RSC Chemical Biology最新文献

Enzymatically activated prodrugs can enable context-specific target inhibition. AKR1C3 is an NADPH-dependent aldo-ketoreductase involved in androgen metabolism, prostaglandin synthesis, and cell proliferation that is overexpressed in tumors, making it an ideal candidate for tumor-specific prodrug activation. Reported prodrugs that exploit AKR1C3 catalytic activity release DNA-intercalating toxins or other non-selective poisons upon enzymatic activation. OBI-3424, a prodrug of a DNA alkylating agent, is a prominent example of this strategy. To extend this concept to selective enzymatic inhibitors, we have developed AKR1C3-activated prodrugs of OTS964, a CDK11 inhibitor. We have probed the activities of the compounds with biochemical and cellular assays, finding specific activation of the lead prodrug by AKR1C3. Upon enzymatic conversion, the compound recapitulates the cellular activity of the parent compound. These results demonstrate that the AKR1C3-activated prodrug strategy can be used to convert selective kinase inhibitors into context-dependent prodrugs. Extension of this approach may enable synthesis of prodrugs for targeted therapies that spare normal tissue, further improving their therapeutic windows.

Imaging the activities of hydrolases using molecular imaging probes can reveal underlying molecular mechanisms in the context of cells to organisms and their correlation with different pathological conditions can be used in diagnosis. Due to the nature of hydrolases, substrate-based probes can take advantage of their catalytic cycles to free reporter moieties that can generate amplified signal. This is less ideal in the context of cell- or organism-based detection, as the reporter moiety can easily diffuse away from the target site upon activation. Therefore, spatial resolution is a key factor for probe sensitivity. One strategy to improve the spatial resolution is to form a covalent linkage between the reporter moiety and intracellular proteins upon probe activation by the enzyme via a reactive intermediate. In this work, we tuned the reactivity of the quinone methide intermediate by synthesizing fluorescent probes containing different modifications to the phenol linker for β-galactosidase activation. The labeling efficacy of these probes was evaluated using fluorescence gel electrophoresis, flow cytometry, and fluorescence cell imaging. This study provides insights into the further development of hydrolase-targeting probes for cell- or organism-based imaging with enhanced efficiency via in situ labeling.

Changing the backbone connectivity of proteins can impart useful new traits while maintaining essential structural and functional features. In design of artificial proteomimetic agents, backbone modification is usually isolated to sites that are solvent-exposed in the folded state, as similar changes at buried residues can alter the fold. Recent work has shown that core backbone modification without structural perturbation is possible; however, the modifications in that study were consistently destabilizing and made in a prototype of exceptionally high conformational stability. Here, we report efforts to broaden the scope and improve the efficacy of core backbone engineering by applying it to the C-terminal subdomain of villin headpiece. A series of variants are prepared in which different artificial residue types are incorporated at core positions throughout the sequence, including a crucial aromatic triad. Impacts on folding energetics are quantified by biophysical methods, and high-resolution structures of several variants determined by NMR. We go on to construct a variant with ∼40% of its core modified that adopts a fold identical to the prototype while showing enhanced thermodynamic stability.

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Phosphonate natural products (P-NPs) represent a unique and underexplored class of bioactive compounds with significant pharmaceutical and biotechnological potential. Many novel P-NPs with promising bioactivities were identified in recent years by genome mining of actinomycetes. The DSMZ strain collection harbors more than 6000 actinobacterial strains including an increasing number of genome sequenced strains. In this study, 940 genome-sequenced actinomycetes from the DSMZ and University of Tübingen strain collections were screened for the presence of phosphonate biosynthetic gene clusters (P-BGCs) by searching for the conserved pepM gene. This effort led to the identification of 54 potential phosphonate producer strains. Subsequent bioassays with a phosphonate-sensitive E. coli test strain showed activity for 17 strains, and ³¹P NMR spectroscopic analysis of culture supernatants confirmed phosphonate production for 21 strains, including the rare actinomycete Kitasatospora fiedleri DSM 114396^T. The functionality of the unique K. fiedleri P-BGC was verified by pepM gene deletion, which abolished phosphonate production in K. fiedleri, whereas overexpression of a cluster-situated LuxR-like regulator improved phosphonate production. These findings highlight the P-NP biosynthetic potential of actinomycetes and pave the way for discovering novel bioactive phosphonates.

Iterative polyketide synthases (iPKSs) rely on communication between acyl carrier protein (ACP) and acyltransferase (AT) domains to ensure efficient delivery of starter and extender substrates during biosynthesis. However, the molecular determinants governing the AT:ACP interface remain poorly understood. Here, we use the fungal highly reducing PKS, SimG, a component of the cyclosporin biosynthetic pathway, as a model system to dissect the AT:ACP interface. Using alanine scanning mutagenesis combined with a high-throughput intact protein mass spectrometry assay, we identified interface residues that affect AT:ACP interaction. These experimental constraints were used to guide docking and molecular dynamics simulations to produce a data-driven structural model of the SimG AT:ACP complex in a catalytically competent geometry. We also demonstrate that the SimG AT domain transacylates ACP domains from a range of fungal PKS architectural classes, highlighting significant interface plasticity. These insights advance our fundamental understanding of domain communication in these enigmatic megasynthases and provide a foundation for rational engineering to expand substrate scope towards novel polyketide scaffolds.

Fluorescent probes for measuring the structure, dynamics, cellular localization, and biochemistry of DNA and RNA are useful for determining the regulatory mechanisms of gene expression. Intrinsically fluorescent, Watson–Crick-capable nucleobase analogues are especially powerful because they can precisely probe desired loci while minimally perturbing native nucleic acid function. Here, we study the fluorescent responses of the tricyclic pyrimidine analogue ^DEAtC to base pairing with adenine, guanine, and a set of noncanonical nucleobases in duplex DNA oligonucleotides. We find that single-stranded oligonucleotides containing one ^DEAtC exhibit up to a fivefold fluorescence increase upon hybrid duplex formation and base pairing with G, and a lesser degree of fluorescence turn-on when base pairing with inosine. Other purine nucleobases do not induce significant fluorescence turn-on. Solvent kinetic isotope effect measurements, excitation–emission matrix (EEM) analysis, and spectral comparisons indicate that fluorescence turn-on originates from base pairing-templated tautomerism. The non-emissive T-like form predominates in the single strand and in duplexes paired with A, whereas the emissive C-like tautomer is selectively stabilized upon duplex formation when paired with G. Density functional theory (DFT) calculations further support this tautomeric control model. Although base stacking influences overall brightness, it does not alter the mechanism or specificity of fluorescence turn-on. Modulation of emission through tautomeric control offers a powerful strategy for designing nucleobase analogues with base pairing-specific fluorescence responses.

Protein glycosylation is a very common post-translational modification seen in all branches of biology. The functional roles for protein glycosylation are many and varied, essential in eukaryotes but seemingly dispensable in bacteria. One group of bacteria where protein glycosylation has been looked at for at least 50 years are the actinobacteria, a large and diverse group of bacteria which include well know pathogens like Mycobacteria tuberculosis, Corynebacterium diphtheriae, and well know species important in biotechnology like Streptomyces lividans and Corynebacterium glutamicum. Actinobacterial protein glycosylation is a form of protein O-mannosylation which is found widely in eukaryotes from single celled yeast to complex multicellular organisms but is much less understood at the functional level. Very few direct roles for protein O-mannosylation have been described in the literature. This review examines newer findings from the actinobacterial research literature which with the help of glycoprotein models suggests how the glycans might play a role in actinobacterial growth and physiology.

We report here the use of Tris-BODIPY-OH as a scaffold for the multivalent display of sugar heads. A chloroacetyl thioether ligation reaction easily yields mannosylated BODIPYs, named Man₉-BODIPY and (Man-TEG)₉-BODIPY, which display nine mannose residues. Regardless of the linker length, both glycoBODIPYs provide an arrangement of mannose heads that allows for proper recognition by the carbohydrate binding domain of concanavalin A (ConA). Moreover, the interactions of Man₉-BODIPY with relevant human lectins, i.e. dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and langerin, were further investigated. The approach proposed is versatile and paves the way for the development of multivalent and fluorescent glyco-BODIPY probes useful to interrogate carbohydrate-lectin interactions in different biological contexts.

Streptomyces albus J1074 (now S. albidoflavus J1074) is a widely used heterologous host for natural product discovery due to its capacity to express biosynthetic gene clusters (BGCs) from diverse organisms. A derivative of this strain, S. albus Del14, enhances heterologous expression by reducing background metabolite production enabling the identification of the previously hidden BGC responsible for producing mansouramycins. In this study, we demonstrate the biosynthetic crosstalk between the native mansouramycin BGC in S. albus Del14 and introduced BGCs from three different organisms results in the production of novel compounds, some featuring rare and complex chemical scaffolds. These include malevonin, which combines NRPS- and mansouramycin-derived building blocks forming a fluorene scaffold, as well as 5′-chloromansouramycin D, a halogenated derivative of mansouramycin D. Additionally, we identified mansevorone, a compound structurally similar to mansouramycin D but utilizing a different tryptophan-derived C7 precursor. This precursor likely arises from the activation of native genes in the host S. albus Del14, triggered by SARP regulators present on the introduced BGC. These findings highlight the evolutionary significance of BGC interactions and underscore their potential as a powerful tool for discovering novel natural products, providing insights that could inform innovative strategies in biosynthetic engineering and the guided evolution of new bioactive compounds.