ACS Chemical Biology最新文献

Rhynchosporium commune, the causal agent of barley scald disease, poses a major threat to global barley production. Despite its significant impact, the molecular mechanisms underlying R. commune’s infection process remain largely unexplored. To address this, we analyzed the differential gene expression data of R. commune WAI453 cultivated under both in planta and in vitro conditions, aiming to identify secondary metabolite biosynthetic gene clusters that are potentially involved in the pathogenicity of R. commune. Our analysis revealed increased expression of a polyketide-terpene gene cluster (the rhy cluster), containing a specific myeloblastosis (MYB)-type transcription factor gene rhyM, during in planta growth. Overexpression of rhyM in an axenic culture activated the expression of the rhy cluster, resulting in the production of a series of new meroterpenoid metabolites, which we named rhynchospenes A–E. Their structures were elucidated through a combination of spectroscopic methods and single crystal X-ray diffraction analysis. Infiltration of rhynchospenes into barley leaves resulted in strong necrosis, with rhynchospene B demonstrating the highest phytotoxicity and causing necrosis at a minimum concentration of 50 ppm. Silencing rhyM in R. commune WAI453 confirmed the role of rhynchospenes as virulence factors in barley disease. The resulting mutant showed significantly reduced expression of the rhy cluster in planta compared to the wild-type strain and decreased virulence in seedling pathogenicity assays on barley. The characterization of the rhy cluster and rhynchospenes provided insights into the role of secondary metabolites in R. commune virulence and barley scald disease development. The study also highlights the potential use of MYB-type transcription factor overexpression in uncovering cryptic SMs involved in pathogenicity and host adaptations.

Allosteric regulation is achieved by regulatory domains that sense stimuli and induce conformational changes in the functional domain that performs the catalytic activity of the enzyme. Poly-ADP-ribose polymerases (PARPs) are modular enzymes present across all domains of life including Archaea, Bacteria, and Eukarya. A typical domain architecture of PARPs consists of a conserved C-terminal catalytic domain (CAT) associated with multiple distinct N-terminal sensory and/or regulatory domains which together serve as regulatory region (REG). In this study, we investigated whether REG of different orthologs and paralogs of PARPs from mammals (hPARP1 and hPARP2), plants (atPARP2), and bacteria (haPARP) can assemble with CAT of each other to generate functional chimeric assemblies. We have employed qualitative and quantitative enzyme activity assays along with binding studies to examine these in vitro chimeric assemblies. The cis-complemented REG and CAT of hPARP2 exhibited micromolar binding affinity, suggesting that these domains can interact independent of allosteric ligands. Also, our results show that REG and CAT of PARP proteins can assemble in a functionally active conformation in the presence of DNA implying that REG and CAT are not required to be present on a single polypeptide for catalytic activity stimulation. Interestingly, only CAT of atPARP2 displayed functional complementation with REG of the other studied PARPs. Conversely, REG of hPARP1 and atPARP2 failed to cross-complement CAT of other PARPs while REG of hPARP2 showed robust cross-complementation. Our novel studies on chimeric PARP assemblies can be developed as a powerful synthetic biology tool to interrogate and control their activities in living cells. In addition, by co-engineering non-complementing REG and CAT domains of different PARPs, new functional chimeric PARPs can be developed for selective allosteric ligand-dependent regulation of PARP systems. Furthermore, our study can facilitate the understanding of the coevolution of REG and CAT domains in PARP enzymes.

Proteolysis targeting chimeras (PROTACs) have gained considerable attention as a new modality in drug discovery. The development of PROTACs has been mainly focused on using CRBN (Cereblon) and VHL (Von Hippel-Lindau ligase) E3 ligase ligands. However, the considerable size of the human E3 ligase family, newly developed E3 ligase ligands, and the favorable druggability of some E3 ligase families hold the promise that novel degraders with unique pharmacological properties will be designed in the future using this large E3 ligase space. Here, we developed a workflow aiming to improve and streamline the evaluation of E3 ligase ligand efficiency for PROTAC development and the assessment of the corresponding “degradable” target space using broad-spectrum kinase inhibitors and the well-established VHL ligand VH032 as a validation system. Our study revealed VH032 linker attachment points that are highly efficient for kinase degradation as well as some of the pitfalls when using protein degradation as a readout. For instance, cytotoxicity was identified as a major mechanism leading to PROTAC- and VHL-independent kinase degradation. The combination of E3 ligase ligand negative controls, competition by kinase parent compounds, and neddylation and proteasome inhibitors was essential to distinguish between VHL-dependent and -independent kinase degradation events. We share here the findings and limitations of our study and hope that this study will provide guidance for future evaluations of new E3 ligase ligand systems for degrader development.

Acyclic and cyclic N-acyl O-aminophenol prodrugs of duocarmycin analogues were reported as members of a unique class of reductively cleaved prodrugs that map seamlessly onto the duocarmycin family of natural products. Although these prodrugs were explored with the expectations that they may be cleaved selectively within hypoxic tumor environments that have intrinsically higher concentrations of reducing nucleophiles, the remarkable stability of some such prodrugs suggests another mechanism of free drug release is operative. The prototype of such chemically unreactive N-acyl O-aminophenol prodrugs is 1, which proved remarkably efficacious in vivo in vertebrate tumor models; was found to lack the toxicity that is characteristic of traditional chemotherapeutic drugs as well as the free drugs in the class (e.g., myelosuppression); and displayed a preferential site (intracellular), a slow and sustained rate, and a potentially unique mechanism of free drug release. Herein, we detail studies that provide insights into this stereoselective mechanism of free drug release. Combined, the results of the studies are consistent with an exclusive protein-mediated (enantio)selective activation and free drug release from prodrug 1 by N–O bond cleavage preferentially in cancer cell lines versus cultured normal human cell lines effected by a cytosolic cysteine-based enzyme and suggest that the activating protein is one that is selectively expressed, upregulated, or preferentially activated in cancer cell lines, potentially constituting a new oncology targeted precision therapy.

With the growing number and diversity of known genome sequences, there is an increasing opportunity to regulate gene expression through synthetic, cell-permeable small molecules. Enhancing the DNA sequence recognition abilities of minor groove compounds has the potential to broaden their therapeutic applications with significant implications for areas such as modulating transcription factor activity. While various classes of minor groove binding agents can selectively identify pure AT and mixed AT and GC base pair(s) containing sequences, there remains a lack of compounds capable of distinguishing between different AT sequences. In this work, we report on the design compounds that exhibit selective binding to -TTAA- or -TATA- containing DNA minor groove sequences compared with other AT ones. Several studies have shown that the -AATT- and -TTAA- sequences have distinct physical and interaction properties, especially in terms of their different requirements for recognition in the minor groove. Achieving strong, selective minor groove binding at -TTAA- sequences has been challenging, but DB1003, a benzimidazole–furan–furan diamidine, has demonstrated cooperative dimeric binding activity at -TTAA-. It has significantly less binding preference for AATT. To better understand and modify the selectivity, we synthesized a set of rationally designed analogs of DB1003 by altering the position of the five-membered heterocyclic structure. Binding affinities and stoichiometries obtained from biosensor-surface plasmon resonance experiments show that DB1992, a benzimidazolefuran–thiophene diamidine, binds strongly to -TTAA- as a positive cooperative dimer with high cooperativity. The high-resolution crystal structure of the TTAA–DNA–DB1992 complex reveals that DB1992 binds as an antiparallel π-stacked dimer with numerous diverse contacts to the DNA minor groove. This distinctive binding arrangement and the properties of diamidines at the -TTAA- minor groove demonstrate that benzimidazole–furan–thiophene is a unique DNA binding pharmacophore. Competition mass spectroscopy and circular dichroism studies confirmed the binding stoichiometry and selectivity preference of the compounds for the -TTAA- sequence.

Proteasomes catalyze protein degradation in cells and play an integral role in cellular homeostasis. Its activity decreases with age alongside the load of defective proteins, resulting from mutations or oxidative stress-induced damage. Such proteins are prone to aggregation and, if not efficiently degraded, can form toxic oligomers and amyloid plaques. Developing an effective way to activate the proteasome could prevent such pathologies. Designing activators is not easy because they do not bind in the active site, which is well-defined and highly conserved, but away from it. The structures of proteasome complexes with natural activators can help here, but these are large proteins, some even multimeric, whose activity is difficult to replace with a small-molecule compound. Nevertheless, the use of fragments of such proteins makes it possible to accumulate knowledge about the relevance of various structural elements for efficient and selective activation. Here, we presented peptidic activators of the 20S proteasome, which were designed based on both the C-terminal sequence of the yeast proteasome activator, Blm10 protein, and the interactions predicted by molecular modeling. These Blm analogs were able to stimulate human 20S proteasome to more efficiently degrade both small fluorogenic substrates and proteins. The best activators also demonstrated their efficacy in cell lysates. X-ray crystallography indicated that an effective modulator can bind to several sites on the surface of the proteasome without causing permanent structural changes in its immediate vicinity but affecting the active sites.

Histone deacetylase (HDAC) enzymes remove acetyl groups from acetyllysine-containing proteins, including nucleosomal histones to control gene expression. Beyond fundamental cell biology, HDAC activity is linked to various cancers, with many HDAC inhibitors developed as anticancer therapeutics. Among the 11 metal-dependent HDAC proteins, the four class IIa isoforms (HDAC4, 5, 7, and 9) are “pseudodeacetylases” without measurable enzymatic activity due to mutation of a catalytic tyrosine. Deacetylase-related activities of class IIa HDAC proteins are attributed to scaffolding functions, where recruitment of an active HDAC isoform leads to bound substrate deacetylation. Scaffolding of class IIa proteins beyond simple recruitment of an active HDAC is only starting to emerge. This review explores the various scaffolding roles of HDAC7, including recently reported acetylation-mediated reversible scaffolding, which is a form of acetyllysine-binding reader function. Studying the functional roles of HDAC7 will provide molecular insight into normal and pathological conditions, which could facilitate drug design.

Second-generation siRNA-mimetic ratiometric pH probes (sMiRpH-2) were developed by hybridizing a 3′-FAM-labeled 2′-OMe RNA strand with a 3′-Cy5-labeled 25mer RNA strand. These duplexes demonstrated the silencing of cytoplasmic mRNA targets in HeLa cells as measured by RT-qPCR and supported by western blot analysis. Fluorescence intensity and lifetime measurements revealed that a single guanosine (G) positioned adjacent to FAM achieves substantial static quenching at pH 5, with additional collisional quenching rendering the dye almost nonemissive. A FAM-G π–π stacking interaction was evidenced by a red-shifted absorbance spectrum for FAM. Decreased quenching at near-neutral pH enhances the FAM dynamic range in the physiologic pH window and improves the differentiation in cells between endocytic entrapment and cytoplasmic release. Flow cytometric analysis of intracellular pH and uptake using sMiRpH-2 was corroborated by live cell confocal microscopy and found to be predictive of knockdown efficacy. A sMiRpH-2 probe successfully predicted the relative efficacy of two transfection agents in more challenging SK-OV-3 cells, which highlights its use for the rapid assessment of nonviral siRNA delivery vectors.

The National Institutes of Health molecular probe ML283 was synthesized as a potent, selective inhibitor of the helicase encoded by the hepatitis C virus. Because modeling with AutoDock Vina predicted that ML283 might bind the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein 13 (nsp13) helicase, the effects of a collection of ML283 analogs and other hepatitis C virus (HCV) helicase inhibitors on the SARS-CoV-2 helicase were analyzed. Only modest impacts on nsp13-catalyzed ATP hydrolyses were observed with some compounds, most of which were analogs of the drug ebselen, not ML283. In contrast, a new molecular-beacon-based helicase assay revealed that ML283 and many ML283 analogs are potent SARS-CoV-2 helicase inhibitors. Analog potencies correlate with the binding energies predicted by modeling, which suggests that a pocket surrounded by the carboxy-terminal nsp13 RecA-like helicase motor domain might be exploitable for antiviral drug development.

8-Oxoguanine (8-oxoG) is not only a biomarker of oxidative DNA damage but also an epigenetic-like regulator in mammalian cells. The identification and characterization of 8-oxoG-binding proteins would be crucial for further understanding the biological consequences of 8-oxoG. Here, we identified human Y-box-binding protein 1 (YBX1) as a novel binding protein for 8-oxoG modification in DNA by using a quantitative proteomic approach. Moreover, we found that the deficiency of YBX1 can substantially decrease the cellular sensitivity to oxidative stress and facilitate the repair of 8-oxoG embedded in DNA. These findings provided new insight into the biological significance of the functional interplay between YBX1 and 8-oxoG modification in DNA.