ACS Chemical Biology最新文献

Cytidine analogues have conferred highly efficacious antimetabolites with broad utility as antiviral and anticancer agents. However, in many cases, human cytidine deaminase (CDA) converts the cytidine-based inhibitor into an inactive uridine metabolite with diminished potency. Inhibitors of CDA are useful agents to boost the efficacy of cytosine- and cytidine-containing drugs by inhibiting their rapid degradation. Toward the goal of developing CDA inhibitors, and our overarching interest in cytosine deaminase enzymes in general, we developed a real-time fluorescence-based deamination activity assay for CDA using isomorphic nucleoside analogues. Base-modified pyrimidine nucleosides that exhibit differential fluorescence properties as either the cytosine or uracil nucleobase were developed. We found that 5-benzo-2-furyl-2′-deoxycytidine is the best fluorescence reporter when implemented in a CDA enzyme activity assay, which permits detailed measurements of the kinetics of CDA activity in the presence or absence of inhibitors. Utilizing this assay, we then screened our in-house collection of 1054 fragments and found 23 hits that were further studied. Two fragment-sized CDA inhibitors with low micromolar potency (200–300 μM) and good ligand efficiency (>0.3) were identified, thereby conferring promising starting points for future inhibitor development.

Riboswitches are structured RNA elements that regulate gene expression by sensing and binding small molecules. The guanidine-III riboswitch, a critical bacterial regulator responding to guanidine toxicity, undergoes precise conformational changes that remain poorly characterized at a dynamic, mechanistic level. In this study, we employed single-molecule Förster Resonance Energy Transfer (smFRET) coupled with molecular dynamics (MD) simulations to delineate how the guanidine-III riboswitch transitions among distinct conformational states. We identify three principal states─an extended (E-state), a compacted partially folded intermediate (I-state), and a folded pseudoknot structure (F-state)─with rapid interconversion in the absence of ligand. Mg²⁺ ions shift the conformational equilibrium toward the I- and F-states, reducing the reverse transition rates by up to 20-fold and enhancing guanidine binding affinity. Guanidine binding further suppresses the reverse transitions, kinetically trapping the riboswitch into its active folded state primarily through a conformational selection mechanism, with additional induced-fit contributions observed for the E-F transition. This work provides insight into the dynamic pathway by which the guanidine-III riboswitch integrates ionic and ligand cues, supporting its role in gene regulatory responses in bacteria.

Cystic fibrosis (CF) is caused by loss-of-function mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel predominantly expressed on the apical membrane of epithelial cells. Reduced Cl^– and HCO₃^– secretion due to dysfunctional CFTR results in a decrease in lung function and is the leading cause of morbidity in individuals with CF. Recent therapies, known as highly effective CFTR modulator therapy (HEMT), help improve the lung function in individuals with specific CF-causing mutations by enhancing the folding, trafficking, and gating of CFTR. However, variability in HEMT responsiveness leads to suboptimal clinical outcomes in some people with CF undergoing modulator therapy. A potential strategy is to complement their function with a CFTR-independent mechanism. One possibility is the use of ion channel-forming small molecules such as amphotericin B, which has shown promise in restoring function and host defenses in CF airway disease models. Amphotericin B functions as a molecular prosthetic for CFTR and may thus complement CFTR modulators. Here, we show that cotreatment of CF airway epithelia with HEMT and amphotericin B results in greater increases in both HCO₃^– secretory flux and ASL pH compared to treatment with either agent alone. These findings suggest that coadministration of CFTR modulators and molecular prosthetics may provide additive therapeutic benefits for individuals with CF.

Nucleoside analogues have been extensively used to treat viral and bacterial infections and cancer for more than 60 years. However, their chemical synthesis is complex and often requires multiple steps and a dedicated synthetic route for every new nucleoside to be produced. Wild type nucleoside 2′-deoxyribosyltransferase enzymes are promising for biocatalysis. Guided by the structure of the enzyme from the thermophilic organism Chroococcidiopsis thermalis PCC 7203 (CtNDT) bound to the ribonucleoside analogue Immucillin-H, we designed mutants of CtNDT and the psychrotolerant Bacillus psychrosaccharolyticus (BpNDT) to improve catalytic efficiency with 3′-deoxynucleosides and ribonucleosides, while maintaining nucleobase promiscuity to generate over 100 distinct nucleoside products. Enhanced catalytic efficiency toward ribonucleosides and 3′-deoxyribonucleosides occurred via gains in turnover rate, rather than improved substrate binding. We determined the crystal structures of two engineered variants as well as kinetic parameters with different substrates, unveiling molecular details underlying their expanded substrate scope. Our rational approach generated robust enzymes and a roadmap for reaction conditions applicable to a wide variety of substrates.

Candida albicans is an opportunistic fungal pathogen that causes millions of infections per year, for which more efficacious treatments are needed. Observations that azole antifungals incite C. albicans to adjust a variety of metal-dependent processes led us to hypothesize that vulnerabilities in metallohomeostasis incurred by drug stress could be leveraged by compounds that interrupt metal trafficking. Here, we show that tetrathiomolybdate (TTM), a copper (Cu) chelator that interferes with Cu trafficking and use, inhibits growth of C. albicans on its own and synergizes with select azoles to enhance antifungal activity. Proteomic and biochemical experiments revealed that TTM causes differential expression and stabilization of proteins involved in fermentation and oxidative stress responses in C. albicans. The synergy between TTM and azoles was found to arise from increased expression and stability of the nitric oxide dioxygenase Yhb1, a response driven by the decreased stability and activity incurred by TTM of CuZn superoxide dismutase 1. Addition of imidazole-based antifungals highjacks this stress response by inhibiting Yhb1. This study highlights the centrality of Cu homeostasis as a regulatory hub connecting energy production, oxidative stress management, and overall cellular fitness in ways that can be pharmacologically manipulated to enhance efficacy of existing antifungal agents.

Guanine nucleotide dissociation inhibitors (GDIs) proteins, including RhoGDI2, regulate the functions of Ras superfamily proteins that are known to be important cancer drug targets. Given the challenges in directly targeting Ras superfamily proteins with small molecules, targeting GDIs represents a unique opportunity but has seen limited success. In this work, we discovered HR3119 as the first ligand of RhoGDI2 with low-micromolar affinity (K_d = 8 μM) starting from a millimolar binding affinity fragment hit (K_d = 714 μM). HR3119 and its derivatives were rationally designed based on a series of ligand-bound RhoGDI2 crystal structures. HR3119 occupies the protein–protein interaction interface between RhoGDI2 and its endogenous ligand Rac1 to disrupt RhoGDI2–Rac1 binding. Interestingly, the complex structure suggests that (6R)-HR3119 preferentially bound to RhoGDI2 when crystallized with a racemic mixture. The purified (6R)-HR3119 demonstrated a nearly 100-fold binding affinity advantage compared to (6S)-HR3119. Finally, (6R)-HR3119 engaged with RhoGDI2 in cells and suppressed the migration of aggressive breast cancer cells. Our work provides insights into the discovery of small-molecule compounds targeting RhoGDI2 in terms of methodology, chemistry starting points, compound design, and phenotype studies, underscoring exciting new perspectives in early drug discovery.

Protein N-glycosylation contributes to folding and quality control of secretory proteins involved in protein misfolding diseases. A central quality control machinery of nascent glycoproteins in the endoplasmic reticulum (ER) is the calnexin/calreticulin (CNX/CRT) cycle. This cycle assists and checks protein folding by monitoring glycan structure, however how terminally misfolded glycoproteins are discharged from the cycle has remained unclear. Here, we leveraged chemical probes to identify a previously uncharacterized ER endo-α-mannosidase complex (ER-EM) that provides this missing release step. ER-EM selectively cleaves the terminal Glc-Man disaccharide from glucosylated high-mannose glycans only when the glycan is attached to a hydrophobic aglycone─an intrinsic marker of misfolded proteins─thereby converting Glc₁Man₉GlcNAc₂ to Man_8AGlcNAc₂ glycans that cannot bind CNX/CRT. This activity is allosterically stimulated by hydrophobic ligands and shares the same aglycone preference as the folding sensor UDP-glucose: glycoprotein glucosyltransferase 1 (UGGT1), creating a two-tier surveillance system in which UGGT1 reglucosylates incompletely folded proteins, whereas ER-EM ejects those that fail to mature. Proteomic and native-gel analyses revealed that ER-EM is an ∼ 800 kDa assembly composed of at least carboxylesterase 1D (Ces1d), ERp57 and UGGT1; the lack of activity of recombinant Ces1d alone underscores that the catalytic function arises only through the concerted action of this multisubunit complex. ER-EM therefore acts as a folding-status-dependent triage factor that liberates terminally misfolded glycoproteins from the CNX/CRT cycle and targets them for degradation, adding a critical new branch to the ER quality-control network.

Dopamine D1 receptor (D1R) plays key roles in health and disease. D1R is broadly expressed throughout the brain and body and is dynamically activated in response to endogenous dopamine, making it difficult to target this receptor with sufficient precision. We previously developed a robust light-activatable, tetherable agonist for D1R, wherein a temporally precise photoswitch (the P compound) binds to a genetically encoded membrane anchoring protein (the M protein) in specific brain locations and cell types. Here we extended our approach by developing a complementary antagonist P compound that could be used to block specific populations of D1R in the brain with precise timing. Together, we have generated a robust toolkit for interrogating D1R function in the brain with unprecedented precision.

Protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification, known as O-GlcNAcylation, is an essential post-translational modification (PTM) that plays critical roles in regulating various cellular processes, ranging from transcription and signal transduction to protein degradation. O-GlcNAcylation levels are dynamically regulated by a single pair of human enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Dysregulation of O-GlcNAcylation has been implicated in many diseases, including cancer, diabetes, neurodegeneration, and cardiovascular disorders. In the past decade, remarkable progress has been achieved regarding the structures of OGT and OGA proteins, as well as a series of innovative chemical and engineered tools that inhibit or induce the activities of these enzymes. While initial studies mainly focused on the catalytic domains of these enzymes, recent research has begun to uncover the structural and functional roles of non-catalytic regions. Notably, domains such as OGT’s tetratricopeptide repeat (TPR) and intervening domain (Int-D), as well as OGA’s stalk domain and pseudo histone acetyltransferase (pHAT) domain, have emerged as critical contributors to enzyme functions. This Account discusses recent progress in studying these essential enzymes, especially highlighting their unique structural features and intrinsic flexibility as potential mechanisms underlying their substrate recognition and functional regulation. New perspectives and research directions are also discussed. Such information is expected to facilitate the rational design of novel modulators of OGT and OGA to enable more specific functional control and potential treatment of disease.