ACS Chemical Biology最新文献

ABCG2 is a multidrug transporter that protects tissues from xenobiotics, affects drug pharmacokinetics, and contributes to multidrug resistance of cancer cells. Here, we present tetracyclic fumitremorgin C analog Ko143 derivatives, evaluate their in vitro modulation of purified ABCG2, and report four high-resolution cryo-EM structures and computational analyses to elucidate their interactions with ABCG2. We found that Ko143 derivatives that are based on a ring-opened scaffold no longer inhibit ABCG2-mediated transport activity. In contrast, closed-ring, tetracyclic analogs were highly potent inhibitors. Strikingly, the least potent of these compounds, MZ82, bound deeper into the central ABCG2 cavity than the other inhibitors and it led to partial closure of the transmembrane domains and increased flexibility of the nucleotide-binding domains. Minor structural modifications can thus convert a potent inhibitor into a compound that induces conformational changes in ABCG2 similar to those observed during binding of a substrate. Molecular dynamics simulations and free energy binding calculations further supported the correlation between reduced potency and distinct binding pose of the compounds. We introduce the highly potent inhibitor AZ99 that may exhibit improved in vivo stability.

Chronic neuropathic pain is an increasingly prevalent societal issue that responds poorly to existing therapeutic strategies. The α9α10 nicotinic acetylcholine receptor (nAChR) has emerged as a potential target to treat neuropathic pain. However, challenges in expressing functional α9α10 nAChRs in mammalian cell lines have slowed the discovery of α9α10 ligands and studies into the relationship between α9α10 nAChRs and neuropathic pain. Here, we develop a cell line in the HEK293 background that stably expresses functional α9α10 nAChRs. By also developing cell lines expressing only α9 and α10 subunits, we identify distinct receptor pharmacology between homomeric α9 or α10 and heteromeric α9α10 nAChRs. Moreover, we demonstrate that incubation with nAChR ligands differentially regulates the expression of α9- or α10-containing nAChRs, suggesting a possible mechanism by which ligands may modify receptor composition and trafficking in α9- and α10-expressing cells. We then apply our α9α10 cell line in a screen of FDA-approved and investigational drugs to identify α9α10 ligands that provide new tools to probe α9α10 nAChR function. We demonstrate that one compound from this screen, diphenidol, possesses antinociceptive activity in a murine model of neuropathic pain. These results expand our understanding of α9α10 receptor pharmacology and provide new starting points for developing efficacious neuropathic pain treatments.

Mutations in creatine transporter SLC6A8 cause creatine transporter deficiency (CTD), which is responsible for 2% of all cases of X-linked intellectual disability. CTD has no current treatments and has a high unmet medical need. Inspired by the transformational therapeutic impact of small molecule "correctors" for the treatment of cystic fibrosis, which bind to mutated versions of the CFTR ion channel to promote its trafficking to the cell surface, we sought to identify small molecules that could stabilize SLC6A8 as a potential treatment for CTD. We leveraged a novel chemoproteomic technology for ligand discovery, reactive affinity probe interaction discovery, to identify small-molecule fragments with photoaffinity handles that bind to SLC6A8 in a cellular environment. We synthesized a library of irreversible covalent analogs of these molecules to characterize in functional assays, which revealed molecules that could promote the trafficking of mutant SLC6A8 variants to the cell surface. Further medicinal chemistry was able to identify reversible drug-like small molecules that both promoted trafficking of the transporter and also rescued creatine uptake. When profiled across the 27 most prevalent SLC6A8 missense variants, we found that 10-20% of patient mutations were amenable to correction by our molecules. These results were verified in an endogenous setting using the CRISPR knock-in of selected missense alleles. We established in vivo proof-of-mechanism for correctors in a novel CTD mouse model with the P544L patient-defined variant knocked in to the SLC6A8 locus, where treatment with our orally bioavailable and brain penetrant tool corrector increased brain creatine levels in heterozygous female mice, validating correctors as a potential therapeutic approach for CTD.

A cryptic cluster of genes, known as the ebo cluster, has been found in a variety of genomic contexts among bacteria and algae. In Pseudomonas fluorescens NZI7, the ebo cluster (a.k.a. EDB cluster) is involved in the bacterial repellent mechanism against nematode grazing. In cyanobacteria, the cluster plays a role in the transport of the scytonemin monomer from the cytosol to the periplasm. Despite their broad distribution and interesting phenotypes, neither the pathway nor the functions of the enzymes are known. Here we show that EboD proteins from the ebo clusters in Nostoc punctiforme and Sporocytophaga myxococcoides catalyze the cyclization of mannose 6-phosphate to a novel cyclitol, 2-deoxy-4-epi-scyllo-inosose. The enzyme product is postulated to be a precursor of a signaling molecule or a transporter in the organisms. This study sheds the first light onto ebo/EDB pathways and established a functionally distinct enzyme that extends the diversity of sugar phosphate cyclases.

Pleurotin is a meroterpenoid specialized metabolite made by the fungus Hohenbuehelia grisea, and it is a lead anticancer molecule due to its irreversible inhibition of the thioredoxin-thioredoxin reductase system. Total synthesis of pleurotin has been achieved, including through a stereoselective route; however, its biosynthesis has not been characterized. In this study, we used isotope-labeled precursor feeding to show that the nonterpenoid quinone ring of pleurotin and its congeners is derived from phenylalanine. We sequenced the genome of H. grisea and used comparative transcriptomics to identify putative genes involved in pleurotin biosynthesis. We heterologously expressed a UbiA-like prenyltransferase from H. grisea that led to the accumulation of the first predicted pleurotin biosynthetic intermediate, 3-farnesyl-4-hydroxybenzoic acid. This work sets the foundation to fully elucidate the biosynthesis of pleurotin and its congeners, with long-term potential to optimize their production for therapeutic use and engineer the pathway toward the biosynthesis of valuable analogues.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a major global health concern. TetR family repressors (TFRs) are important for Mtb's adaptation to the human host environment. Our study focuses on one notable Mtb repressor, Mce3R, composed of an unusual double TFR motif. Mce3R-regulated genes encode enzymes implicated in cholesterol metabolism, resistance against reactive oxygen species, and lipid transport activities important for Mtb survival and persistence in the host and for the cellular activity of a 6-azasteroid derivative. Here, we present the structure of Mce3R bound to its DNA operator, unveiling a unique asymmetric assembly previously unreported. We obtained a candidate DNA-binding motif through MEME motif analysis, comparing intergenic regions of mce3R orthologues and identifying nonpalindromic regions conserved between orthologues. Using an electrophoretic mobility shift assay (EMSA), we confirmed that Mce3R binds to a 123-bp sequence that includes the predicted motif. Using scrambled DNA and DNA oligonucleotides of varying lengths with sequences from the upstream region of the yrbE3A (mce3) operon, we elucidated the operator region to be composed of two Mce3R binding sites, each a 25-bp asymmetric sequence separated by 53 bp. Mce3R binds with a higher affinity to the downstream site with a K_d of 2.4 ± 0.7 nM. The cryo-EM structure of Mce3R bound to the 123-bp sequence was refined to a resolution of 2.51 Å. Each Mce3R monomer comprises 21 α-helices (α1-α21) folded into an asymmetric TFR-like structure with a core asymmetric four-helix bundle. This complex has two nonidentical HTH motifs and a single ligand-binding domain. The two nonidentical HTHs from each TFR bind within the high-affinity, nonpalindromic operator motif, with Arg53 and Lys262 inserted into the major groove. Site-directed mutagenesis of Arg53 to alanine abrogated DNA binding, validating the Mce3R/DNA structure obtained. Among 811,645 particles, 63% were Mce3R homodimer bound to two duplex oligonucleotides. Mce3R homodimerizes primarily through α15, and each monomer binds to an identical site in the DNA duplex oligonucleotide.

Protein cysteine residues are sensitive to redox-regulating molecules, including reactive sulfur species (RSS). As an important member of the RSS family, polysulfides are known to react with protein cysteines to form persulfides and disulfides, both affecting protein functions. In this work, we studied how polysulfides could impact cysteine proteases through careful mechanistic and kinetic studies. The model protein papain was treated with different polysulfides to elucidate the efficacy of polysulfides as inhibitors for this protein. We also explored the effects of different reductants that could regenerate papain activity after polysulfide-mediated inhibition. A triarylphosphine reagent, TXPTS, was found to be efficient in differentiating between papain persulfidation and disulfide formation.

Protein mycoloylation is a recently identified unusual post-translational modification (PTM) exclusively observed in Mycobacteriales, an order of bacteria that includes several human pathogens. These bacteria possess a distinctive outer membrane, known as the mycomembrane, composed of very long-chain fatty acids called mycolic acids. It has been demonstrated that a few mycomembrane proteins undergo covalent modification with mycolic acids in the model organism Corynebacterium glutamicum through the action of mycoloyltransferase MytC. This PTM represents the first example of protein O-acylation in prokaryotes and also the first example of protein modification by mycolic acid. Many questions about the specificity of protein O-mycoloylation remain crucial for understanding its evolutionary significance in Mycobacteriales and its role in cell physiology. We have developed the first bioorthogonal mycolate donor featuring the natural mycolic acid pattern, enabling direct, unambiguous transfer of the lipid moiety to its acceptors and efficient metabolic labeling and enrichment of MytC protein substrates. Mass spectrometry analysis of the labeled proteins and comparative proteomic analysis of the cell envelope proteome between wild-type and ΔmytC strains identified an unbiased list of 21 proteins likely mycoloylated in the cell. The robustness of our approach is demonstrated by the successful biological validation of mycoloylation in 6 candidate proteins within wild-type cells, revealing the characteristic profile of proteins modified with natural mycolates. These findings provide interesting insights into the significance of this new lipidation pathway and pave the way for understanding their function, especially concerning the mycoloyltransferase family that includes the essential Antigen85 enzymes in Mycobacteria.

Gemcitabine-based monotherapy or combination therapy has become the standard treatment for locally advanced and metastatic pancreatic cancer. However, the emergence of resistance within weeks of treatment severely compromises therapeutic efficacy. The intricate biological process of gemcitabine resistance in pancreatic cancer presents a complex challenge, as the underlying mechanisms remain unclear. Identifying the target protein of gemcitabine is crucial for studying its drug-resistance mechanism. An activity-based probe is a powerful tool for studying drug target proteins, but the current lack of activity-based gemcitabine probes with robust biological activity hinders research on gemcitabine. In this study, we developed three active probes based on gemcitabine, among which Gem-3 demonstrated excellent stability and labeling efficacy. We utilized Gem-3 in conjunction with chemical proteomics to identify intracellular target proteins. We identified 79 proteins that interact with gemcitabine, most of which were previously unknown and represented various functional classes. Additionally, we validated the increased expression of IFIT3 and MARCKS in drug-resistant cells, along with the activation of the NF-κB signaling pathway. These findings substantially contribute to our comprehension of gemcitabine's target proteins and further our understanding of the mechanisms driving gemcitabine resistance in pancreatic cancer cells.

Estrogen receptor α (ERα)-positive breast cancer patients are typically treated with ERα inhibitors, including selective estrogen receptor modulators (SERMs) and selective estrogen receptor degraders (SERDs). However, the distinct pharmacological properties of various ERα inhibitors remain incompletely understood. In this study, we employed formaldehyde cross-linking followed by ERα immunoprecipitation and mass spectrometry to reveal that fulvestrant, the first FDA-approved SERD, induces the interaction between ERα and SUMO E3 ligases PIAS1 and PIAS2. Biochemical and genomic assays confirmed that fulvestrant induces SUMOylation of ERα, which inhibits ERα's binding to chromatin DNA. In addition, raloxifene (a SERM) and elacestrant (the first FDA-approved oral SERD) were identified as compounds that similarly induce ERα SUMOylation and inhibit its chromatin interaction. Our findings reveal a mechanism by which select ERα inhibitors disrupt ERα function through SUMOylation, offering insights for the development of next-generation ERα-targeted therapies.