricular arrhythmias combined variably with epilepsy and delayed neurodevelopment. Mutations altered residues in or adjacent to critical calcium binding loops in the calmodulin carboxyl-terminal domain. Recombinant mutant calmodulins exhibited several-fold reductions in calcium binding affinity. Conclusions— Human calmodulin mutations disrupt calcium ion binding to the protein and are associated with a life-threatening condition in early infancy. Defects in calmodulin function will disrupt important calcium signaling events in heart, affecting membrane ion channels, a plausible molecular mechanism for potentially deadly disturbances in heart rhythm during infancy.

ETAA1 acts at stalled replication forks to maintain genome integrity

The ATR checkpoint kinase coordinates cellular responses to DNA replication stress. Budding yeast contain three activators of Mec1 (the ATR orthologue); however, only TOPBP1 is known to activate ATR in vertebrates. We identified ETAA1 as a replication stress response protein in two proteomic screens. ETAA1-deficient cells accumulate double-strand breaks, sister chromatid exchanges, and other hallmarks of genome instability. They are also hypersensitive to replication stress and have increased frequencies of replication fork collapse. ETAA1 contains two RPA-interaction motifs that localize ETAA1 to stalled replication forks. It also interacts with several DNA damage response proteins including the BLM/TOP3α/RMI1/RMI2 and ATR/ATRIP complexes. It binds ATR/ATRIP directly using a motif with sequence similarity to the TOPBP1 ATR-activation domain; and like TOPBP1, ETAA1 acts as a direct ATR activator. ETAA1 functions in parallel to the TOPBP1/RAD9/HUS1/RAD1 pathway to regulate ATR and maintain genome stability. Thus, vertebrate cells contain at least two ATR-activating proteins.

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.

Structural analysis of replication protein A recruitment of the DNA damage response protein SMARCAL1.

SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like1 (SMARCAL1) is a recently identified DNA damage response protein involved in remodeling stalled replication forks. The eukaryotic single-strand DNA binding protein replication protein A (RPA) recruits SMARCAL1 to stalled forks in vivo and facilitates regression of forks containing leading strand gaps. Both activities are mediated by a direct interaction between an RPA binding motif (RBM) at the N-terminus of SMARCAL1 and the C-terminal winged-helix domain of the RPA 32 kDa subunit (RPA32C). Here we report a biophysical and structural characterization of the SMARCAL1–RPA interaction. Isothermal titration calorimetry and circular dichroism spectroscopy revealed that RPA32C binds SMARCAL1-RBM with a Kd of 2.5 μM and induces a disorder-to-helix transition. The crystal structure of RPA32C was refined to 1.4 Å resolution, and the SMARCAL1-RBM binding site was mapped on the structure on the basis of nuclear magnetic resonance chemical shift perturbations. Conservation of the interaction surface to other RBM-containing proteins allowed construction of a model for the RPA32C/SMARCAL1-RBM complex. The implications of our results are discussed with respect to the recruitment of SMARCAL1 and other DNA damage response and repair proteins to stalled replication forks.

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.

NMR studies of the interaction of calmodulin with IQ motif peptides.

Calmodulin (CaM) is a ubiquitous EF-hand calcium sensor protein that transduces calcium signals in a wide range of signaling pathways. Structural analysis of complexes with peptides has provided valuable insights into the remarkable variety in the way in which CaM interacts with and activates its targets. Among these various targets, CaM has been shown to be an essential component of a calcium-sensing regulatory apparatus for a number of voltage-gated ion channels. NMR spectroscopy has proven to be a powerful tool for the structural characterization of CaM-peptide complexes, in particular for the study of IQ motifs, which bind CaM at the basal level of calcium in cells and thereby serve to localize CaM to its sites of action. We describe here methods for the robust expression and purification of CaM isotopically enriched for NMR analysis, as well as for the complex of CaM with a peptide derived from the IQ motif sequence of the human cardiac sodium channel Na(V)1.5. We also describe methods for NMR analysis of titrations of CaM with IQ motif peptides to determine the stoichiometry of the complex and to identify the residues at the binding interface.

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.

Simian virus Large T antigen interacts with the N-terminal domain of the 70 kD subunit of Replication Protein A in the same mode as multiple DNA damage response factors.

Simian virus 40 (SV40) serves as an important model organism for studying eukaryotic DNA replication. Its helicase, Large T-antigen (Tag), is a multi-functional protein that interacts with multiple host proteins, including the ubiquitous ssDNA binding protein Replication Protein A (RPA). Tag recruits RPA, actively loads it onto the unwound DNA, and together they promote priming of the template. Although interactions of Tag with RPA have been mapped, no interaction between Tag and the N-terminal protein interaction domain of the RPA 70kDa subunit (RPA70N) has been reported. Here we provide evidence of direct physical interaction of Tag with RPA70N and map the binding sites using a series of pull-down and mutational experiments. In addition, a monoclonal anti-Tag antibody, the epitope of which overlaps with the binding site, blocks the binding of Tag to RPA70N. We use NMR chemical shift perturbation analysis to show that Tag uses the same basic cleft in RPA70N as multiple of DNA damage response proteins. Mutations in the binding sites of both RPA70N and Tag demonstrate that specific charge reversal substitutions in either binding partner strongly diminish the interaction. These results expand the known repertoire of contacts between Tag and RPA, which mediate the many critical roles of Tag in viral replication.

Abstract 3232: Optimization of a potent stapled helix peptide that binds to Replication Protein A

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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 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 can serve as useful tools for inhibiting protein-protein interactions. However, their utility can be limited due to difficulties often encountered during attempts to improve the binding affinity to the target. Here, we report the discovery and optimization 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. Alanine scanning, charge abrogation, and rational sequence optimization resulted in a peptide with a 100-fold potency gain over the native sequence and improved physical characteristics. In addition to the application of these traditional strategies, we describe a novel approach for efficiently designing peptides containing unnatural amino acids. This method involves the incorporation of an unnatural amino acid inspired by the structure activity relationships of small molecules that bind to the same site on the protein. Use of this approach produced stapled peptides with dramatic increases in binding affinity to RPA70N relative to aooIn al peptide containing only natural amino acids. The optimized peptides are cell penetrant, able to enter the nucleus, and co-localize with RPA in the nucleus at sites of DNA damage. Such a peptide may serve as a probe molecule to explore both the effects of RPA inhibition on the DNA damage response and the therapeutic potential of RPA inhibition as a cancer target. Citation Format: Alex G. Waterson, Andreas O. Frank, Bhavatarini Vandgamudi, Michael D. Feldkamp, Elaine M. Souza-Fagundes, Jessica W. Luzwick, David Cortez, Edward T. Olejniczak, Olivia W. Rossanese, Walter J. Chazin, Stephen W. Fesik. Optimization of a potent stapled helix peptide that binds to Replication Protein A. [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 3232. doi:10.1158/1538-7445.AM2014-3232

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