RecQ Research Articles

Synergistic protection of nascent DNA at stalled forks by MSANTD4 and BRCA1/2-RAD51.

The regressed arms of reversed replication forks exhibit structural similarities to one-ended double-stranded breaks and need to be protected against uncontrolled nucleolytic degradation. Here, we identify MSANTD4 (Myb/SANT-like DNA-binding domain-containing protein 4), a functionally uncharacterized protein that uniquely counters the replication protein A (RPA)-Bloom (BLM)/Werner syndrome helicase (WRN)-DNA replication helicase/nuclease 2 (DNA2) complex to safeguard reversed replication forks from detrimental degradation, independently of the breast cancer susceptibility proteins (BRCA1/2)-DNA repair protein RAD51 pathway. MSANTD4 specifically interacts with the junctions between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in DNA substrates harboring a 3' overhang, which resemble the structural features of regressed arms processed by WRN-DNA2. This DNA-binding capability allows MSANTD4 to accumulate at reversed forks, strategically antagonizing the RPA-BLM/WRN-DNA2 complex by impeding its access to the ssDNA-dsDNA junction of the regressed arms. Loss of MSANTD4 exacerbates genome instability induced by replication stress in BRCA1/2-deficient cells. Our findings unveil a collaborative defense mechanism orchestrated by MSANTD4 and BRCA1/2-RAD51, effectively counteracting nucleolytic attacks on the regressed arms and synergistically preserving the integrity of reversed forks.

Functions of the Bloom Syndrome Helicase N-terminal Intrinsically Disordered Region.

Bloom Syndrome helicase (Blm) is a RecQ family helicase involved in DNA repair, cell-cycle progression, and development. Pathogenic variants in human BLM cause the autosomal recessive disorder Bloom Syndrome, characterized by predisposition to numerous types of cancer. Prior studies of Drosophila Blm mutants lacking helicase activity or protein have shown sensitivity to DNA damaging agents, defects in repairing DNA double-strand breaks (DSBs), female sterility, and improper segregation of chromosomes in meiosis. Blm orthologs have a well conserved and highly structured RecQ helicase domain, but more than half of the protein, particularly in the N-terminus, is predicted to be intrinsically disordered. Because this region is poorly conserved across metazoa, we compared closely related species to identify regions of conservation that might be associated with important functions. We deleted two Drosophila-conserved regions in D. melanogaster using CRISPR/Cas9 gene editing and assessed the effects on several Blm functions. Each deletion had distinct effects. Deletion of either conserved region 1 (CR1) or conserved region 2 (CR2) compromised DSB repair through synthesis-dependent strand annealing and resulted in increased mitotic crossovers. In contrast, CR2 is critical for embryonic development but CR1 is less important. Loss of CR1 leads to defects in meiotic crossover designation and patterning but does not impact meiotic chromosome segregation, whereas deletion of CR2 does not result in significant meiotic defects. Thus, while the two regions have overlapping functions, there are distinct roles facilitated by each. These results provide novel insights into functions of the N-terminal region of Blm helicase.

Novel FANCI and RAD54B Variants and the Observed Clinical Outcomes in a Hungarian Melanoma Cohort.

Accumulating evidence suggests that inherited melanoma is not rare and approx. one in seven individuals with melanoma has clinically relevant hereditable cancer-predisposing and/or -susceptibility variant(s). Concerning its germline genetic background, genetic screening aims to identify either variants of predisposing genes with high penetrance or variants of susceptibility genes with medium or low penetrance. However, less attention is paid to genetic testing of germline variants of genes influencing patients' survival outcomes or enhancing the design of new therapies. We aimed to investigate whether the germline genetic background of a Hungarian melanoma cohort (n = 17) contains any pathogenic or likely pathogenic variants of the BRCA2, POLE, WRN, FANCI, PALB2, and RAD54B genes and if the presence of these variants correlate with the clinical findings of the patients, including the advanced stage of melanoma, poor prognosis, and poor survival. We identified three novel variants in the FANCI gene and one novel variant in the RAD54B gene. We detected rapid disease progression, unfavorable outcome, and therapeutic resistance in the patient carrying the likely pathogenic FANCI variant. Our study highlights the importance of screening germline variants of genes influencing melanoma progression, therapy resistance, and survival of patients.

Novel pof1 mutation suppresses the sensitivity to DNA replication inhibitor in fission yeast RecQ helicase mutant.

Homologous recombination is vital for DNA double-strand break repair. Dysfunction in homologous recombination can lead to cell death, mutations, and cancer. In fission yeast (Schizosaccharomyces pombe), RecQ helicase Rqh1 resolves recombination intermediates. We found that rqh1-hd strain impaired growth in media containing hydroxyurea and thiabendazole. Using this condition, we identified a novel pof1 mutation (pof1-A81T) that suppress the poor growth of the rqh1-hd strain on the plate containing hydroxyurea and thiabendazole. Compared to rqh1-hd, rqh1-hd pof1-A81T cells displayed reduced Replication Protein A foci on chromosome bridges after hydroxyurea treatment. This suggests that pof1-A81T mutation suppresses the accumulation of recombination intermediates in hydroxyurea-treated rqh1-hd cells. Additionally, pof1-A81T mutation rescued the segregation defect of nucleolar protein Gar2 observed in hydroxyurea-treated rqh1-hd cells, potentially by mitigating recombination intermediate accumulation in rDNA. These results suggest that the pof1-A81T mutation suppresses the accumulation of recombination intermediates, particularly in rDNA, and alleviates the rqh1 deficiency phenotype in S. pombe.

Application and research progress of synthetic lethality in the development of anticancer therapeutic drugs.

With the tremendous success of the PARP inhibitor olaparib in clinical practice, synthetic lethality has become an important field for the discovery and development of anticancer drugs. More and more synthetic lethality targets have been discovered with the rapid development of biotechnology in recent years. Currently, many drug candidates that were designed and developed on the basis of the concept of synthetic lethality have entered clinical trials. Taking representative synthetic lethal targets Poly ADP-ribose polymerase 1 (PARP1), Werner syndrome helicase (WRN) and protein arginine methyltransferase 5 (PRMT5) as examples, this article briefly discusses the application and research progress of synthetic lethality in the development of anticancer drugs.

Quinazoline derivatives inhibit cell growth of prostate cancer as a WRN helicase dependent manner by regulating DNA damage repair and microsatellite instability

WRN helicase is a crucial target of synthetic death in cancer and has a unique advantage in the treatment of microsatellite unstable cancers. Our previous studies have found that quinazoline derivatives showed the WRN-dependent antiproliferative activity. In this study, a series of new quinazoline derivatives were designed and synthesized by optimizing the structure, and evaluating the targeting and sensitivity to WRN helicase. Cell growth inhibition experiments on WRN overexpressing PC3 cells (PC3-WRN) showed that the antiproliferative activity of some compounds was significantly dependent on WRN helicase. Moreover, the antitumor activity of 9in vivo was significantly decreased in the nude mouse model constructed with WRN knockdown PC3 cells (PC3-shWRN) compare (P < 0.01) to the control group. The molecular docking and CETSA results showed that 9 directly binds to WRN protein. Mechanism studies have confirmed that 9 targeted WRN, and may affect the binding between WRN and other key regulators, to destroy the repair function and regulate genomic stability. In addition, 9 also has suitable pharmacokinetic parameters and low toxicity in vivo. This result indicates that the quinazoline derivative 9 could be a novel WRN function inhibitor for the treatment of prostate cancer.

Characterization and organization of telomeric-linked helicase (tlh) gene families in Fusarium oxysporum.

Telomere-linked RecQ helicase (tlh) genes have been reported in several fungi and a choanoflagellate in the regions adjacent to the terminal telomere repeats. In this study, we explored the Telomere-linked RecQ helicase (tlh) genes in four strains of Fusarium oxysporum, offering new insights into their genomic structure, functional motifs, and impact on chromosomal ends. We conducted a comprehensive analysis, comparing the tlh genes of F. oxysporum with those previously identified in other organisms and uncovering significant similarities. Through comparative genomics, we identified conserved protein motifs across these genes, including a TLH domain, C2H2, and RecQ helicase motifs. Our phylogenetic analysis positions the F. oxysporum tlh genes in a cluster with other known tlhs, suggesting a shared evolutionary origin. Mutation analysis revealed a relatively low level of deleterious mutations in tlh gene paralogs, with a notable proportion of full-size structures maintained across strains. Analysis of subtelomeric sequences indicates that a region with almost identical sequences flanks the majority of chromosome ends, termed tlh-containing region (TLHcr), across these strains. The presence of TLHcrs at chromosome ends, either as single entities or in arrays, underscores their potential role in telomere function and genome stability. Our findings provide a detailed examination of tlh genes in four strains of F. oxysporum, laying the groundwork for future studies on their biological significance and evolutionary history in fungal genomes.

Design, synthesis, and structure−activity relationship studies of triazolo-pyrimidine derivatives as WRN inhibitors for the treatment of MSI tumors

Werner syndrome RecQ helicase (WRN), a member of the RecQ helicase family, has recently been identified as a synthetic lethal target in microsatellite instability (MSI) tumors. The triazolo-pyrimidine compound HRO761 is the first WRN inhibitor to enter clinical trials, but research on this scaffold remains limited. Here, we designed a series of derivatives to systematically study the structure-activity relationship (SAR) of triazolo-pyrimidine scaffolds, leading to the discovery of compound S35. S35 exhibited excellent WRN helicase inhibitory activity (ADP-Glo kinase assay IC50 = 16.1 nM, fluorometric helicase assay IC50 = 23.5 nM). Additionally, S35 exhibited excellent cellular selectivity, with antiproliferative activity against multiple MSI cell lines (GI50 = 36.4−306 nM), while the GI50 values for multiple microsatellite stability (MSS) cell lines were greater than 20,000 nM. Furthermore, we observed that compound S35 induced DNA damage and caused G2/M cell cycle arrest in MSI cells, which did not occur in MSS cells. S35 demonstrated favorable oral pharmacokinetic properties, with oral administration resulting in dose-dependent tumor growth inhibition in the SW48 xenograft model. These findings provide a promising outlook for the development of WRN inhibitors for the treatment of MSI tumors.

Germline Variants in DNA Interstrand-Cross Link Repair Genes May Contribute to Increased Susceptibility for Serrated Polyposis Syndrome.

Serrated polyposis syndrome (SPS) is characterized by the development of multiple colorectal serrated polyps and increased predisposition to colorectal cancer (CRC). However, the molecular basis of SPS, especially in cases presenting family history of SPS and/or polyps and/or CRC in first-degree relatives (SPS-FHP/CRC), is still poorly understood. In a previous study, we proposed the existence of two molecular entities amongst SPS-FHP/CRC families, proximal/whole-colon and distal SPS-FHP/CRC, according to the preferential location of lesions and somatic events involved in tumor initiation. In the present study, we aimed to investigate these distinct subgroups of SPS patients in a larger cohort at the germline level and to identify the genetic defects underlying an inherited susceptibility for these two entities. Next-generation sequencing was performed using multigene analysis with a custom-designed panel in a Miseq platform in 60 SPS patients (with and without/unknown FHP/CRC). We found germline pathogenic variants in 6/60 patients (ATM, FANCM, MITF, RAD50, RAD51C, and RNF43). We also found variants of unknown significance (VUS), with prediction of probable damaging effect in 23/60 patients (ATM, BLM, BRCA1, FAN1, ERCC2, ERCC3, FANCA, FANCD2, FANCL, MSH2, MSH6, NTHL1, PALB2, PDGFRA, PMS2, PTCH1, RAD51C, RAD51D, RECQL4, TSC2, WRN, and XRCC5 genes). Most variants were detected in gene coding for proteins of the Fanconi Anemia (FA) pathway involved in the DNA Interstrand-Cross Link repair (ICLR). Notably, variants in ICLR genes were significantly more frequent in the proximal/whole-colon than in the distal subgroup [15/44 (34%) vs 1/16 (6%), p = 0.025], as opposed to the non-ICLR genes that were slightly more frequent in the distal group [8/44 (18%) vs. 5/16 (31%), p > 0.05]. Germline defects in the DNA-ICLR genes may contribute to increased serrated colorectal polyps/carcinoma risk in SPS patients, particularly in proximal/whole-colon SPS. The inclusion of DNA-ICLR genes in the genetic diagnosis of SPS patients, mainly in those with proximal/whole-colon lesions, should be considered and validated by other studies. In addition, patients with germline defects in the DNA-ICLR genes may be more sensitive to treatment with platinum-based therapeutics, which can have implications in the clinical management of these patients.

Werner syndrome RECQ helicase participates in and directs maintenance of the protein complexes of constitutive heterochromatin in proliferating human cells.

Werner syndrome of premature aging is caused by mutations in the WRN RECQ helicase/exonuclease, which functions in DNA replication, repair, transcription, and telomere maintenance. How the loss of WRN accelerates aging is not understood in full. Here we show that WRN is necessary for optimal constitutive heterochromatin levels in proliferating human fibroblasts. Locally, WRN deficiency derepresses SATII pericentromeric satellite repeats but does not reduce replication fork progression on SATII repeats. Globally, WRN loss reduces a subset of protein-protein interactions responsible for the organization of constitutive heterochromatin in the nucleus, namely, the interactions involving Lamin B1 and Lamin B receptor, LBR. Both the mRNA level and subcellular distribution of LBR are affected by WRN deficiency, and unlike the former, the latter phenotype does not require WRN catalytic activities. The phenotypes of heterochromatin disruption seen in WRN-deficient proliferating fibroblasts are also observed in WRN-proficient fibroblasts undergoing replicative or oncogene-induced senescence. WRN interacts with histone deacetylase 2, HDAC2; WRN/HDAC2 association is mediated by heterochromatin protein alpha, HP1α, and WRN complexes with HP1α and HDAC2 are downregulated in senescing cells. The data suggest that the effect of WRN loss on heterochromatin is separable from senescence program, but mimics at least some of the heterochromatin changes associated with it.

Comprehensive in silico characterization of Arabidopsis thaliana RecQl helicases through structure prediction and molecular dynamics simulations

Helicases are ubiquitous enzymes with specific functions that contribute to almost all nucleic acid metabolic processes. The RecQ helicase family is essential for integrity in all organisms through DNA replication, repair, and recombination. This study investigated five RecQ-like helicases in Arabidopsis thaliana (AtRecQl) that exhibit diverse structural and physiochemical attributes and functions. Cis-regulatory element analysis identified stress, hormone, cell cycle, and development-responsive modules involved in various events in plant growth and development. Gene ontology analysis revealed that the five AtRecQl were associated with various cellular components, molecular functions, and biological processes. Protein-protein interaction analysis also implicated some in various abiotic stress processes. Structural analysis and molecular dynamics (MD) simulations were performed to examine conformational stability through root means square deviation and radius of gyration, showing stable AtRecQl protein structures. Free energy landscape analysis validated thermodynamically stable structures throughout the MD simulation. Principle component analysis and probability density functions from MD simulations provided satisfactory structural variational data for the complexes and limited coordinate movements. These insights might greatly benefit future studies.

Expression patterns of Arabidopsis thaliana RecQ-like (AtRecQl) genes and the roles of AtRecQl2 and AtRecQl3 in response to abiotic stress.

Helicases are involved in almost every nucleic acid metabolism process. Within this family, RecQ helicase proteins protect genome integrity across all organisms through DNA recombination, repair, and replication. This study focused on five Arabidopsis thaliana RecQ-like (AtRecQl) genes with diverse functionalities. Analysis of ProAtRecQl: GUS expression during vegetative and reproductive development stages revealed organ- and tissue-specific patterns. Changes in AtRecQls transcript levels in response to abiotic stressors suggest their involvement in diverse stimuli responses. Notably, germination and growth rates were lower in atrecql2 and atrecql3 mutants under various salt concentrations and cold conditions. These findings indicate that AtRecQl2 and AtRecQl3 act as positive regulators of abiotic stress tolerance during the germinative and postgerminative phases.

Switch-like phosphorylation of WRN integrates end-resection with RAD51 metabolism at collapsed replication forks.

Replication-dependent DNA double-strand breaks are harmful lesions preferentially repaired by homologous recombination (HR), a process that requires processing of DNA ends to allow RAD51-mediated strand invasion. End resection and subsequent repair are two intertwined processes, but the mechanism underlying their execution is still poorly appreciated. The WRN helicase is one of the crucial factors for end resection and is instrumental in selecting the proper repair pathway. Here, we reveal that ordered phosphorylation of WRN by the CDK1, ATM and ATR kinases defines a complex regulatory layer essential for correct long-range end resection, connecting it to repair by HR. We establish that long-range end resection requires an ATM-dependent phosphorylation of WRN at Ser1058 and that phosphorylation at Ser1141, together with dephosphorylation at the CDK1 site Ser1133, is needed for the proper metabolism of RAD51 foci and RAD51-dependent repair. Collectively, our findings suggest that regulation of WRN by multiple kinases functions as a molecular switch to allow timely execution of end resection and repair at replication-dependent DNA double-strand breaks.

USP50 suppresses alternative RecQ helicase use and deleterious DNA2 activity during replication

Mammalian DNA replication relies on various DNA helicase and nuclease activities to ensure accurate genetic duplication, but how different helicase and nuclease activities are properly directed remains unclear. Here, we identify the ubiquitin-specific protease, USP50, as a chromatin-associated protein required to promote ongoing replication, fork restart, telomere maintenance, cellular survival following hydroxyurea or pyridostatin treatment, and suppression of DNA breaks near GC-rich sequences. We find that USP50 supports proper WRN-FEN1 localisation at or near stalled replication forks. Nascent DNA in cells lacking USP50 shows increased association of the DNA2 nuclease and RECQL4 and RECQL5 helicases and replication defects in cells lacking USP50, or FEN1 are driven by these proteins. Consequently, suppression of DNA2 or RECQL4/5 improves USP50-depleted cell resistance to agents inducing replicative stress and restores telomere stability. These data define an unexpected regulatory protein that promotes the balance of helicase and nuclease use at ongoing and stalled replication forks.

PROTAC-mediated conditional degradation of the WRN helicase as a potential strategy for selective killing of cancer cells with microsatellite instability

Multiple studies have demonstrated that cancer cells with microsatellite instability (MSI) are intolerant to loss of the Werner syndrome helicase (WRN), whereas microsatellite-stable (MSS) cancer cells are not. Therefore, WRN represents a promising new synthetic lethal target for developing drugs to treat cancers with MSI. Given the uncertainty of how effective inhibitors of WRN activity will prove in clinical trials, and the likelihood of tumours developing resistance to WRN inhibitors, alternative strategies for impeding WRN function are needed. Proteolysis-targeting chimeras (PROTACs) are heterobifunctional small molecules that target specific proteins for degradation. Here, we engineered the WRN locus so that the gene product is fused to a bromodomain (Bd)-tag, enabling conditional WRN degradation with the AGB-1 PROTAC specific for the Bd-tag. Our data revealed that WRN degradation is highly toxic in MSI but not MSS cell lines. In MSI cells, WRN degradation caused G2/M arrest, chromosome breakage and ATM kinase activation. We also describe a multi-colour cell-based platform for facile testing of selective toxicity in MSI versus MSS cell lines. Together, our data show that a degrader approach is a potentially powerful way of targeting WRN in MSI cancers and paves the way for the development of WRN-specific PROTAC compounds.

Single-strand DNA-binding protein suppresses illegitimate recombination in Escherichia coli, acting in synergy with RecQ helicase

Single-strand DNA-binding proteins SSB/RPA are ubiquitous and essential proteins that bind ssDNA in bacteria/eukaryotes and coordinate DNA metabolic processes such as replication, repair, and recombination. SSB protects ssDNA from degradation by nucleases, while also facilitating/regulating the activity of multiple partner proteins involved in DNA processes. Using Spi− assay, which detects aberrantly excised λ prophage from the E. coli chromosome as a measure of illegitimate recombination (IR) occurrence, we have shown that SSB inhibits IR in several DSB resection pathways. The conditional ssb-1 mutation produced a higher IR increase at the nonpermissive temperature than the recQ inactivation. A double ssb-1 recQ mutant had an even higher level of IR, while showing reduced homologous recombination (HR). Remarkably, the ssb gene overexpression complemented recQ deficiency in suppressing IR, indicating that the SSB function is epistatic to RecQ. Overproduced truncated SSBΔC8 protein, which binds to ssDNA, but does not interact with partner proteins, only partially complemented recQ and ssb-1 mutations, while causing an IR increase in otherwise wild-type bacteria, suggesting that ssDNA binding of SSB is required but not sufficient for effective IR inhibition, which rather entails interaction with RecQ and likely some other protein(s). Our results depict SSB as the main genome caretaker in E. coli, which facilitates HR while inhibiting IR. In enabling high-fidelity DSB repair under physiological conditions SSB is assisted by RecQ helicase, whose activity it controls. Conversely, an excess of SSB renders RecQ redundant for IR suppression.

Chromosomal breakage and sister chromatid exchange analysis in breast cancer patients with heterozygous BLM gene variants

Abstract Objectives BLM, a member of the RecQ helicase family, plays an important role in DNA repair, and its biallelic mutations cause autosomal recessive Bloom syndrome, a disease characterized by elevated levels of sister chromatid exchange (SCE) in affected individuals and hereditary cancer susceptibility in carriers. This study aims to investigate genomic instability in breast cancer patients carrying heterozygous variants in the BLM gene. Methods Spontaneous chromosome breakage count and SCE counting were performed on newly drawn blood cultures, both spontaneous and stimulated. The spontaneous breakage count was conducted alongside control samples. In SCE analysis, 0–10 per metaphase was considered normal, 10–40 borderline, and counts above 40 were considered high. Results The study included 26 patients and one healthy control at each session. The clinical and pathological characteristics of the patients were evaluated. The analyses revealed borderline-level increased SCE rates in only one patient. No increase in spontaneous breakage count or SCE analysis was observed in other individuals compared to controls. Conclusions Increased genomic instability was not observed in the analyzed patient group. These results can lead to multiple interpretations. The variants carried in the BLM gene in the patient group may be of low pathogenicity, or increased instability compared to controls may not be necessary for heterozygous variants. Additionally, our patient group may not have been exposed to a genotoxic effect causing genomic instability. These results could also indicate a favorable position in terms of avoiding chemotherapy and radiotherapy complications.

Rare Germline Variants in DNA Repair Genes Detected in BRCA-Negative Finnish Patients with Early-Onset Breast Cancer.

Breast cancer is the most common malignancy, with a mean age of onset of approximately 60 years. Only a minority of breast cancer patients present with an early onset at or before 40 years of age. An exceptionally young age at diagnosis hints at a possible genetic etiology. Currently, known pathogenic genetic variants only partially explain the disease burden of younger patients. Thus, new knowledge is warranted regarding additional risk variants. In this study, we analyzed DNA repair genes to identify additional variants to shed light on the etiology of early-onset breast cancer. Germline whole-exome sequencing was conducted in a cohort of 63 patients diagnosed with breast cancer at or before 40 years of age (median 33, mean 33.02, range 23-40 years) with no known pathogenic variants in BRCA genes. After filtering, all detected rare variants were sorted by pathogenicity prediction scores (CADD score and REVEL) to identify the most damaging genetic changes. The remaining variants were then validated by comparison to a validation cohort of 121 breast cancer patients with no preselected age at cancer diagnosis (mean 51.4 years, range 28-80 years). Analysis of novel exonic variants was based on protein structure modeling. Five novel, deleterious variants in the genes WRN, RNF8, TOP3A, ERCC2, and TREX2 were found in addition to a splice acceptor variant in RNF4 and two frameshift variants in EXO1 and POLE genes, respectively. There were also multiple previously reported putative risk variants in other DNA repair genes. Taken together, whole-exome sequencing yielded 72 deleterious variants, including 8 novel variants that may play a pivotal role in the development of early-onset breast cancer. Although more studies are warranted, we demonstrate that young breast cancer patients tend to carry multiple deleterious variants in one or more DNA repair genes.

WRN Helicase: Is There More to MSI-H than Immunotherapy?

In this issue, Picco and colleagues provide further evidence that WRN inhibitors are synthetically lethal in microsatellite instability-high (MSI-H) cancers and function by blocking the helicase domain of select WRN residues. They demonstrate that WRN inhibitors may be even more effective in a subset of MSI-high tumors with (TA)n repeat expansions, which represents a possible strategy in clinical development. See related article by Picco et al., p. 1457 (1).

High-Throughput Covalent Modifier Screening with Acoustic Ejection Mass Spectrometry.

Interests in covalent drugs have grown in modern drug discovery as they could tackle challenging targets traditionally considered "undruggable". The identification of covalent binders to target proteins typically involves directly measuring protein covalent modifications using high-resolution mass spectrometry. With a continually expanding library of compounds, conventional mass spectrometry platforms such as LC-MS and SPE-MS have become limiting factors for high-throughput screening. Here, we introduce a prototype high-resolution acoustic ejection mass spectrometry (AEMS) system for the rapid screening of a covalent modifier library comprising ∼10,000 compounds against a 50 kDa-sized target protein─Werner syndrome helicase. The screening samples were arranged in a 1536-well format. The sample buffer containing high-concentration salts was directly analyzed without any cleanup steps, minimizing sample preparation efforts and ensuring protein stability. The entire AEMS analysis process could be completed within a mere 17 h. An automated data analysis tool facilitated batch processing of the sample data and quantitation of the formation of various covalent protein-ligand adducts. The screening results displayed a high degree of fidelity, with a Z' factor of 0.8 and a hit rate of 2.3%. The identified hits underwent orthogonal testing in a biochemical activity assay, revealing that 75% were functional antagonists of the target protein. Notably, a comparative analysis with LC-MS showcased the AEMS platform's low risk of false positives or false negatives. This innovative platform has enabled robust high-throughput covalent modifier screening, featuring a 10-fold increase in library size and a 10- to 100-fold increase in throughput when compared with similar reports in the existing literature.