Identification of Potent, Broad-Spectrum Coronavirus Main Protease Inhibitors for Pandemic Preparedness.

The COVID-19 pandemic highlights the ongoing risk of zoonotic transmission of coronaviruses to global health. To prepare for future pandemics, it is essential to develop effective antivirals targeting a broad range of coronaviruses. Targeting the essential and clinically validated coronavirus main protease (Mpro), we constructed a structurally diverse Mpro panel by clustering all known coronavirus sequences by Mpro active site sequence similarity. Through screening, we identified a potent covalent inhibitor that engaged the catalytic cysteine of SARS-CoV-2 Mpro and used structure-based medicinal chemistry to develop compounds in the pyrazolopyrimidine sulfone series that exhibit submicromolar activity against multiple Mpro homologues. Additionally, we solved the first X-ray cocrystal structure of Mpro from the human-infecting OC43 coronavirus, providing insights into potency differences among compound-target pairs. Overall, the chemical compounds described in this study serve as starting points for the development of antivirals with broad-spectrum activity, enhancing our preparedness for emerging human-infecting coronaviruses.

Rational Chemical Design of Molecular Glue Degraders.

Targeted protein degradation with molecular glue degraders has arisen as a powerful therapeutic modality for eliminating classically undruggable disease-causing proteins through proteasome-mediated degradation. However, we currently lack rational chemical design principles for converting protein-targeting ligands into molecular glue degraders. To overcome this challenge, we sought to identify a transposable chemical handle that would convert protein-targeting ligands into molecular degraders of their corresponding targets. Using the CDK4/6 inhibitor ribociclib as a prototype, we identified a covalent handle that, when appended to the exit vector of ribociclib, induced the proteasome-mediated degradation of CDK4 in cancer cells. Further modification of our initial covalent scaffold led to an improved CDK4 degrader with the development of a but-2-ene-1,4-dione ("fumarate") handle that showed improved interactions with RNF126. Subsequent chemoproteomic profiling revealed interactions of the CDK4 degrader and the optimized fumarate handle with RNF126 as well as additional RING-family E3 ligases. We then transplanted this covalent handle onto a diverse set of protein-targeting ligands to induce the degradation of BRD4, BCR-ABL and c-ABL, PDE5, AR and AR-V7, BTK, LRRK2, HDAC1/3, and SMARCA2/4. Our study undercovers a design strategy for converting protein-targeting ligands into covalent molecular glue degraders.

Deubiquitinase-targeting chimeras for targeted protein stabilization.

Many diseases are driven by proteins that are aberrantly ubiquitinated and degraded. These diseases would be therapeutically benefited by targeted protein stabilization (TPS). Here we present deubiquitinase-targeting chimeras (DUBTACs), heterobifunctional small molecules consisting of a deubiquitinase recruiter linked to a protein-targeting ligand, to stabilize the levels of specific proteins degraded in a ubiquitin-dependent manner. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48-ubiquitin-specific deubiquitinase OTUB1. We showed that a DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR), robustly stabilized ΔF508-CFTR protein levels, leading to improved chloride channel conductance in human cystic fibrosis bronchial epithelial cells. We also demonstrated stabilization of the tumor suppressor kinase WEE1 in hepatoma cells. Our study showcases covalent chemoproteomic approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.

A polyomavirus peptide binds to the capsid VP1 pore and has potent antiviral activity against BK and JC polyomaviruses.

In pursuit of therapeutics for human polyomaviruses, we identified a peptide derived from the BK polyomavirus (BKV) minor structural proteins VP2/3 that is a potent inhibitor of BKV infection with no observable cellular toxicity. The thirteen-residue peptide binds to major structural protein VP1 with single-digit nanomolar affinity. Alanine-scanning of the peptide identified three key residues, substitution of each of which results in ~1000 fold loss of binding affinity with a concomitant reduction in antiviral activity. Structural studies demonstrate specific binding of the peptide to the pore of pentameric VP1. Cell-based assays demonstrate nanomolar inhibition (EC50) of BKV infection and suggest that the peptide acts early in the viral entry pathway. Homologous peptide exhibits similar binding to JC polyomavirus VP1 and inhibits infection with similar potency to BKV in a model cell line. Lastly, these studies validate targeting the VP1 pore as a novel strategy for the development of anti-polyomavirus agents.

Structural basis of indisulam-mediated RBM39 recruitment to DCAF15 E3 ligase complex.

The anticancer agent indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor, leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which indisulam mediates the DCAF15-RBM39 interaction, we solved the DCAF15-DDB1-DDA1-indisulam-RBM39(RRM2) complex structure to a resolution of 2.3 Å. DCAF15 has a distinct topology that embraces the RBM39(RRM2) domain largely via non-polar interactions, and indisulam binds between DCAF15 and RBM39(RRM2), coordinating additional interactions between the two proteins. Studies with RBM39 point mutants and indisulam analogs validated the structural model and defined the RBM39 α-helical degron motif. The degron is found only in RBM23 and RBM39, and only these proteins were detectably downregulated in indisulam-treated HCT116 cells. This work further explains how indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade additional targets.

First Crystal Structure of a Nonstructural Hepatitis E Viral Protein Identifies a Putative Novel Zinc-Binding Protein.

Hepatitis E virus (HEV) is a 7.2-kb positive-sense, single-stranded RNA virus containing three partially overlapping reading frames, ORF1 to ORF3. All nonstructural proteins required for viral replication are encoded by ORF1 and are transcribed as a single transcript. Computational analysis of the complete ORF1 polyprotein identified a previously uncharacterized region of predicted secondary structure bordered by two disordered regions coinciding partially with a region predicted as a putative cysteine protease. Following successful cloning, expression, and purification of this region, the crystal structure of the identified protein was determined and identified to have considerable structural homology to a fatty acid binding domain. Further analysis of the structure revealed a metal binding site, shown unambiguously to specifically bind zinc via a nonclassical, potentially catalytic zinc-binding motif. Based on the structural homology of the HEV protein with known structures, along with the presence of a catalytic zinc-binding motif, it is possible that the identified protein corresponds to the HEV protease, which could require activation or repression through the binding of a fatty acid. This represents a significant step forward in the characterization and the understanding of the molecular mechanisms of the HEV genome. We present analysis for the first time of this identified nonstructural protein, expanding the knowledge and understanding of the complex mechanisms of HEV biology.IMPORTANCE Hepatitis E virus (HEV) is an emerging virus found predominately in developing countries; it causes an estimated 20 million infections, which result in approximately 57,000 deaths a year. Although it is known that the nonstructural proteins of HEV ORF1 are expressed as a single transcript, there is debate as to whether ORF1 functions as a single polyprotein or if it is processed into separate domains via a viral or endogenous cellular protease. Here we present the first structural and biophysical characterization of an HEV nonstructural protein using a construct that has partially overlapping boundaries with the predicted putative cysteine protease.

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.

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.

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 RPA's interaction with ssDNA.

Discovery of Protein-Protein Interaction Inhibitors of Replication Protein A.

Replication Protein A (RPA) is a ssDNA binding protein that is essential for DNA replication and repair. The initiation of the DNA damage response by RPA is mediated by protein-protein interactions involving the N-terminal domain of the 70 kDa subunit with partner proteins. Inhibition of these interactions increases sensitivity towards DNA damage and replication stress and may therefore be a potential strategy for cancer drug discovery. Towards this end, we have discovered two lead series of compounds, derived from hits obtained from a fragment-based screen, that bind to RPA70N with low micromolar affinity and inhibit the binding of an ATRIP-derived peptide to RPA. These compounds may offer a promising starting point for the discovery of clinically useful RPA inhibitors.

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.

A high-throughput fluorescence polarization anisotropy assay for the 70N domain of replication protein A.

Replication protein A (RPA) interacts with multiple checkpoint proteins and promotes signaling through the ATR kinase, a key regulator of checkpoint pathways in the mammalian response to DNA damage. In cancer cells, increased DNA repair activity contributes to resistance to chemotherapy. Therefore, small molecules that block binding of checkpoint proteins to RPA may inhibit the DNA damage response and, thus, sensitize cancer cells to DNA-damaging agents. Here we report on the development of a homogeneous, high-throughput fluorescence polarization assay for identifying compounds that block the critical protein-protein interaction site in the basic cleft of the 70N domain of RPA (RPA70N). A fluorescein isothiocyanate (FITC)-labeled peptide derived from the ATR cofactor, ATRIP, was used as a probe in the binding assay. The ability of the assay to accurately detect relevant ligands was confirmed using peptides derived from ATRIP, RAD9, MRE11, and p53. The assay was validated for use in high-throughput screening using the Spectrum collection of 2000 compounds. The FPA assay was performed with a Z' factor of ≥ 0.76 in a 384-well format and identified several compounds capable of inhibiting the RPA70N binding interface.

The impact of amino acid side chain mutations in conformational design of peptides and proteins.

Local energetic effects of amino acid replacements are often considered to have only a moderate influence on the backbone conformation of proteins or peptides. As these effects are difficult to determine experimentally, no comparison has yet been performed. However, knowledge of the influence of side chain mutations is essential in protein homology modeling and in optimizing biologically active peptide ligands in medicinal chemistry. Furthermore, the tool of N-methylation of peptides is of increasing importance for the design of peptidic drugs to gain oral availability or receptor selectivity. However, N-methylation is often accompanied by considerable population of cis-peptide bond structures, resulting in completely different conformations compared with the parent peptide. To retain a favored structure, it might be important to understand the effect of different side chains on the backbone conformation and to enable the introduction of an N-methylation at the right position without disturbing a biologically active conformation. In order to detect even small energetic effects due to side chain mutations, we employed a trick to investigate the structural equilibrium of a selected cyclic pentapeptide in which two conformations are equally populated. Very small energetic differences between both conformations could easily be determined experimentally by identifying shifts in the population of both isomers.