Bifunctional glycosylases, OGG1 for purines and NTH1 for pyrimidines, repair oxidized DNA bases via consecutive glycosylase and AP-lyase reactions, yet their catalytic relationships and lyase activity's biological relevance remain unresolved. Here, we solved crystal structures of archaeal and human Ogg1 and Nth1 captured in key damage-recognition and catalysis-ready states, complemented by ab initio molecular dynamics simulations of their complete reaction trajectories. We thereby define a unified catalytic mechanism for OGG1 and NTH1 conserved over three billion years, distinct from the canonical oxocarbenium-ion mechanism of monofunctional glycosylases. While divergent in their oxidized substrate recognition, their chemistry converged on ribose protonation and ring opening that precede the deglycosylation step. Acid-base catalysis mediated by a conserved aspartate-lysine dyad lowers the C-N bond cleavage barrier, while the excised 8-oxoG base in OGG1 or a conserved aspartate in NTH1 facilitates the AP-lyase reaction. Moreover, structures of human OGG1 bound to product DNA and to product DNA plus a potent small-molecule agonist F51, within the catalytic pocket, reveal that agonists accelerate enzyme turnover by promoting product release. Together, these findings clarify the catalytic logic of bifunctional glycosylases, enabling the development of chemical tools to interrogate lyase activity and therapeutics for oxidative damage in cancer and aging.

Exonuclease/endonuclease/phosphatase (EEP)-fold hydrolases are canonically monomeric phosphodiesterases exemplified by APE1, DNase I, and TDP2 nucleases. While EEP family domain containing protein 1 (EEPD1) acts in DNA stress responses, its proposed nuclease activities are enigmatic. Here, we integrate hybrid structural methods, evolution, biochemistry, cancer genomics, plus molecular and cell biology to define EEPD1 structure, assembly, and function at stalled DNA replication forks. Results imply EEPD1 surprisingly requires both unique EEP domain dimer and distinctive tandem Helix-hairpin-Helix [(HhH)2] domains to clamp double-stranded (ds) DNA at reversed DNA replication forks for fork protection. Small-angle X-ray Scattering (SAXS), crystal, and cryo-EM structures unveil an unprecedented tryptophan handshake dimer, conserved interface di-Trp-Pro pocket, and adjustable "wrist" enabling an open-closed conformational switch. EEPD1 dimer cooperatively binds complex dsDNA replication fork intermediates but alone lacks nuclease activity due to loss of key EEP catalytic residues during Metazoan evolution and atmospheric oxygen buildup. Instead, EEPD1 prevents nucleolytic degradation of reversed replication forks by MRE11. Furthermore, cancer bioinformatics support oxidative damage-dependent EEPD1 association as a significant modulator of overall patient survival. Collective findings uncover unexpected EEP dimer and fork protection function in clamping, not cleaving, reversed replication forks for metazoan oxidative stress responses controlling genome stability and cancer outcomes.

Unresolved DNA lesions trigger replication stress, forcing cancer cells to hijack DNA damage tolerance (DDT) networks, specifically translesion synthesis (TLS) and template switching, to sustain replication. While DDT prevents lethal fork collapse, error-prone TLS drives mutagenesis, tumor evolution, chemoresistance and radioresistance. Proliferating cell nuclear antigen post-translational modifications dynamically govern pathway selection. Cancer cells exploit this plasticity, creating actionable vulnerabilities such as postreplicative single-stranded DNA gaps. Emerging inhibitors targeting TLS polymerases, upstream regulators such as ubiquitin-specific peptidase 1 (USP1), and critical protein-protein interactions offer unprecedented opportunities for precision oncology. By integrating DDT inhibition with biomarkers such as homologous recombination deficiency and tumor mutational burden, we can drive synthetic lethality, sensitize tumors to genotoxic agents, suppress treatment-induced mutagenesis, and potentially enhance responses to immunotherapy.

Radiotherapy is one of the most important adjuvant treatment methods for gastric cancer (GC). However, radioresistance remains a major clinical obstacle. In this study, APOL2 is identified as a key player in promoting non-homologous end joining (NHEJ)-mediated double-strand break (DSB) repair and enhancing radioresistance in GC. Bioinformatics and clinical data revealed that high APOL2 expression is correlated with poor prognosis in GC patients. Functional experiments showed that APOL2 overexpression enhances genomic stability by accelerating DSB repair via the NHEJ pathway, while APOL2 knockout impairs repair capacity. Mechanistically, APOL2 binds to and stabilizes Ku80 by enhancing USP7-mediated deubiquitylation, thereby increasing Ku80 protein levels to promote NHEJ repair, ultimately conferring radioresistance. Moreover, high-throughput screening identified formononetin (FN) as a small molecule capable of disrupting the APOL2-Ku80 interaction, thereby restoring radiosensitivity in GC cells. Our findings underscore the role of APOL2 in mediating radioresistance through Ku80 stabilization and highlight FN as a potential therapeutic agent to counteract radioresistance in GC treatment.

TDP43 is an RNA/DNA-binding protein increasingly recognized for its role in neurodegenerative conditions, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As characterized by its aberrant nuclear export and cytoplasmic aggregation, TDP43 proteinopathy is a hallmark feature in over 95% of ALS/FTD cases, leading to detrimental cytosolic aggregates and a reduction in nuclear functionality in neurons. Building on our prior work linking TDP43 proteinopathy to the accumulation of DNA double-strand breaks (DSBs) in neurons, the present investigation uncovers a novel regulatory relationship between TDP43 and DNA mismatch repair (MMR) gene expression. Here, we show that TDP43 depletion or overexpression directly affects the expression of key MMR genes. Alterations include changes in MLH1, MSH2, MSH3, MSH6, and PMS2 levels across various primary cell lines, independent of their proliferative status. Our results specifically establish that TDP43 selectively influences the expression of MLH1 and MSH6 by influencing their alternative transcript splicing patterns and stability. We furthermore find that aberrant MMR gene expression is linked to TDP43 proteinopathy in two distinct ALS mouse models and in post-mortem brain and spinal cord tissues of ALS patients. Notably, MMR depletion resulted in the partial rescue of TDP43 proteinopathy-induced DNA damage and signaling. Moreover, bioinformatics analysis of the TCGA cancer database reveals significant associations between TDP43 expression, MMR gene expression, and mutational burden across multiple cancers. Collectively, our findings implicate TDP43 as a critical regulator of the MMR pathway and unveil its broad impact on the etiology of both neurodegenerative and neoplastic pathologies.

Cyclin-dependent kinase 5 (CDK5), long considered atypical and activated by non-cyclin cofactors in neurons, also functions in mitosis. To resolve its activation mechanism during mitosis, we determined the first high-resolution crystal structures of CDK5–cyclin B1 in apo and nucleotide-bound states. Contrary to AlphaFold predictions, we find CDK5 structures unexpectedly mirrors CDK1-cyclin B1 assembly and activation, establishing CDK5 as a bona fide mitotic kinase acting in parallel with CDK1. ### Competing Interest Statement The authors have declared no competing interest. Claudia Adams Barr Program for Innovative Basic Cancer Research JCRT Foundation grant National Institutes of Health, P01 CA092584, R35 CA2204300, R01 CA208244, R01 CA264900 Robert Welch Chemistry Chair, G-0010 Gray Foundation Team Science Award DOD Ovarian Cancer Award, W81XWH-15-0564/OC140632 Tina’s Wish Foundation V Foundation Award American Cancer Society, PF-23-1145928-01-DMC

Activating signal co-integrator complex 1 (ASCC1) acts with ASCC-ALKBH3 complex in alkylation damage responses. ASCC1 uniquely combines two evolutionarily ancient domains: nucleotide-binding K-Homology (KH) (associated with regulating splicing, transcriptional, and translation) and two-histidine phosphodiesterase (PDE) (associated with hydrolysis of cyclic nucleotide phosphate bonds). Germline mutations link loss of ASCC1 function to spinal muscular atrophy with congenital bone fractures 2 (SMABF2). Herein analysis of The Cancer Genome Atlas (TCGA) suggests ASCC1 RNA overexpression in certain tumors correlates with poor survival, Signatures 29 and 3 mutations, and genetic instability markers. We determined crystal structures of Alvinella pompejana (Ap) ASCC1 and Human (Hs) PDE domain revealing high resolution details and features conserved over 500 million years of evolution. Extending understanding of the KH domain Gly-X-X-Gly sequence motif, we define a novel structural Helix-Clasp-Helix (HCH) nucleotide binding motif and show ASCC1 sequence–specific binding to CGCG-containing RNA. The V-shaped PDE nucleotide binding channel has two His-Φ-Ser/Thr-Φ (HXT) motifs (Φ being hydrophobic) positioned to initiate cyclic phosphate bond hydrolysis. A conserved atypical active-site histidine torsion angle implies a novel PDE substrate. Flexible active site loop and arginine-rich domain linker appear regulatory. Small angle X-ray scattering (SAXS) revealed aligned KH-PDE RNA binding sites with limited flexibility in solution. Quantitative evolutionary bioinformatic analyses of disease and cancer-associated mutations support implied functional roles for RNA binding, phosphodiesterase activity, and regulation. Collective results inform ASCC1 roles in transactivation and alkylation damage responses, its targeting by structure-based inhibitors, and how ASCC1 mutations may impact inherited disease and cancer.

Non-B DNA G-quadruplex (G4) structures with guanine (G) runs of 2 to 4 repeats can trigger opposing experimental transcriptional impacts. Here, we used bioinformatic algorithms to comprehensively assess correlations of steady-state RNA transcript levels with all putative G4 sequence (pG4) locations genome-wide in three mammalian genomes and in normal and tumor human tissues. The human pG4-containing gene set displays higher expression levels than the set without pG4, supporting and extending some prior observations. pG4 enrichment at transcription start sites (TSSs) in human, but not chimpanzee and mouse genomes, suggests possible positive selection pressure for pG4 at human TSS, potentially driving genome rewiring and gene expression divergence between human and chimpanzee. Comprehensive bioinformatic analyses revealed lower pG4-containing gene set variability in humans and among different pG4 genes in tumors. As G4 stabilizers are under therapeutic consideration for cancer and pathogens, such distinctions between human normal and tumor G4s along with other species merit attention. Furthermore, in germline and cancer sequences, the most mutagenic pG4 mapped to regions promoting alternative DNA structures. Overall findings establish high pG4 at TSS as a human genome attribute statistically associated with robust well-coordinated transcription and reduced cancer transcriptome variation with implications for biology, model organisms, and medicine.

The pre-mRNA life cycle requires intron processing; yet, how intron-processing defects influence splicing and gene expression is unclear. Here, we find that TTDN1/MPLKIP, which is encoded by a gene implicated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to spliceosomal function. The conserved TTDN1 C-terminal region directly binds lariat debranching enzyme DBR1, whereas its N-terminal intrinsically disordered region (IDR) binds the intron-binding complex (IBC). TTDN1 loss, or a mutated IDR, causes significant intron lariat accumulation, as well as splicing and gene expression defects, mirroring phenotypes observed in NP-TTD patient cells. A Ttdn1-deficient mouse model recapitulates intron-processing defects and certain neurodevelopmental phenotypes seen in NP-TTD. Fusing DBR1 to the TTDN1 IDR is sufficient to recruit DBR1 to the IBC and circumvents the functional requirement for TTDN1. Collectively, our findings link RNA lariat processing with splicing outcomes by revealing the molecular function of TTDN1.

Abstract Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition. Using hydrogen deuterium exchange-mass spectrometry (HX-MS), biophysical, biochemical, computational and cellular assays, we found NVB is a non-competitive inhibitor of ATP hydrolysis. NVB sugar group deletion resulted in decreased potency and reduced HX-MS interactions, supporting a specific NVB binding orientation. Collective results revealed that NVB binds to an allosteric site to block DNA binding, both in vitro and in cells. Comparisons of The Cancer Genome Atlas (TCGA) tumors and matched controls implied that POLQ upregulation in tumors stems from its role in replication stress responses to increased cell proliferation: this can now be tested in fifteen tumor types by NVB blocking ssDNA-stimulation of ATPase activity, required for Polθ function at replication forks and DNA damage sites. Structural and functional insights provided in this study suggest a path for developing NVB derivatives with improved potency for Polθ inhibition by targeting ssDNA binding with entropically constrained small molecules.

RAD51C is an enigmatic predisposition gene for breast, ovarian, and prostate cancer. Currently, missing structural and related functional understanding limits patient mutation interpretation to homology-directed repair (HDR) function analysis. Here we report the RAD51C-XRCC3 (CX3) X-ray co-crystal structure with bound ATP analog and define separable RAD51C replication stability roles informed by its three-dimensional structure, assembly, and unappreciated polymerization motif. Mapping of cancer patient mutations as a functional guide confirms ATP-binding matching RAD51 recombinase, yet highlights distinct CX3 interfaces. Analyses of CRISPR/Cas9-edited human cells with RAD51C mutations combined with single-molecule, single-cell and biophysics measurements uncover discrete CX3 regions for DNA replication fork protection, restart and reversal, accomplished by separable functions in DNA binding and implied 5' RAD51 filament capping. Collective findings establish CX3 as a cancer-relevant replication stress response complex, show how HDR-proficient variants could contribute to tumor development, and identify regions to aid functional testing and classification of cancer mutations.

From initiation through progression, cancer cells are subjected to a magnitude of endogenous and exogenous stresses, which aid in their neoplastic transformation. Exposure to these classes of stress induces imbalance in cellular homeostasis and, in response, cancer cells employ informative adaptive mechanisms to rebalance biochemical processes that facilitate survival and maintain their existence. Different kinds of stress stimuli trigger epigenetic alterations in cancer cells, which leads to changes in their transcriptome and metabolome, ultimately resulting in suppression of growth inhibition or induction of apoptosis. Whether cancer cells show a protective response to stress or succumb to cell death depends on the type of stress and duration of exposure. A thorough understanding of epigenetic and molecular architecture of cancer cell stress response pathways can unveil a plethora of information required to develop novel anticancer therapeutics. The present view highlights current knowledge about alterations in epigenome and transcriptome of cancer cells as a consequence of exposure to different physicochemical stressful stimuli such as reactive oxygen species (ROS), hypoxia, radiation, hyperthermia, genotoxic agents, and nutrient deprivation. Currently, an anticancer treatment scenario involving the imposition of stress to target cancer cells is gaining traction to augment or even replace conventional therapeutic regimens. Therefore, a comprehensive understanding of stress response pathways is crucial for devising and implementing novel therapeutic strategies.

Whilst DNA repeat expansions cause numerous heritable human disorders, their origins and underlying pathological mechanisms are often unclear. We collated a dataset comprising 224 human repeat expansions encompassing 203 different genes, and performed a systematic analysis with respect to key topological features at the DNA, RNA and protein levels. Comparison with controls without known pathogenicity and genomic regions lacking repeats, allowed the construction of the first tool to discriminate repeat regions harboring pathogenic repeat expansions (DPREx). At the DNA level, pathogenic repeat expansions exhibited stronger signals for DNA regulatory factors (e.g. H3K4me3, transcription factor-binding sites) in exons, promoters, 5′UTRs and 5′genes but were not significantly different from controls in introns, 3′UTRs and 3′genes. Additionally, pathogenic repeat expansions were also found to be enriched in non-B DNA structures. At the RNA level, pathogenic repeat expansions were characterized by lower free energy for forming RNA secondary structure and were closer to splice sites in introns, exons, promoters and 5′genes than controls. At the protein level, pathogenic repeat expansions exhibited a preference to form coil rather than other types of secondary structure, and tended to encode surface-located protein domains. Guided by these features, DPREx ( http://biomed.nscc-gz.cn/zhaolab/geneprediction/# ) achieved an Area Under the Curve (AUC) value of 0.88 in a test on an independent dataset. Pathogenic repeat expansions are thus located such that they exert a synergistic influence on the gene expression pathway involving inter-molecular connections at the DNA, RNA and protein levels.

Microdeletions and gross deletions are important causes (~20%) of human inherited disease. Their genomic locations are strongly influenced by the local DNA sequence environment. Yet no systematic study has examined the generative mechanisms. Here, we obtained 42,098 pathogenic microdeletions and gross deletions from the Human Gene Mutation Database (HGMD) that together form a continuum of germline deletions ranging in size from 1 bp to 28,394,429 bp. We analyzed the sequence within 1-kb of the breakpoint junctions and found the frequencies of non-B DNA-forming repeats, GC content, and the presence of seven of 78 specific sequence motifs in the vicinity of pathogenic deletions correlated with deletion length for deletions of length ≤30 bp. Furthermore, we found the repeats of DR, GQ, and STR appear to be important for the formation of longer deletions (>30 bp) but not for the formation of shorter deletions (≤30 bp) and significantly (Chi-square test P-value < 2E-16) more microhomologies were identified in flanking short deletions than long deletions (length >30 bp). We provide evidence to support a functional distinction between microdeletions and gross deletions. A deletion length cut-off of 25-30 bp may serve as an objective means to functionally distinguish microdeletions from gross deletions.

Topoisomerase 1 (Top1) removes transcription-associated helical stress to suppress G4-formation and its induced recombination at genomic loci containing guanine-run containing sequences. Interestingly, Top1 binds tightly to G4 structures, and its inhibition or depletion can cause elevated instability at these genomic loci. Top1 is targeted by the widely used anti-cancer chemotherapeutic camptothecin (CPT) and its derivatives, which stabilize Top1 covalently attached on a DNA nick and prevent the re-ligation step. Here we investigated how CPT-resistance conferring Top1 mutants, which emerge in cancer patients and cells treated with CPT, affect G4-induced genomic instability in S. cerevisiae. We found that Top1 mutants form stable complexes with G4 DNA and that expression of Top1 cleavage-defective mutants but not a DNA-binding-defective mutant lead to significantly elevated instability at a G4-forming genomic locus. Elevated recombination rates were partly suppressed by their proteolytic removal by SPRTN homolog Wss1 SUMO-dependent metalloprotease in vivo. Furthermore, interaction between G4-DNA binding protein Nsr1, a homolog to clinically-relevant human nucleolin, and Top1 mutants lead to a synergistic increase in G4-associated recombination. These results in the yeast system are strengthened by our cancer genome data analyses showing that functionally detrimental mutations in Top1 correlate with an enrichment of mutations at G4 motifs. Our collective experimental and computational findings point to cooperative binding of Top1 cleavage-defective mutants and Nsr1 as promoting DNA replication blockage and exacerbating genomic instability at G4-motifs, thus complicating patient treatment.

Summary The pre-mRNA life cycle requires intron processing; yet, how intron processing defects influence splicing and gene expression is unclear. Here, we find TTDN1, which is frequently mutated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to the spliceosome. The conserved TTDN1 C-terminal region directly binds lariat debranching enzyme DBR1, while its N-terminal intrinsically disordered region (IDR) binds the intron binding complex (IBC). The IDR forms condensates in vitro and is needed for IBC interaction. TTDN1 loss causes significant intron lariat accumulation, as well as splicing and gene expression defects, mirroring phenotypes observed in NP-TTD patient cells. Ttdn1 Δ/Δ mice recapitulate intron processing defects and neurodevelopmental phenotypes seen in NP-TTD. A DBR1-IDR fusion recruits DBR1 to the IBC and circumvents the requirement for TTDN1, indicating this tethering role as its major molecular function. Collectively, our findings unveil key functional connections between lariat processing, splicing outcomes, and NP-TTD molecular pathology.

The massive amount of experimental DNA and RNA sequence information provides an encyclopedia for cell biology that requires computational tools for efficient interpretation. The ability to write and apply simple computing scripts propels the investigator beyond the boundaries of online analysis tools to more broadly interrogate laboratory experimental data and to integrate them with all available datasets to test and challenge hypotheses. Here we describe robust prototypic bash and C++ scripts with metrics and methods for validation that we have made publicly available to address the roles of non-B DNA-forming motifs in eliciting genetic instability and to query The Cancer Genome Atlas. Importantly, the methods presented provide practical data interpretation tools to examine fundamental relationships and to enable insights and correlations between alterations in gene expression patterns and patient outcome. The exemplary source codes described are simple and can be efficiently modified, elaborated, and applied to other relationships and areas of investigation.

Human genome stability requires efficient repair of oxidized bases, which is initiated via damage recognition and excision by NEIL1 and other base excision repair (BER) pathway DNA glycosylases (DGs). However, the biological mechanisms underlying detection of damaged bases among the million-fold excess of undamaged bases remain enigmatic. Indeed, mutation rates vary greatly within individual genomes, and lesion recognition by purified DGs in the chromatin context is inefficient. Employing super-resolution microscopy and co-immunoprecipitation assays, we find that acetylated NEIL1 (AcNEIL1), but not its non-acetylated form, is predominantly localized in the nucleus in association with epigenetic marks of uncondensed chromatin. Furthermore, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) revealed non-random AcNEIL1 binding near transcription start sites of weakly transcribed genes and along highly transcribed chromatin domains. Bioinformatic analyses revealed a striking correspondence between AcNEIL1 occupancy along the genome and mutation rates, with AcNEIL1-occupied sites exhibiting fewer mutations compared to AcNEIL1-free domains, both in cancer genomes and in population variation. Intriguingly, from the evolutionarily conserved unstructured domain that targets NEIL1 to open chromatin, its damage surveillance of highly oxidation-susceptible sites to preserve essential gene function and to limit instability and cancer likely originated ∼500 million years ago during the buildup of free atmospheric oxygen.

All tumors have DNA mutations, and a predictive understanding of those mutations could inform clinical treatments. However, 40% of the mutations are variants of unknown significance (VUS), with the challenge being to objectively predict whether a VUS is pathogenic and supports the tumor or whether it is benign. To objectively decode VUS, we mapped cancer sequence data and evolutionary trace (ET) scores onto crystallography and cryo-electron microscopy structures with variant impacts quantitated by evolutionary action (EA) measures. As tumors depend on helicases and nucleases to deal with transcription/replication stress, we targeted helicase-nuclease-RPA complexes: (1) XPB-XPD (within TFIIH), XPF-ERCC1, XPG, and RPA for transcription and nucleotide excision repair pathways and (2) BLM, EXO5, and RPA plus DNA2 for stalled replication fork restart. As validation, EA scoring predicts severe effects for most disease mutations, but disease mutants with low ET scores not only are likely destabilizing but also disrupt sophisticated allosteric mechanisms. For sites of disease mutations and VUS predicted to be severe, we found strong co-localization to ordered regions. Rare discrepancies highlighted the different survival requirements between disease and tumor mutations, as well as the value of examining proteins within complexes. In a genome-wide analysis of 33 cancer types, we found correlation between the number of mutations in each tumor and which pathways or functional processes in which the mutations occur, revealing different mutagenic routes to tumorigenesis. We also found upregulation of ancient genes including BLM, which supports a non-random and concerted cancer process: reversion to a unicellular, proliferation-uncontrolled, status by breaking multicellular constraints on cell division. Together, these genes and global analyses challenge the binary "driver" and "passenger" mutation paradigm, support a gradient impact as revealed by EA scoring from moderate to severe at a single gene level, and indicate reduced regulation as well as activity. The objective quantitative assessment of VUS scoring and gene overexpression in the context of functional interactions and pathways provides insights for biology, oncology, and precision medicine.

Arrival of the novel SARS-CoV-2 has launched a worldwide effort to identify both pre-approved and novel therapeutics targeting the viral proteome, highlighting the urgent need for efficient drug discovery strategies. Even with effective vaccines, infection is possible, and at-risk populations would benefit from effective drug compounds that reduce the lethality and lasting damage of COVID-19 infection. The CoV-2 MacroD-like macrodomain (Mac1) is implicated in viral pathogenicity by disrupting host innate immunity through its mono (ADP-ribosyl) hydrolase activity, making it a prime target for antiviral therapy. We therefore solved the structure of CoV-2 Mac1 from non-structural protein 3 (Nsp3) and applied structural and sequence-based genetic tracing, including newly determined A. pompejana MacroD2 and GDAP2 amino acid sequences, to compare and contrast CoV-2 Mac1 with the functionally related human DNA-damage signaling factor poly (ADP-ribose) glycohydrolase (PARG). Previously, identified targetable features of the PARG active site allowed us to develop a pharmacologically useful PARG inhibitor (PARGi). Here, we developed a focused chemical library and determined 6 novel PARGi X-ray crystal structures for comparative analysis. We applied this knowledge to discovery of CoV-2 Mac1 inhibitors by combining computation and structural analysis to identify PARGi fragments with potential to bind the distal-ribose and adenosyl pockets of the CoV-2 Mac1 active site. Scaffold development of these PARGi fragments has yielded two novel compounds, PARG-345 and PARG-329, that crystallize within the Mac1 active site, providing critical structure-activity data and a pathway for inhibitor optimization. The reported structural findings demonstrate ways to harness our PARGi synthesis and characterization pipeline to develop CoV-2 Mac1 inhibitors targeting the ADP-ribose active site. Together, these structural and computational analyses reveal a path for accelerating development of antiviral therapeutics from pre-existing drug optimization pipelines.