ACS Chemical Biology最新文献

Bacteria produce natural products for multiple activities including cell signaling, competition, and virulence. Another important role is nutrient acquisition, which is carried out by a diverse pool of iron-sequestering molecules called siderophores. Siderophores can be produced by nonribosomal peptide synthetases (NRPSs), NRPS-independent siderophore (NIS) synthetases, or both pathways, as shown in the biosynthesis of the recently described hybrid siderophore nocardichelin. Nocardichelin and the hydroxamate siderophore desferrioxamine B both contain N-hydroxy-N-succinylcadaverine (HSC) and N-hydroxy-N-acylcadaverine (HRC) biosynthetic intermediates. Some desferrioxamine pathways contain DesG, an N-terminal nucleophile (Ntn) hydrolase, that has been proposed to hydrolyze HSC to create N-hydroxycadaverine and succinate, which may help to modulate levels of the two building blocks, HSC and HRC. The biosynthetic gene cluster of nocardichelin also harbors an Ntn hydrolase and homologue of DesG. To explore the biochemical function of NcdG and its role in nocardichelin biosynthesis, we examined its structure and function. We demonstrate that NcdG cleaves the hydroxamate bond of HSC and HSC dimers and also determine the structure of NcdG, an acyl hydroxamate-cleaving Ntn hydrolase.

Inositols are a family of cyclic sugar alcohols comprising nine stereoisomers. myo-Inositol is the most abundant isomer found in humans and has been studied most extensively. It plays an important role in osmoregulation and is incorporated into membrane-anchored phosphatidylinositols. scyllo-Inositol is the second most abundant inositol isomer in the human brain, and aberrant concentrations are associated with various diseases; however, its biological functions remain unclear. Here, the development and application of [¹³C₆]scyllo-inositol as an isotopic tracer to study its metabolism are reported. A concise and robust synthetic route was established to obtain [¹³C₆]scyllo-inositol from [¹³C₆]myo-inositol in good yield. The uptake of [¹³C₆]scyllo-inositol at concentrations used in clinical trials and the responses of endogenous inositol isomers were measured in multiple immortalized mammalian cell lines by hydrophilic liquid interaction (HILIC)-MS/MS. [¹³C₆]scyllo-Inositol proved to be a versatile isotopic tracer when coupled with mass spectrometry (MS)-based lipidomics and 2D nuclear magnetic resonance (NMR) experiments. These experiments provide evidence that scyllo-inositol is incorporated into phosphatidylinositols in different immortalized mammalian cell lines and suggest a previously underappreciated role of scyllo-inositol. Future research utilizing [¹³C₆]scyllo-inositol can elucidate scyllo-inositol metabolism and its role in physiological and diseased states.

A subset of endogenous steroids can be formed in the brain and exhibit brain-specific activities linked to neuronal health and disease. Despite the biological relevance of these so-called neurosteroids, molecular profiling of their effects is incomplete and their signature in the nuclear hormone receptor family that mediates signals of classical steroid hormones is elusive. Here, we present a comprehensive characterization of neurosteroids for nuclear receptor modulation and reveal pronounced inverse RORγ agonism as a relevant molecular mechanism of several abundant neurosteroids, with an impact on circadian rhythms and transcriptomic profiles in cellular settings. Structural analysis of the DHEA-RORγ interaction demonstrated orthosteric binding and pronounced rearrangements in the RORγ ligand binding domain to a fully repressed state, rationalizing the strong inverse agonist efficacy.

UBA5 is the E1 enzyme that initiates UFMylation, a ubiquitin-like modification implicated in proteostasis, neurodegeneration, and cancer. Here, we report a chemical biology approach to interrogate UBA5 function through small-molecule inhibition. We developed a robust high-throughput screening assay based on AMP-Glo^TM and screened a library of blood–brain barrier-permeable compounds, identifying five inhibitors spanning three distinct scaffolds with low-micromolar potency. Orthogonal biochemical and gel-based assays confirmed that these compounds directly inhibit UBA5-mediated UFM1 activation and conjugation, with selectivity over other E1 enzymes, including UBA1. In cells, these compounds suppressed endogenous UFMylation without significantly affecting global polyubiquitination, supporting pathway and target engagement. Together, these studies establish UBA5 as a tractable enzymatic target and provide first-in-class chemical tools to probe UFMylation. Given the emerging role of UFMylation in disease, these inhibitors offer a foundation for developing therapeutics targeting proteostasis pathways.

Fe–S clusters are emerging as important cofactors in viral replication but are frequently misassigned as Zn due to their O₂-sensitivity and overlapping cysteine-based coordination chemistry. The Hepatitis B virus regulatory protein HBx, which is essential for viral replication and hepatocarcinogenesis, has long remained mechanistically intractable because of uncertainty surrounding its physiologically relevant metallocofactor. Although HBx can bind either an Fe–S cluster or Zn, its intrinsic disorder and extensive mutational tolerance have hindered precise characterization of its metal-binding environment. Here, we combine chemoproteomics with HYSCORE spectroscopy to define the metal-coordinating ligands in HBx and overcome limitations associated with conventional mutational analysis of disordered proteins. We exclude histidine coordination and identify C61, C69, C143, and C148 as the primary cysteine ligands for Fe–S cluster binding, with C137 functioning as a conditional auxiliary ligand. These residues also support Zn binding and overlap with regions implicated in HBx transactivation and clinically relevant variants. In addition, HBx engages the host cytosolic Fe–S cluster assembly machinery and displays sensitivity to Fe–S-targeting reagents, behavior consistent with Fe–S cluster acquisition and cofactor lability. Together, these findings propose HBx as an Fe–S cluster-associated viral protein and expand the growing class of viral Fe–S proteins that are critical for infection.

Human mucin-1 (MUC1) is an attractive tumor antigen due to its high expression levels on tumor cells and its important roles in tumor development. Due to the aberrant glycosylation of MUC1 on tumor cells, tumor-associated MUC1 (^tMUC1) has a shortened O-glycan attached on the peptide backbone, distinguishing it from its counterpart on normal cells. Preclinical evaluation of ^tMUC1-based vaccine candidates typically involved the immunization of mice that are tumor-free. However, tumor development is often associated with immune suppression. To better mimic clinical conditions encountered in cancer treatment, a breast cancer mouse model was built by crossing mouse mammary tumor virus (MMTV)-polyoma middle T (PyMT) mice with human MUC1 transgenic (MUC1.Tg) mice. The resulting female double-transgenic mice (MUC1/MMTV) spontaneously develop palpable breast cancer from 6 weeks of age. A promising vaccine candidate composed of a short 9-amino-acid Thomsen-nouveau (Tn) antigen containing ^tMUC1 glycopeptide conjugated with bacteriophage Qβ was evaluated in MUC1/MMTV mice. The vaccine was found to be immunogenic and capable of inducing high levels of anti-^tMUC1 IgG antibody responses. The antibodies were selective toward both the glycan and the peptide sequence of ^tMUC1, as deciphered from a glycopeptide microarray study. Furthermore, the induced antibodies bound tumor cells in a MUC1-dependent manner and could kill a range of tumor cells via complement-dependent cytotoxicity. Vaccination with a Qβ-^tMUC1 conjugate significantly prolonged the survival of MUC1/MMTV mice, with 83% of treated MUC1/MMTV mice outliving the control group. The Qβ-^tMUC1 vaccine exhibited promising results in an aggressive and rapidly growing spontaneous tumor model, laying the groundwork for its clinical translation to human patients.

Irreversible covalent inhibitors have garnered significant attention in recent years. Despite encouraging progress, the vast majority contain electrophiles that target the least abundant amino acid, cysteine, substantially limiting target inhibitor design for therapeutic intervention. Here, we generalize 2-ethynylbenzaldehyde as a proximity-induced electrophile for generating irreversible covalent peptide and protein inhibitors that specifically target native lysine residues. Leveraging this warhead, we designed a covalent de novo peptide that potently engages MCL1 to block its interaction with Bak. We show it is faster, more site-selective, and increases potency by 61-fold for MCL1 relative to a sulfonyl fluoride warhead. Additionally, with the guide of a computational script to predict "reactive hotspots" at the protein level, we developed a minibinder that labels PD-L1 in vitro and in live cells, displays a slower off-rate, and potently blocks the native PD-1 and PD-L1. These results establish isoquinolinium capture as a promising strategy to inhibit protein–protein interactions and for the development of novel covalent peptide and protein therapeutics.

Activity-based ubiquitin probes have emerged as powerful tools for dissecting protein ubiquitination. In this work, we discovered that an α-amidomethyl-substituted acrylate electrophile exhibits markedly enhanced reactivity. Based on this finding, we designed and synthesized a new activity-based probe, ubiquitin-α-amidomethyl acrylate (Ub-AMA). In vitro biochemical assays demonstrated that Ub-AMA effectively crosslinked with deubiquitinases, E1 and E3s. In addition, compared to the classic Ub-VME and Ub-PA probes, Ub-AMA achieves improved labeling efficiency for E2 enzymes. Finally, proteomic profiling in cell lysates confirmed that Ub-AMA can capture E1, E2, and E3 enzymes as well as DUBs in a complex biological environment. In summary, the Ub-AMA probe developed here provides a new tool for in-depth analysis of the dynamic regulatory mechanisms of ubiquitination. Moreover, this work offers valuable insights for the design of other activity-based probes based on α,β-unsaturated carbonyl scaffolds.

Nonribosomal peptide synthetases (NRPSs) are modular assembly-line enzymes that generate structurally diverse and biologically active natural products. Although adenylation (A) domain selectivity has been extensively characterized and engineered, the specificity of condensation (C) domains, which catalyze peptide bond formation and can constrain NRPS reprogramming, remains less well understood. Here, we report a systematic functional analysis of VibH, a stand-alone C domain VibH from the vibriobactin biosynthetic pathway. By integrating VibH with wild-type and engineered variants of the upstream aryl acid A domain EntE, we bypassed intrinsic A-domain constraints and independently interrogated donor- and acceptor-site substrate tolerance. The donor site of VibH exhibited stringent specificity, accepting only mono- and disubstituted benzoic acid derivatives closely resembling 2,3-dihydroxybenzoic acid (DHB), whereas bulkier aryl substrates were not processed. In contrast, the acceptor site displayed broad tolerance toward structurally diverse amines. Monoamines showed clear chain-length dependence, with productive turnover restricted to medium-chain substrates, whereas diamines were more broadly accepted. Systematic analysis further revealed distance-dependent steric tolerance, in which bulky substituents were accommodated when positioned distal to the reactive amine but were restricted beyond an upper steric threshold. This engineered reconstitution strategy enabled the synthesis of numerous non-native amide conjugates and uncovered an asymmetric substrate-recognition architecture characterized by a stringent donor site and a permissive yet spatially constrained acceptor site. Molecular docking analysis provided a structural rationale for these trends by suggesting distinct binding modes for native and non-native donor substrates and distance-dependent accommodation of bulky amine acceptors. These findings provide mechanistic insights into C-domain specificity and establish a framework for rational NRPS reprogramming.

Backbone-cyclized conotoxin Vc1.1 is a potent GABA_B receptor agonist and analgesic drug lead for treating neuropathic pain. Here, we describe two yeast-based strategies for the direct recombinant production of this cyclic, disulfide-rich peptide. In the first approach, using Pichia pastoris, a fusion protein comprising the conotoxin precursor and a full-length asparaginyl ligase connected by a poly-His linker was secreted into the culture medium. Cyclization of the secreted α-conotoxin was initiated by a two-step pH adjustment protocol for generating the correctly folded conotoxin. In the second approach, Saccharomyces cerevisiae was engineered to display the activated ligase on its cell surface while secreting Vc1.1 precursors extracellularly. Cyclized peptides were detected in the culture supernatant without further processing. These two strategies provide efficient routes for the direct recombinant production of disulfide-rich and backbone-cyclized peptides.