ACS Chemical Biology最新文献

Estrogen receptor α (ERα)-positive breast cancer patients are typically treated with ERα inhibitors, including selective estrogen receptor modulators (SERMs) and selective estrogen receptor degraders (SERDs). However, the distinct pharmacological properties of various ERα inhibitors remain incompletely understood. In this study, we employed formaldehyde cross-linking followed by ERα immunoprecipitation and mass spectrometry to reveal that fulvestrant, the first FDA-approved SERD, induces the interaction between ERα and SUMO E3 ligases PIAS1 and PIAS2. Biochemical and genomic assays confirmed that fulvestrant induces SUMOylation of ERα, which inhibits ERα's binding to chromatin DNA. In addition, raloxifene (a SERM) and elacestrant (the first FDA-approved oral SERD) were identified as compounds that similarly induce ERα SUMOylation and inhibit its chromatin interaction. Our findings reveal a mechanism by which select ERα inhibitors disrupt ERα function through SUMOylation, offering insights for the development of next-generation ERα-targeted therapies.

Didemnins are a class of cyclic depsipeptides derived from sea tunicates that exhibit potent anticancer, antiviral, and immunosuppressive properties. Although certain Tistrella species can produce didemnins, their complete biosynthetic potential remains largely unexplored. In this study, we utilize feature-based molecular networking to analyze the metabolomics of Tistrella mobilis and Tistrella bauzanensis, focusing on the production of didemnin natural products. In addition to didemnin B, we identify nordidemnin B and [hysp²]didemnin B, as well as several minor didemnin analogs. Heterologous expression of the didemnin biosynthetic gene cluster in a Streptomyces host results in the production of only didemnin B and nordidemnin B in limited quantities. Isotope-labeling studies reveal that the substrate promiscuity of the adenylation domains during biosynthesis leads to the accumulation of nordidemnin B and [hysp²]didemnin B. Additionally, precursor-directed biosynthesis is applied to generate eight novel didemnin derivatives by supplementing the culture with structurally related amino acids. Furthermore, we increased the titers of nordidemnin B and [hysp²]didemnin B by supplementing the fermentation medium with l-valine and l-isoleucine, respectively. Finally, both compounds undergo side-chain oxidation to enhance their biological activity, with their anticancer properties found to be as potent as plitidepsin.

Split-intein circular ligation of proteins and peptides (SICLOPPS) is a method for generating intracellular libraries of cyclic peptides that has yielded several first-in-class inhibitors. Here, we detail a revised high-content, high-throughput SICLOPPS screening protocol that utilizes next-generation sequencing, biopanning, and computational tools to identify hits against a given protein-protein interaction. We used this platform for the identification of inhibitors of the HIF-1α/HIF-1β protein-protein interaction. The revised platform resulted in a significantly higher positive hit rate than that previously reported for SICLOPPS screens, and the identified cyclic peptides were more active in vitro and in cells than our previously reported inhibitors. The platform detailed here may be used for the identification of inhibitors of a wide range of other targets.

Among bacteria used as anticancer vaccines, attenuated Listeria monocytogenes (Lm^at) stands out, because it spreads from one infected cancer cell to the next, induces a strong adaptive immune response, and is suitable for repeated injection cycles. Here, we use click chemistry to functionalize the Lm^at cell wall and turn the bacterium into an "intelligent carrier" of the chemotherapeutic drug doxorubicin. Doxorubicin-loaded Lm^at retains most of its biological properties and, compared to the control fluorophore-functionalized bacteria, shows enhanced cytotoxicity against melanoma cells both in vitro and in a xenograft model in zebrafish. Our results show that drugs can be covalently loaded on the Lm^at cell wall and pave the way to the development of new two-in-one therapeutic approaches combining immunotherapy with chemotherapy.

Tumor-selective degradation of target proteins has the potential to offer superior therapeutic benefits with maximized therapeutic windows and minimized off-target effects. However, the development of effective lysosome-targeted degradation platforms for achieving selective protein degradation in tumors remains a substantial challenge. Cancer cells depend on certain solute carrier (SLC) transporters to acquire extracellular nutrients to sustain their metabolism and growth. This current study exploits facilitative glucose transporters (GLUTs), a group of SLC transporters widely overexpressed in numerous types of cancer, to drive the endocytosis and lysosomal degradation of target proteins in tumor cells. GLUT-targeting chimeras (GTACs) were generated by conjugating multiple glucose ligands to an antibody specific for the target protein. We demonstrate that the constructed GTACs can induce the internalization and lysosomal degradation of the extracellular and membrane proteins streptavidin, tumor necrosis factor-alpha (TNF-α), and human epidermal growth factor receptor 2 (HER2). Compared with the parent antibody, the GTAC exhibited higher potency in inhibiting the growth of tumor cells in vitro and enhanced tumor-targeting capacity in a tumor-bearing mouse model. Thus, the GTAC platform represents a novel degradation strategy that harnesses an SLC transporter for tumor-selective depletion of secreted and membrane proteins of interest.

Deciphering the functional relevance of every protein is crucial to developing a better (patho)physiological understanding of human biology. The discovery and use of quality chemical probes propel exciting developments for developing drugs in therapeutic areas with unmet clinical needs. Myosin light-chain kinase (MLCK) serves as a possible therapeutic target in a plethora of diseases, including inflammatory diseases, cancer, etc. Recent years have seen a substantial increase in interest in exploring MLCK biology. However, there is only one widely used MLCK modulator, namely, ML-7, that too with a narrow working concentration window and high toxicity profile leading to limited insights. Herein, we report the identification of a potent and highly selective chemical probe, Myokinasib-II, from the synthesis and structure-activity relationship studies of a focused indotropane-based compound collection. Notably, it is structurally distinct from ML-7 and hence meets the need for an alternative inhibitor to study MLCK biology as per the recommended best practices. Moreover, our extensive benchmarking studies demonstrate that Myokinasib-II displays better potency, better selectivity profile, and no nonspecific interference in relevant assays as compared to other known MLCK inhibitors.

Peptidyl arginine deiminases (PADs) are important enzymes in many diseases, especially those involving inflammation and autoimmunity. Despite many years of effort, developing isoform-specific inhibitors has been a challenge. We describe herein the discovery of a potent, noncovalent PAD2 inhibitor, with selectivity over PAD3 and PAD4, from a DNA-encoded library. The biochemical and biophysical characterization of this inhibitor and two noninhibitory binders indicated a novel, Ca²⁺ competitive mechanism of inhibition. This was confirmed via X-ray crystallographic analysis. Finally, we demonstrate that this inhibitor selectively inhibits PAD2 in a cellular context.

Fungal unspecific peroxygenases (UPOs) are gaining momentum in synthetic chemistry. Of special interest is the UPO from Marasmius rotula (MroUPO), which shows an exclusive repertoire of oxyfunctionalizations, including the terminal hydroxylation of alkanes, the α-oxidation of fatty acids and the C-C cleavage of corticosteroids. However, the lack of heterologous expression systems to perform directed evolution has impeded its engineering for practical applications. Here, we introduce a close ortholog of MroUPO, a UPO gene from Marasmius wettsteinii (MweUPO-1), that has a similar reaction profile to MroUPO and for which we have set up a directed evolution platform based on tandem-yeast expression. Recombinant MweUPO-1 was produced at high titers in the bioreactor (0.7 g/L) and characterized at the biochemical and atomic levels. The conjunction of soaking crystallographic experiments at a resolution up to 1.6 Å together with the analysis of reaction patterns sheds light on the substrate preferences of this promiscuous biocatalyst.

Recent advancements in AI-driven computational modeling, especially AlphaFold2, have revolutionized the prediction of biological macromolecule structures. AlphaFold2 enabled accurate predictions of structural domains and complex arrangements. However, computational models lack a clear metric for accuracy. This study explores whether computational models can match the crystallographic resolution of crystal structures. By comparing distances between atoms in models and crystal structures using t tests, it was found that AlphaFold2 models are comparable to high-resolution crystal structures (1.1 to 1.5 Å). While these models exhibit exceptional quality, their accuracy is lower than the crystal structure with resolutions better than 1 Å.