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. 2025 Nov 17;10(47):57741–57752. doi: 10.1021/acsomega.5c09571

Inhibition of Clostridioides difficile-Specific DNA Adenine Methyltransferase CamA by Analogs of S‑Adenosyl‑l‑methionine

Jujun Zhou †, Youchao Deng ‡, Dan Yu †, Taraneh Hajian §, Masoud Vedadi §,∥, Xing Zhang †, Robert M Blumenthal ⊥, Rong Huang ‡,*, Xiaodong Cheng †,*
PMCID: PMC12676344  PMID: 41358085

Abstract

Epigenetically targeted therapies, especially those inhibiting S-adenosyl-l-methionine (SAM)-dependent methylations of DNA, mRNA, and histones, have advanced rapidly in cancer treatment. However, these therapies remain underexplored for antibiotic development, despite the growing threat of antimicrobial resistance. Here, we screened a focused library of SAM analogs against the DNA adenine methyltransferase CamA specific to the enteric pathogen Clostridioides difficile. At the same time, we examined six other adenine methyltransferases, including two bacterial DNA methyltransferases and four human RNA methyltransferases having distinct RNA substrates. Compound 113 selectively inhibited CamA (IC50 = 0.15 μM). In addition, compound 67 inhibited Caulobacter crescentus CcrM (IC50 = 1.8 μM), which has orthologs present in pathogens such as Brucella, while compounds 77 and 37 inhibited the human RNA methyltransferase complexes MettL3-MettL14 and MettL5-Trm112, respectively, at 7–8 μM concentrations. These results provide chemical probes for exploring the role of CamA in sporulation and colonization with potential as antivirulence agents against C. difficile infection. Our study also introduces the first chemical probes for inhibiting bacterial CcrM and human MettL5, each of which plays key roles in their respective hosts.

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Introduction

Clostridioides difficile infection (CDI) is a leading cause of community-associated infection in children and of hospital-acquired diarrhea in older patients. − CDI can have life-threatening consequences and is considered to be an urgent threat to public health. According to a recent report by the Centers for Disease Control and Prevention, the incidence rate of CDI increases with age, is higher in women than in men, and is more prevalent among Caucasian individuals compared to other racial groups. At present, no vaccine is available, and one of the major treatmentsFecal Microbiota Transplantation (FMT)is still in the process of being characterized and optimized. , Phage therapy and monoclonal antibody treatments are also under development. In addition, several small-molecule therapeutics targeting C. difficile have progressed to phase I–III clinical trials. These include CRS3123 (a tRNAMet synthetase inhibitor), ibezapolstat (a DNA polymerase IIIC inhibitor), , and two DNA minor groove bindersMGB-BP-3 and ridinilazole. Thus, while several potentially beneficial therapeutic approaches are under active development, the fact remains that there is no consistently efficacious C. difficile treatment at present. Furthermore, antivirulence drugswhich target the pathogenesis rather than the growth of a bacteriumare less likely to select for antibiotic resistance, and can synergize with more traditional antibiotics.

Genomic analyses of sequenced C. difficile strains have identified a DNA adenine methyltransferase (CamA) unique to, and widespread within, this species. , Unlike canonical bacterial restriction-modification systems, which couple a methyltransferase (MTase) with a restriction endonuclease, CamA is an “orphan” MTase, lacking a cognate restriction enzyme partner. CamA is essential for normal sporulation and is required for persistent CDI in an animal model, but not for C. difficile growth. Because recurrent CDI is often associated with antibiotic use within the prior 12 weeks, , and because C. difficile itself is increasingly antibiotic resistant, , there is a pressing need for nonconventional therapeutic agents to effectively control CDI, and CamA appears to be a potentially valuable target.

CamA is a member of the S-adenosyl-l-methionine (SAM)-dependent methyltransferase (MTase) family, which catalyzes methyl transfers to a broad range of substrates, including DNA, RNA, proteins, and small molecules such as histamine and thiopurine. SAM is a metabolite synthesized in nearly all species, , with the exceptions being obligate parasites that obtain SAM from their hosts. , SAM is by far the major methyl donor in transmethylation reactions, , and also participates in radical SAM reactions, − polyamine biosynthesis, and SAM-sensing riboswitch regulation. − Given the importance of nucleic acid and histone methylation in diseases such as cancer and immune disorders, − SAM-dependent MTases have become attractive therapeutic targets. − SAM analogs are versatile tools for probing and inhibiting MTase activity, − with some of them already in use in clinical trials or preclinical studies that target human epigenetic enzymes , and viral RNA MTases. −

We previously demonstrated that several SAM analogs, originally designed as selective inhibitors of human protein arginine MTases (PRMTs) ,, and of the histone H3 lysine 79 MTase DOT1L, also inhibit CamA in vitro at low micromolar concentrations. Subsequent optimization of adenosine analogs yielded CamA inhibitors with half-maximal inhibitory concentration (IC50) values in the submicromolar range. ,

In the present study, we leveraged a focused library of SAM analogs to screen against three bacterial DNA adenine MTases: our primary target of interest, C. difficile CamA, which modifies the AT-rich CAAAA A sequence, Escherichia coli Dam, which methylates G A TC sites and is widespread among γ-Proteobacteria, , and Caulobacter crescentus cell cycle-regulated DNA MTase (CcrM), which targets the G A nTC motif (n = any nucleotide). While C. crescentus is not itself a human pathogen, CcrM orthologs play key regulatory roles in other α-Proteobacteria, including pathogens like Brucella , and the colorectal cancer-associated Fusobacterium nucleatum. To test for selectivity, we also screened this SAM analogue library against four human RNA adenine MTases, each acting on distinct RNA substrates. Finally, we discuss the potential of small-molecule therapeutics targeting C. difficile in relation to CamA inhibition.

Results

A Library of SAM Analogs

We began with a set of pan-PRMT inhibitors , and a focused library of adenosine analogs, which led to the identification of a PRMT4-selective inhibitor with an IC50 < 0.5 nM, and ultimately yielded a curated library of 114 compounds (Figure ). All compounds share a 5′-thioadenosine (S-Ad) scaffold with a flexible 2–3 carbon linker (Figure A–J). Compounds in groups A and B incorporate a moiety that resembles the guanidino moiety of the arginine side chain (Figure A,B), while groups C and D replace the guanidino with a urea moiety (Figure C,D). Groups E and F feature a benzylamine with a single hydrogen bond donor (NH) (Figure E,F). Group G compounds contain a dimethoxyquinazolin-4-amine moiety (Figure G), group H includes a phenylether moiety (Figure H), and group I introduces a phenylpropane moiety at the N6 position of the adenosine ring and a 3-carbon linker (Figure I), whereas compound 113 contains a 2-carbon linker (Figure J). The groups I and J compounds were developed based on our previous work with adenosine analogs carrying a 3-phenylpropy substituent at the N6 (C6-amino) position. As a control, sinefungin (114), a pan-inhibitor of SAM-dependent MTases, was included in the screen (Figure K), bringing the total library size to 114 compounds (Supporting Information Table S1). As compounds 38 (YD1120) and 42 (7q), as well as 81 and 87, were synthesized from different batches but had the same structure, they served as an internal quality control. Thus, there are a total of 112 unique structures in this library.

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A library of 114 SAM analogs sharing a 5′-thioadenosine scaffold with a 2–3 carbon linker (see Supporting Information Table S1). (A, B) Incorporation of a guanidino moiety. (C, D) Compounds featuring a urea moiety. We note that compounds 38 and 42, as well as 81 and 87, are the same compound synthesized twice. (E, F) Compounds featuring a benzylamine. (G) Compounds containing a dimethoxyquinazolin-4-amine moiety. (H) Compounds including a phenylether moiety. (I) Compounds introducing a phenylpropane moiety at the N6 position of the adenosine ring. (J) Compound 113. (K) Sinefungin (114).

Library Screening against Seven DNA or RNA Adenine Methyltransferases

We tested the SAM analogue library against three bacterial DNA MTases and four human RNA MTases that act on distinct RNA substrates (Figure A). All seven enzymes catalyze the methylation of the exocyclic amino group of adenine at the N6 position, producing N6-methyladenine. We reasoned that, during the methylation reaction, the adenine ring’s exocyclic amino group may undergo chemical transformation similar to that of the planar guanidino group of arginine that is targeted by PRMTs. An initial inhibition screen was conducted at a compound concentration of 10 μM using the MTase-Glo biochemical assay, which measures the conversion of the methyl donor SAM to S-adenosyl-l-homocysteine (SAH). ,

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Inhibition study of seven MTase activities at a single inhibitor concentration of 10 μM. (A) Heatmaps of relative inhibition by compounds against four human RNA MTases and three bacterial DNA MTases, where redder shades indicate increasing inhibition. (B) Relative inhibition by compounds against C. difficile CamA in a bar graph. N = 2 replicates. (C) Relative inhibition by compounds against C. crescentus CcrM in the bar graph. N = 2 replicates. The three panels are vertically aligned by the compound number. For other inhibition data, see Table S1.

Among the three bacterial DNA MTases tested (Dam, CcrM, and CamA), no compounds meaningfully inhibited (i.e., >50%) E. coli Dam, which methylates the G A TC sequence. In contrast, seven compounds inhibited C. crescentus CcrM (which targets G A nTC motifs) by 50% or more. Surprisingly, 18 compounds inhibited CamA activity by more than 90%. Based on these initial results, E. coli Dam was excluded from further analysis.

Inhibition of CamA

The 18 compounds that (among tested MTases) specifically inhibited CamA activity by more than 90% at 10 μM are 27 and 58, and all 16 compounds from groups I and J (Figure B). Groups I and J compounds share a common structural feature: a phenylpropane moiety attached at the N6 position of the adenosine ring (Figure I,J). This observation suggests that analog N6 substitution, as opposed to modifications at the SAM homocysteine moiety, provides greater potency and selectivity for targeting CamA. This result aligns with prior structural insights showing that SAM binding to CamA involves a conformational rearrangement of two N-terminal helices and that the solvent-exposed edge of the SAM adenosine moiety can be derivatized to make inhibitory analogs. Compared to parent compounds in Group B, N6-substituted analogs showed greater inhibition, indicating the important contribution from 3-phenylpropyl group for interacting with CamA. Selective inhibition of CamA may also reflect the fact that CamA has an unusually low binding affinity for SAM, relative to most other MTases (K m in the range of 17–25 μM). , For comparison, E. coli Dam has a SAM K m of 3–6 μM, − while the human enzymes PCIF1 and MettL5 exhibit SAM K m values of 0.7 and 1.0 μM, respectively. However, this rationale does not explain the lack of inhibition of MettL16, which has a reported SAM K m > 400 μM. Compared to Groups A–D, a guanidine moiety appears to be preferred over a urea. There is no hit from Group H except 85, which showed ∼50% inhibition at 10 μM, suggesting that substitution at the para-position of the phenyl ring is disfavored.

Next, we selected four SAM analogs (compounds 27, 58, 67, and 113 in Figure A) for more detailed inhibition studies, along with the previously characterized adenosine analogue MC4741 for comparison. MC4741 lacks the homocysteine moiety but, like compound 113, carries a 3-phenylpropyl group at the N6-amino position of adenosine. Compound 67 was also chosen for its overlapping activity against CcrM. Under the defined conditions with 0.05 μM CamA, 40 μM SAM, and 2.5 μM DNA substrates, the five compounds exhibited IC50 values ranging from 9 to 0.15 μM (Figure B). Reducing the linker length from three carbons (67) to two carbons (58) more than doubled the potency. Substituting the secondary amine in compounds 67 and 58 with a guanidino-containing moiety (compound 27) further increased potency 2.7×. MC4741, despite lacking the homocysteine moiety (Figure A), showed twice the potency of compound 27 (Figure C), suggesting that the N6 substitution plays a key role. Compound 113, which contains both the guanidino group and the 3-phenylpropyl moiety, exhibited the highest potency with an IC50 of 0.15 μM. Structural comparison revealed that compounds 113 (PDB 8FS1) and MC4741 (PDB 8CXZ) occupy the SAM-binding pocket in a manner similar to that of sinefungin (PDB 7RFK), with the adenosine ring aligned closely among these structures (RMSD ≤ 1 Å between the protein components; Figure D–F).

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Inhibition of CamA. (A) Chemical structure of the five SAM analogs used most in this study. (B) IC50 measurements as a function of inhibitor concentrations. Error bars indicate the mean ± SD of N = 3 independent determinations. (C) Summary of the IC50 values. (D) Structure of CamA-sinefungin-DNA (PDB entry 7RFK). (E) Structure of CamA-MC4741-DNA (PDB entry 8CXZ). (F) Overlap of sinefungin and MC4741.

Inhibition of CcrM

As shown in Figure C, a single-dose screen at 10 μM revealed that in order of decreasing potency, compound 67 inhibited C. crescentus CcrM activity by 72%, followed by 112 (56%), 74 (50%), 77 (50%), 58 (47%), and 13 (44%). Besides compound 67, all five others share potency similar to that of Sinefungin (114). The IC50 of compound 67 was determined to be 1.8 μM, approximately 1.7× more potent than sinefungin (Figure A,B). Unlike CamA, which functions as a monomer, CcrM is a β-class MTase and operates as a homodimer: one subunit binds and recognizes the target DNA strand and catalyzes the methyl transfer, while the second subunit interacts with the nontarget strand (Figure C). Interestingly, compound 67 exhibited 5× stronger inhibition of CcrM compared to CamA, with IC50 values of 1.8 and 9 μM, respectively. Using the Protenix server, an open-source reproduction inspired by AlphaFold3, we modeled the binding of compound 67 to CcrM with high confidence scores of both predicted template modeling (pTM) and interface predicted template modeling (iPTM) metrics (Figure D). Among the top five predicted models, compound 67 consistently occupies the SAM-binding pocket, showing alternative conformations of its biphenyl moiety (Figure D).

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Inhibition of CcrM. (A, B) IC50 measurements of SAM analog 67 and sinefungin (114). Error bars indicate the mean ± SD of N = 3 independent determinations. (C) Structure of CcrM-sinfungin-DNA (PDB entry 6PBD). (D) AlphaFold3 predicted models of compound 67 bound with CcrM.

Inhibition of Human RNA MTases

Among the four human RNA enzymes tested, MettL5 targets rRNA and MettL16 modifies snRNA, with MettL3 and PCIF1 methylating mRNA at different sites. Dysregulation of these RNA modifications can alter gene expression, particularly in cancer cells. MettL16 methylates adenine within a conserved UAC A GAGAA sequence, found in hairpins of the 3′ UTR of the SAM synthetase (MAT2A) mRNA and in U6 snRNA. ,− Among all tested compounds, only one (compound 87 at 10 μM) showed ∼50% inhibition of MettL16 (Figure A).

PCIF1 (phosphorylated RNA polymerase II CTD interacting factor 1) methylates adenosine when it is the first transcribed nucleotide after the mRNA cap. − Two compounds at 10 μM, 46 and 68, showed ∼40% inhibition of PCIF1 (Figure A) and contain a para-chlorobenzene moiety. Notably, compound 46 inhibited PCIF1 (42% inhibition), MettL5 (46%), and MettL3 (58%) to similar extents, while compound 68 also showed overlapping inhibition of PCIF1 (42% inhibition), MettL3 (60%), and MettL5 (35%) (Figure A).

Inhibition of MettL5-Trm112

MettL5 forms a heterodimer with Trm112 and catalyzes adenine methylation at position 1832 of 18S rRNA, ,− modulating translation of mRNA. Of the 114 compounds tested, three compounds37 (66% inhibition), 77 (53%), and 112 (57%)showed greater than 50% inhibition of MettL5-Trm112 at 10 μM (Figure A). We determined an IC50 value of 8 μM for compound 37 against MettL5-Trm112 activity (Figure A). We note that compound 37 is a selective inhibitor of MettL5-Trm112 among the six enzymes tested (Figure A). Given that MettL5 affects differentiation of embryonic stem cells and promotes tumorigenesis in multiple cancer models, ,,− compound 37 may serve as a promising lead for further optimization as an anticancer therapeutic. AlphaFold3-based prediction from the Protenix server positioned compound 37 within the SAM-binding site of MettL5 (Figure B). Notably, four out of the five top models show a bent conformation with their 1,3-benzodioxole moiety extending into the substrate-binding site (Figure C). This orientation effectively bridges between the SAM-binding and substrate-binding pockets, consistent with the intended design of a bisubstrate inhibitor.

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Inhibition of MettL5-Trm112 and MettL3-MettL14. (A) IC50 measurement of compound 37 against MettL5-Trm112. Error bars indicate the mean ± SD of N = 3 independent determinations. (B) AlphaFold3 predicted models of compound 37 bound with MettL5. (C) Four of the five top models adopt a bent conformation, while one model (green) aligns with the homocysteine moiety of SAM. The arrow indicates the direction of methyl transfer to the substrate. (D) IC50 measurements of compounds 77 and STM2457 against MettL3-MettL14 were made by varying inhibitor concentrations. Error bars indicate the mean ± SD of N = 3 independent determinations. (E) AlphaFold3 predicted models of compound 77 bound with MettL3. (F) Five top models of 77 bridge the SAM-binding and substrate-binding pockets. The arrow indicates the direction of methyl transfer to the substrate.

Inhibition of MettL3-MettL14

Like MettL5-Trm112 heterodimer, MettL3 also forms a heterodimer, pairing with MettL14, , and catalyzing adenine-N6 methylation in mRNA (in this case at the degenerate consensus sequence RR A CH, where R = purine and H ≠ G). Also like MettL5-Trm112, MettL3-MettL14 affects embryonic stem cell differentiation and can promote tumorigenesis. − Unlike MettL5-Trm112, the MettL3-MettL14 complex is also active on DNA substrates, at least in vitro. , Seven SAM analogs exhibited 50–60% inhibitory activity against MettL3-MettL14 using an RNA substrate (Table ): 2 (60% inhibition), 27 (52%), 46 (58%), 58 (59%), 68 (54%), 72 (53%), and 75 (57%), while compound 77 displayed 68% inhibition. Other MettL3 inhibitors have been reported. We compared the potency of compound 77 to a known MettL3 inhibitor, STM2457, which targets the SAM-binding site. Compound 77 showed inhibitory activity at an IC50 of 7.4 μM, which was less potent than STM2457 (Figure D). Nevertheless, compound 77 was modeled as a bisubstrate inhibitor occupying both SAM-binding and substrate-binding pockets (Figure E,F).

1. DNA or RNA Methylation Reaction Conditions at Room Temperature (∼21 °C).

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Discussion

Inhibition of Nucleic Acid MTases

Using our focused SAM analog library, we previously identified a selective inhibitor (compound 45) of human PRMT4/CARM1 (Figure A). Compound 45 exhibited >1000-fold selectivity over 38 tested MTases, including 26 protein lysine MTases, which share a SAM cofactor binding site (see Figure 9d in ref ). In the current study, we identified unique scaffolds of SAM analogs that inhibit three additional MTases in vitro with varying potency: compound 113 as a selective inhibitor of C. difficile CamA (Figures and B), compound 67 for C. crescentus CcrM, and compound 37 for human MettL5 (Figure ). These compounds represent promising leads for further optimization toward their respective targets. In the case of 113, this could lead to the development of a therapeutic agent against C. difficile, which can cause lethal infections and is difficult to treat. For 67, this may lead to agents targeting the CcrM orthologs in frank pathogens such as Brucella and the colorectal cancer-associated F. nucleatum. For 37, this might lead to a useful anticancer drug. Among all nine chemical groups, Group H (containing a phenylether group) is less favorable for all tested MTases, except that compounds 85 and 87, respectively, showed a modest inhibition of CamA and MettL16. Groups I and J (with N6-phenylpropyl substitution) clearly, selectively, and potently inhibited CamA, indicating a unique binding pocket in CamA in the studied group. Among all tested MTases, CamA and MettL3-MettL14 are relatively more tolerant for binding these compounds as more hits were identified, while the remaining MTases have stricter preferences. PCIF1 favors a para-chlorophenyl group as shown in both hits (compounds 46 and 68). Compound 67 showed dual inhibition of both CcrM and CamA. Interestingly, its analog 58 (with a 2-C rather than 3-C linker) showed increased potency and selectivity for CamA.

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Comparison with existing antibacterial compounds. (A) Compound 45 for PRMT4 and (B) compound 113 for CamA. (C) CRS3123 for MetRS. (D) Ibezapolstat for polymerase IIIC. (E) Neither CRS3123 nor Ibezapolstat inhibits CamA at 10 μM. Error bars indicate the mean ± SD of N = 3 independent determinations.

SAM Analogs as Antibiotics

A known limitation of SAM analogs is their poor membrane permeability. In mammalian cells, SAM uptake may occur via nucleoside carriers, but SAM crosses cytoplasmic membranes poorly. In bacteria, SAM uptake appears to occur only via specific transporters, typically produced only by obligate parasites. , Prodrug strategies have been employed to improve cellular SAM uptake in human cells; , however, it is unclear whether these strategies will be effective in penetrating bacterial cell walls. In addition, bacteriophages and virus-like particles (VLPs) have been employed as nanocarriers for targeted small-molecule delivery, providing advantages such as cargo protection and improved cellular uptake. ,

Another consideration is the possible development of resistance via mutation. For example, a recent study reported that sinfungin exhibits antifungal activity by disrupting RNA adenine methylation, thereby impairing key pathogenic traits of Candida albicansa fungus commonly found in the gastrointestinal tract and oral cavityincluding hyphal morphogenesis, biofilm formation, and epithelial adhesion. However, an earlier study in the fungus Saccharomyces cerevisiae showed that resistance to sinefungin can arise through mutations in the high-affinity SAM transporter (Sam3) or through upregulation of SAM synthase and mRNA cap MTase. Possible bacterial resistance mechanisms to SAM analogs have not yet been well studied.

Comparison to Other Inhibitors

Two orally active small molecules have been developed and have entered clinical trials for treating C. difficile infection: CRS3123, an inhibitor of bacterial methionyl-tRNA synthetase (MetRS), , and ibezapolstat, which targets DNA polymerase IIIC in Gram-positive bacteria with low G+C content. , CRS3123 is a diaryldiamine compound featuring two bicyclic aromatic rings connected by a flexible diamine bridge (Figure C). The structure of CRS3123 bound to MetRS2 from the multidrug-resistant Gram-negative bacterium Xanthomonas citri has been solved (PDB 6WQT) (Figure C). Ibezapolstat is a guanine analogue with a morphilinoethyl group at the N7 position and a dichlorobenzylamine group at the C2 position (Figure D). Its proposed inhibitory mechanism involves competition with cognate dGTP at the active site of pol IIIC, pairing with the template cytosine; however, no structural data are currently available for ibezapolstat-bound DNA pol IIIC. Neither compound inhibits CamA at 10 μM (Figure E); nevertheless, we aim to incorporate chemical features, particularly from ibezapolstat (a guanine analog), into the design of next-generation adenosine analogs with enhanced cellular permeability.

In summary, our results provide chemical probes for exploring the role of CamA in sporulation and colonization with potential as antivirulence agents against C. difficile infection. Our study also introduces the first chemical probes for inhibiting bacterial CcrM and human MettL5, each of which plays key roles in their respective hosts.

Methods

Chemistry

Compounds 1–113 were synthesized by following our previously reported general methods. The compounds had a purity of at least 95% as determined by HPLC analysis, and their structures were confirmed by 1H and 13C NMR spectroscopy as well as high-resolution mass spectrometry, as previously reported. Sinefungin (114) was purchased from Sigma-Aldrich (Cat. No. S8559); CRS3123 (Cat. No. HY-18323) and ibezapolstat (Cat. No. HY-128357) were purchased from MedChemExpress. Compound MC4741 was reported previously.

General Procedure for the Synthesis of Compounds 1 to 31

To a stirring solution of 1 or 9 (0.07 mmol), phenylboronic acid (0.09 mmol), dioxane (1.6 mL), and H2O (0.4 mL) were added Pd­(PPh3)4 (8.0 mg, 0.007 mmol) and K2CO3 (29 mg, 0.21 mmol). The resulting solution was stirred inside a microwave at 125 °C for 30 min. After the mixture was cooled, the volatiles were removed under vacuum. The residue was dissolved in H2O/methanol (1/1), filtered, and purified by HPLC (MeCN/H2O) to give 2–8 and 10–31 (45–75% yield).

General Procedure for the Synthesis of Compounds 32 to 44

To a stirring solution of NH2-C2-Thioadenosine (163 mg, 0.5 mmol) or NH2-C3-Thioadenosine (170 mg, 0.5 mmol) in 5 mL of anhydrous DMF, TEA (152 mg, 1.5 mmol), and isocyanate (0.6 mmol) were added at room temperature. After being stirred at room temperature for 1–4 h, the mixture was diluted with 100 mL of Et2O and filtered. Then, the residue was dissolved in 5 mL of methanol for purification with prepHPLC (MeCN/H2O) to afford 32–44 (56–88% yield).

General Procedure for the Synthesis of Compounds 45 to 67

Compound 45 and intermediate benzylamine-C3-thioadenosine were synthesized by following our previously reported methods. Compounds 46–67 (43–82% yield) were synthesized from 45 or benzylamine-C3-thioadenosine by following the same method as 2–31.

General Procedure for the Synthesis of Compounds 68 to 78

Compounds 68–78 (31–64% yield) were synthesized from 2-chloro-6,7-dimethoxyquinazolin-4-amineC2-thioadenosine by following the same method as that for 2–31.

General Procedure for the Synthesis of Compounds 79 to 97

Compounds 79–97 (54–81% yield) were synthesized from 4-iodophenoxy-C2-thioadenosine similar to 2–31.

General Procedure for the Synthesis of Compounds 98 to 112

Compounds 98–112 (38–64% yield) were synthesized from N6-phenylpropyl NH2-C3-thioadenosine similar to 1 and 9.

Nucleic Acid Adenine MTases Used in the Study

We characterized and prepared the seven MTases used in the current study in our laboratories: E. coli Dam (pXC1612), , C. crescentus CcrM (pXC2121), C. difficile CamA (pXC2184), human MettL3–MettL14, , human MettL16 (pXC2210), human MettL5–Trm112 (pXC2062–pXC2076), , and human PCIF1 (pXC2055). , MettL3-MettL14 was expressed in Sf9 insect cells, and the other enzymes were expressed in E. coli strain BL21­(DE3).

Inhibition Assays

Methylation inhibition assays were conducted in the presence of 10 μM inhibitors, with the detailed conditions summarized in Table . Assays were performed in low-volume 384-well plates containing 5 μL of reaction mixture per well, and luminescence was measured using a Synergy 4 multimode microplate reader (BioTek). Following the reactions, all samples were quenched by adding trifluoroacetic acid (TFA) to a final concentration of 0.1% (v/v). Methylation activity was assessed using the MTase-Glo bioluminescence assay (Promega), which detects the reaction byproduct SAH. SAH is enzymatically converted to ATP in a two-step reaction, and ATP levels are then quantified via a luciferase-based luminescence readout. For IC50 determination, the same reaction setup was used as described in Table , with the exception that inhibitor concentrations were varied by a 2-fold series dilution.

AlphaFold3 Modeling of Compounds Binding in CcrM, MettL5-Trm112, and MettL3-MettL14

Protenix server (https://protenix-server.com/login) was utilized to generate five top hits of compounds 67, 37, and 77, respectively, with CcrM, MetttL5-Trm112, and MettL3-MettL14. PyMol version 3.1.4.1 (Schrödinger, LLC) was used to prepare the structure images.

Supplementary Material

ao5c09571_si_001.xlsx (380B, xlsx)

Acknowledgments

This work was supported by U.S. National Institutes of Health grant R35GM134744 (to X.C., who is a CPRIT scholar in Cancer Research) and Purdue University Evanson-McCoy Endowment (to R.H.). We thank John R. Horton for providing Dam enzyme, and Dr. Danti Rotili and Dr. Antonello Mai for providing compound MC4741.

Glossary

Abbreviations

CamA

Clostridioides difficile-specific DNA adenine methyltransferase

CDI

Clostridioides difficile infections

IC50

half-maximal inhibitory concentration

K D

dissociation constant

MTase

methyltransferase

PRMT

protein arginine methyltransferase

SAH

S-adenosyl-l-homocysteine

SAM

S-adenosyl-l-methionine

TFA

trifluoroacetic acid

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c09571.

  • Molecular formula strings (SMILES) and associated inhibition data (XLSX)

J.Z. performed inhibition assays, protein purifications of CamA, CcrM, and PCIF1, and AF3-based structural modeling; Y.D. performed compound synthesis. D.Y. performed protein purifications of MettL5-Trm112 and MettL16. T.H. and M.V. provided the recombinant MettL3-MettL14 complex. R.M.B. participated in discussions and assisted in preparing the manuscript; X.Z. provided supervision, conceptualization, and project administration; R.H. and X.C. designed and organized the study, performed writing, reviewing, and editing of the manuscript, conceptualization, and funding acquisition.

The authors declare no competing financial interest.

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