ACS Chemical Biology最新文献

4-Methylazetidinecarboxylic acids (MeAZEs) are unusual nonproteinogenic amino acids found in nonribosomal peptides, such as bonnevillamide D and vioprolide A. The biosynthesis of MeAZEs is thought to proceed from S-adenosyl-l-methionine (SAM) via C-methylation and azetidine ring formation, but the order of these steps has remained unclear. Guided by our previous findings, we proposed that C4″-methylation of SAM by the cobalamin-dependent radical SAM enzyme BnvC, which is encoded in the biosynthetic gene cluster (BGC) of bonnevillamides, precedes azetidine ring formation. In this study, we identified the DUF364-containing enzyme Orf5, which is associated with the bnvC gene in the BGC of bonnevillamides, converts (4″R)-4″-methyl-SAM into cis-MeAZE. Further, we found that VioH, previously shown to catalyze azetidine ring formation from SAM in vioprolide A biosynthesis, transforms (4″S)-4″-methyl-SAM into trans-MeAZE. Structural modeling indicated that the C-methyl group was essential for efficient cyclization. These results elucidated the biosynthetic logic of MeAZEs and established C-methylation as a prerequisite for azetidine ring formation.

Recent studies demonstrated that the third-generation EGFR-TKI (i.e., AZD9291) showed benefits not only for patients with EGFR acquired resistance mutations but also for those with EGFR sensitizing mutations. In particular, AZD9291 showed efficacy superior to that of the first- and second-generation EGFR-TKIs, leading to the extension of its approval for the first-line treatment of EGFR mutant non-small-cell lung cancer (NSCLC). Despite genetic testing being used to detect mutations for identifying patients who may benefit from EGFR-TKIs, it lacks information on the functional status of EGFR kinase, a key determinant of treatment efficacy. Here, we reported a series of chemical probes (3-6) specifically targeting the active site of the EGFR kinase domain by conjugating either an alkyne ligation handle or a fluorescent tag to the third-generation EGFR-TKIs (e.g., AZD9291, PF-06747775). Both in vitro assays and cellular studies demonstrated that PF-06747775-yne (4) showed better selectivity toward EGFR kinase than AZD9291-yne (3). Interestingly, we found that the attachment of a fluorescent tag (i.e., Cy5) to AZD9291 altered the size and chemical properties of the parent compound and enhanced its selectivity. We further showed that PF-06747775-yne (4) was capable of visualizing the active form of EGFR mutants not only in xenograft mouse models but also in clinical specimens from lung cancer patients. These results demonstrate that PF-06747775-based probes hold promise as companion diagnostic tools to guide the development of effective, personalized therapeutic strategies.

S-acylation, often referred to as S-palmitoylation, is a reversible and dynamic posttranslational modification that corresponds to the addition of a long-chain fatty acid to cysteine (Cys) residues. Established mass spectrometry-based chemoproteomics methods have improved our understanding of the S-acylation proteome, notably by identifying hundreds of S-acylated proteins, sometimes with the modified Cys. However, the precise quantification of S-acylation levels for each Cys within a single sample remains challenging at the proteome level. Quantification of S-acylation levels is critical to further our understanding of protein S-acylation in cellular function and its role in health and diseases. We report here the development of an S-acylation quantification workflow based on the sequential labeling of free Cys and S-acylated Cys with isotopic labeling reagents. The workflow was extensively optimized, notably by comparing the number of sites identified with two alkyne-tagged Cys-reactive isotopic probes and four azido-tagged biotin-based capture reagents. By integrating this enhanced workflow with high-field asymmetric waveform ion mobility spectrometry (FAIMS) on LC-MS/MS instruments for the separation of labeled peptides, over 17,000 unique Cys could be quantified in biological samples. Application of the S-acylation quantification workflow to cellular proteomes allowed for the quantification of S-acylation levels in a HeLa proteome. We also identified dynamic S-acylation changes in response to autophagy induction.

Mycobacterium abscessus (Mab) is difficult to treat due to intrinsic and acquired resistance to diverse antibiotics. Among the intrinsic resistance factors is the mycomembrane, a complex structure that limits permeability to several classes of antibiotics. Here, we report new inhibitors of MmpL3, an essential transporter required to build the mycomembrane. Several of the MmpL3 inhibitors have comparable activity in vitro to standard-of-care treatments, exhibit both time- and dose-dependent bactericidal activity, have low eukaryotic cytotoxicity, and are efficacious against Mab growing in macrophages or in biofilms. The inhibitors had varying activities against a panel of 30 different multidrug-resistant clinical isolates and are additive or synergistic with standard-of-care antibiotics, suggesting they could be included in combination therapy. The inhibitors also exhibit a low frequency of resistance, with some of the isolated mutants displaying differential patterns of sensitivity and resistance to the different MmpL3 inhibitors and putative fitness defects. Cross-resistance profiles of 15 structurally related inhibitors against 16 different MmpL3 resistant mutants demonstrate structure-driven clustering patterns of the inhibitors, where those carrying a similar scaffold cluster together and different MmpL3 amino-acid substitutions account for these differences. Cross-resistance profiles were also simulated computationally, showing significant correlation between the computationally calculated parameters and the biological patterns of cross-resistance and emphasizing specific structural-functional associations driving resistance or susceptibility. These inhibitors and their analogs hold promise for clinical translation, and the established structural-functional associations provide mechanistic insights into the function of MmpL3, resistance and susceptibility of MmpL3 inhibitors, and fitness costs associated with MmpL3 resistance.

Pre-mRNA splicing is a core process in eukaryotic gene expression, and splicing dysregulation has been linked to various diseases. However, very few small molecules have been discovered that can modulate spliced mRNA formation or inhibit the splicing machinery itself. This study presents a novel high-throughput screening (HTS) platform for identifying compounds that modulate splicing. Our platform comprises a two-tiered screening approach: A primary screen measuring growth inhibition in sensitized Saccharomyces cerevisiae (yeast) strains and a secondary screen that relies on production of a fluorescent protein as a readout for splicing inhibition. Using this approach, we identified 4 small molecules that cause accumulation of unspliced pre-mRNA in vivo in yeast. In addition, cancer cells expressing a myelodysplastic syndrome-associated splicing factor mutation (SRSF2^P95H) are more sensitive to one of these compounds than those expressing the wild-type version of the protein. Transcriptome analyses showed that this compound causes widespread changes in gene expression in sensitive SRSF2^P95H-expressing cells. Our results demonstrate the utility of using a yeast-based HTS to identify compounds capable of changing pre-mRNA splicing outcomes.

Modular polyketide synthases (PKSs) produce diverse natural products with significant pharmaceutical value, but their protein engineering for drug discovery is hampered by the unpredictable substrate specificity of their ketosynthase (KS) domains. While previous studies have focused on the KS active sites, we conducted extensive alanine scanning of loop regions of a KS domain, most of which are distant from the catalytic center. In vitro screening of 46 point mutants revealed that ∼70% of the mutants retained their activity, whereas ∼25% of the mutants displayed severely reduced activity, and two mutants unexpectedly showed enhanced activity. Interestingly, most mutations with significant impact were located more than 20 Å away from the catalytic center. These findings provide the first clear evidence that KS residues beyond the canonical active site play crucial roles in polyketide biosynthesis. Our results show essential foundational data to understand KS functions, which could be used for developing more effective KS engineering strategies beyond traditional active site modifications.

Sulfation is a fundamental post-translational modification that imparts negative charge and structural complexity to biomolecules, thereby regulating molecular recognition, signaling, and homeostasis across all domains of life. Yet, the ability to interrogate the biological functions of sulfation has long been hindered by the difficulty of constructing molecules with defined sulfation patterns. This Account summarizes our efforts to develop chemical strategies that enable precise control over sulfation in glycans and proteins. We describe an organobase-promoted sulfur(VI) fluoride exchange (SuFEx) chemistry that allows early stage, chemoselective O-sulfation across a broad substrate scope, providing a general solution to sulfate installation in complex settings. Building on this foundation, we introduce an iterative "clickable disaccharide" platform for the programmable assembly of sequence-defined heparan sulfate glycomimetics, enabling systematic dissection of sulfation-dependent glycan-protein interactions. Extending these concepts to the protein realm, we developed a fluorosulfate tyrosine strategy that installs latent sulfates into peptides and proteins, which can be unmasked under physiological conditions or light control via hydroxamic-acid-mediated Lossen rearrangement, offering spatiotemporal control of sulfation in living systems. Collectively, these approaches delineate a unified chemical framework for constructing and manipulating sulfated biomacromolecules with molecular precision, opening new opportunities to elucidate and engineer the biological roles of sulfation.

Methionine aminopeptidase (MAP) is useful in chemical biology research for the N-terminal processing of peptides and proteins and in medicine as a potential therapeutic target. These technologies can benefit from a precise understanding of the enzyme's substrate specificity profiled over a wide chemical space, including not just natural substrates, peptides containing N-terminal Met, but also unnatural peptide substrates containing N-terminal Met analogues that are also cleaved by MAP like homopropargylglycine (HPG) and azidohomoalanine (AHA). A few studies have profiled substrate specificity for cleavage of N-terminal Met, but none have systematically done so using N-terminal Met analogues. Therefore, we devised a high-throughput profiling experiment based on mRNA display and next-generation sequencing to probe MAP's substrate specificity using N-terminal HPG. From subgroup analysis of either single residues or two-residue combinations, we could establish the impact of residue identity at various positions downstream from the cleavage site. To validate the selection results, a collection of short peptides was chemically synthesized and assayed for cleavage efficiency, where we observed reasonable agreement with the selection data. Results generally followed previously reported trends using N-terminal Met, the strongest trend being that the second residue (P1' position) had the greatest impact on MAP cleavage efficiency with moderate impacts discerned for residues further downstream, which could be rationalized through modeling the enzyme-substrate interaction.

Riboswitches are regulatory RNA structures that modulate gene expression in response to a small molecule. Until now, most efforts to design ligand analogs were motivated by their potential antibiotic activity. However, riboswitches are ideally suited as tools for gene therapy, enabling precise control of gene expression without the need for potentially immunogenic regulatory proteins. Developing synthetic RNA switches starting from natural riboswitches will require to engineer both the ligand and the RNA sequence to achieve sensitivity to the designed small molecule modulator but not to the natural ligand. We present the structure-based design of a drug-like small molecule ligand of the thiamine pyrophosphate (TPP) aptamer, BI-5232. BI-5232 is structurally highly diverse from the natural ligand TPP but rivals its binding affinity (K_D = 1.0 nM). Importantly, in our design, the pyrophosphate of TPP was replaced by an uncharged heterocycle that interacts with the PP-helix in an unprecedented way, as revealed by Molecular Dynamics simulations. Subsequently, we altered the aptamer sequence to drastically reduce its affinity to TPP while retaining the binding properties of our designed ligand. Based on the developed small molecule/RNA aptamer interaction, we finally constructed ribozyme-based ON- and OFF-switches of gene expression in human cell lines. Such systems are valuable additions to the synthetic toolbox for conditionally controlling gene expression, with potential applications in next-generation gene therapies.

Side chain-to-side chain peptide macrocyclization or stapling is a chemical modification that is frequently used to increase the metabolic stability, the cell permeability, and/or the binding affinity of peptide drugs. Interestingly, it was recently reported that α-helical stapling can also protect the amyloidogenic peptide hormone islet amyloid polypeptide (IAPP) from aggregation and amyloid-associated toxicity. IAPP is the major component of insoluble amyloid deposits found in diabetic patients, and its derivatives constitute potential therapeutic candidates to treat metabolic disorders. Herein, we investigated the effects of macrocyclization chemistry on amyloid formation and cytotoxicity by comparing different stapling strategies: lactamization, azide-alkyne click chemistry, and formation of thioether link. The (i, i + 4) intramolecular macrocyclization of IAPP between positions 13 and 17 imposed, or not for some derivatives, a local stability of the helical secondary structure, modulating the propensity of the peptide to self-assemble into amyloid fibrils. The helically constrained derivatives inhibited the aggregation of unmodified IAPP and showed a reduced capacity to perturb the cell plasma membrane and to induce cell death. This study offers key molecular insights into the use of stapling strategies as a chemical approach to prevent the aggregation of peptide therapeutics and to inhibit the cytotoxicity of amyloidogenic peptides associated with protein misfolding disorders.