ACS Chemical Biology最新文献

Bufadienolides are a class of steroids with a distinctive α-pyrone ring at C17, mostly produced by toads and consisting of over 100 orthologues. They exhibit potent cardiotonic and antitumor activities and are active ingredients of the traditional Chinese medicine Chansu and Cinobufacini. Direct extraction from toads is costly, and chemical synthesis is difficult, limiting the accessibility of active bufadienolides with diverse modifications and trace content. In this work, based on the transcriptome and genome analyses, using a yeast-based screening platform, we obtained eight cytochrome P450 (CYP) enzymes from toads, which catalyze the hydroxylation of bufalin and resibufogenin at different sites. Moreover, a reported fungal CYP enzyme Sth10 was found functioning in the modification of bufalin and resibufogenin at multiple sites. A total of 15 bufadienolides were produced and structurally identified, of which six were first discovered. All of the compounds were effective in inhibiting the proliferation of tumor cells, especially 19-hydroxy-bufalin (2) and 1β-hydroxy-bufalin (3), which were generated from bufalin hydroxylation catalyzed by CYP46A35. The catalytic efficiency of CYP46A35 was improved about six times and its substrate diversity was expanded to progesterone and testosterone, the common precursors for steroid drugs, achieving their efficient and site-specific hydroxylation. These findings elucidate the key modification process in the synthesis of bufadienolides by toads and provide an effective way for the synthesis of unavailable bufadienolides with site-specific modification and active potentials.

Lanmodulins are small, ∼110-residue proteins with four EF-hand motifs that demonstrate a picomolar affinity for lanthanide ions, making them efficient in the recovery and separation of these technologically important metals. In this study, we examine the thermodynamic and structural complexities of lanthanide ion binding to a 41-residue domain, EF 2–3, that constitutes the two highest-affinity metal-binding sites in the lanmodulin protein from Methylorubrum extorquens. Using a combination of circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), two-dimensional infrared (2D IR) spectroscopy, and molecular dynamics (MD) simulations, we characterize the metal binding capabilities of EF 2–3. ITC demonstrates that binding occurs between peptide and lanthanides with conditional dissociation constants (K_d) in the range 20–30 μM, with no significant differences in the K_d values for La³⁺, Eu³⁺, and Tb³⁺ at pH 7.4. In addition, CD spectroscopy suggests that only one binding site of EF 2–3 undergoes a significant conformational change in the presence of lanthanides. 2D IR spectroscopy demonstrates the presence of both mono- and bidentate binding configurations in EF 2–3 with all three lanthanides. MD simulations, supported by Eu³⁺ luminescence measurements, explore these results, suggesting a competition between water–lanthanide and carboxylate–lanthanide interactions in the EF 2–3 domain. These results underscore the role of the core helical bundle of the protein architecture in influencing binding affinities and communication between the metal-binding sites in the full-length protein.

Cysteine conjugation is widely used to constrain phage displayed peptides for the selection of cyclic peptides against specific targets. In this study, the nontoxic Bi³⁺ ion was used as a cysteine conjugation reagent to cross-link peptide libraries without compromising phage infectivity. We constructed a randomized 3-cysteine peptide library and cyclized it with Bi³⁺, followed by a selection against the maltose-binding protein as a model target. Next-generation sequencing of selection samples revealed the enrichment of peptides containing clear consensus sequences. Chemically synthesized linear and Bi³⁺ cyclized peptides were used for affinity validation. The cyclized peptide showed a hundred-fold better affinity (0.31 ± 0.04 μM) than the linear form (39 ± 6 μM). Overall, our study proved the feasibility of developing Bi³⁺ constrained bicyclic peptides against a specific target using phage display, which would potentially accelerate the development of new peptide–bismuth bicycles for therapeutic or diagnostic applications.

The prevalence of multidrug-resistant (MDR) pathogens combined with a decline in antibiotic discovery presents a major challenge for health care. To refill the discovery pipeline, we need to find new ways to uncover new chemical entities. Here, we report the global genome mining-guided discovery of new lipopeptide antibiotics tridecaptin A₅ and tridecaptin D, which exhibit unusual bioactivities within their class. The change in the antibacterial spectrum of Oct-TriA₅ was explained solely by a Phe to Trp substitution as compared to Oct-TriA₁, while Oct-TriD contained 6 substitutions. Metabolomic analysis of producer Paenibacillus sp. JJ-21 validated the predicted amino acid sequence of tridecaptin A₅. Screening of tridecaptin analogues substituted at position 9 identified Oct-His9 as a potent congener with exceptional efficacy against Pseudomonas aeruginosa and reduced hemolytic and cytotoxic properties. Our work highlights the promise of tridecaptin analogues to combat MDR pathogens.

The Craspase CRISPR-Cas effector consists of the RNA-guided ribonuclease gRAMP and the protease TPR-CHAT, coupling target RNA recognition to protease activation. The natural substrate of Craspase is Csx30, a protein cleaved in two fragments that subsequently activates downstream antiviral pathways. Here, we determined the protease substrate specificity of Craspase from Candidatus “Jettenia caeni” (Jc-Craspase). We find that Jc-Craspase cleaves Jc-Csx30 in a target RNA-dependent fashion in A|S, which is different from the sites found in two other studied Craspases (L|D and M|K for Candidatus “Scalindua brodae” and Desulfonema ishimotonii, respectively). The fact that Craspase cleaves a nonconserved site across orthologs indicates the evolution of specific protein interactions between Craspase and its respective Csx30 target protein. The Craspase family thus represents a panel of proteases with different substrate specificities, which we exploited for the development of a readout for multiplexed RNA detection.

The advent of ultra-large libraries of drug-like compounds has significantly broadened the possibilities in structure-based virtual screening, accelerating the discovery and optimization of high-quality lead chemotypes for diverse clinical targets. Compared to traditional high-throughput screening, which is constrained to libraries of approximately one million compounds, the ultra-large virtual screening approach offers substantial advantages in both cost and time efficiency. By expanding the chemical space with compounds synthesized from easily accessible and reproducible reactions and utilizing a large, diverse set of building blocks, we can enhance both the diversity and quality of the discovered lead chemotypes. In this study, we explore new chemical spaces using reactions of sulfur(VI) fluorides to create a combinatorial library consisting of several hundred million compounds. We screened this virtual library for cannabinoid type II receptor (CB₂) antagonists using the high-resolution structure in conjunction with a rationally designed antagonist, AM10257. The top-predicted compounds were then synthesized and tested in vitro for CB₂ binding and functional antagonism, achieving an experimentally validated hit rate of 55%. Our findings demonstrate the effectiveness of reliable reactions, such as sulfur fluoride exchange, in diversifying ultra-large chemical spaces and facilitate the discovery of new lead compounds for important biological targets.

Neuromodulators play crucial roles in regulating neuronal activity and affecting various aspects of brain functions, including learning, memory, cognitive functions, emotional states, and pain modulation. In this Account, we describe our group’s efforts in designing sensors and tools for studying neuromodulation. Our lab focuses on developing new classes of integrators that can detect neuromodulators across the whole brain while leaving a mark for further imaging analysis at high spatial resolution. Our lab also designed chemical- and light-dependent protein switches for controlling peptide activity to potentially modulate the endogenous receptors of the neuromodulatory system in order to study the causal effects of selective neuronal pathways.

Fungal paracyclophane-decahydrofluorene-containing natural products are complex polycyclic metabolites derived from similar hybrid PKS-NRPS pathways. Herein we studied the biosynthesis of pyrrocidines, one representative of this family, by gene inactivation in the producer Sarocladium zeae coupled to thorough metabolic analysis and molecular modeling of key enzymes. We characterized nine pyrrocidines and analogues as well as in mutants a variety of accumulating metabolites with new structures including rare cis-decalin, cytochalasan, and fused 6/15/5 macrocycles. This diversity highlights the extraordinary plasticity of the pyrrocidine biosynthetic gene cluster. From accumulating metabolites, we delineated the scenario of pyrrocidine biosynthesis. The ring A of the decahydrofluorene is installed by PrcB, a membrane-bound cyclizing isomerase, on a PKS-NRPS-derived pyrrolidone precursor. Docking experiments in PrcB allowed us to characterize the active site suggesting a mechanism triggered by arginine-mediated deprotonation at the terminal methyl of the substrate. Next, two integral membrane proteins, PrcD and PrcE, each predicted as a four-helix bundle, perform hydroxylation of the pyrrolidone ring and paracyclophane formation, respectively. Modelization of PrcE highlights a topological homology with vitamin K oxido-reductase and the presence of a disulfide bond. Our results suggest a previously unsuspected coupling mechanism via a transient loss of aromaticity of tyrosine residue to form the strained paracyclophane motif. Finally, the lipocalin-like protein PrcX drives the exo-cycloaddition yielding ring B and C of the decahydrofluorene to afford pyrrocidine A, which is transformed by a reductase PrcI to form pyrrocidine B. These insights will greatly facilitate the microbial production of pyrrocidine analogues by synthetic biology.

Eremophilanes exhibit diverse biological activities and chemical structures. This study reports the bioinformatics-guided reconstitution of the biosynthetic machinery of fungal eremophilanes, eremofortin C and sporogen-AO1, to elucidate their biosynthetic pathways. Their biosyntheses include P450-catalyzed multistep oxidation and enzyme-catalyzed isomerization by the DUF3237 family protein. Successful characterization of six P450s enabled us to discuss the functions of eremophilane P450s in putative eremophilane biosynthetic gene clusters, providing opportunities to understand the oxidative modification pathways of fungal eremophilanes.

Covalent inhibition has seen a resurgence in the last several years. Although long-plagued by concerns of off-target effects due to nonspecific reactions leading to covalent adducts, there has been success in developing covalent inhibitors, especially within the field of anticancer therapy. Covalent inhibitors can have an advantage over noncovalent inhibitors since the formation of a covalent adduct may serve as an additional mode of selectivity due to the intrinsic reactivity of the target protein that is absent in many other proteins. Unfortunately, many covalent inhibitors form irreversible adducts with off-target proteins, which can lead to considerable side-effects. By designing the inhibitor to form reversible covalent adducts, one can leverage competing on/off kinetics in complex formation by taking advantage of the law of mass action. Although covalent adducts do form with off-target proteins, the reversible nature of inhibition prevents accumulation of the off-target adduct, thus limiting side-effects. In this perspective, we outline important characteristics of reversible covalent inhibitors, including examples and a guide for inhibitor development.