Активность

The sophistication of modulating protein-protein interactions (PPIs) enables the study of PPI networks driving diseases and the development of novel therapeutic agents and diagnostic tools. Standard methods face significant hurdles in targeting the prevalent shallow protein surfaces involved in PPIs, while methods for accessing extended backbone structures remain constrained. A rigid, linear diyne bridge is incorporated between side chains at the i and i+2 positions to furnish a collection of extended-backbone peptide macrocycles with low molecular weights. Detailed NMR and density functional theory analyses show that these elongated peptides retain stable, rigid structures in solution, and their properties can be tailored to explore the extensive range of possible peptide conformations. cyclosporina inhibitor High-throughput synthesis is facilitated by the excellent conversions (greater than 95%) that produce the diyne brace. The core tripeptide, with its minimalist design (under 300 Daltons), is receptive to subsequent synthetic modifications. Inhibitors of bacterial type 1 signal peptidase, stabilized by diyne braces, highlight the method’s effectiveness.

The function of hundreds of phosphorylated clients is regulated by 14-3-3 proteins, which are dimeric hubs for binding. The strategic introduction of stable, functional mimics of phosphorylated amino acids into proteins is a potent tool for exploring 14-3-3 function in cellular contexts; however, a prior genetic code expansion (GCE) technique for translating and introducing non-hydrolyzable phosphoserine (nhpSer), with the oxygen atom substituted by a methylene group, at targeted protein sites has seen limited application. A remarkable 40-fold enhancement in the system’s performance was achieved by incorporating a six-step biosynthetic pathway for nhpSer production from phosphoenolpyruvate into Escherichia coli. This autonomous PermaPhos expression system yields three biologically pertinent proteins containing nhpSer, and we observe that nhpSer mimics the effect of phosphoserine in activating GSK3 phosphorylation of the SARS-CoV-2 nucleocapsid protein, promoting the association of 14-3-3 proteins with client proteins and the conversion of 14-3-3 dimers to monomers. We isolate the interactome of phosphorylated 14-3-3 monomers, featuring nhpSer at Ser58, from HEK293T lysates, and compare it to the interactome of wild-type 14-3-3 to understand their biological functions. Analysis of these data highlights two distinct classes of 14-3-3 client proteins: (i) proteins preferentially bound to dimeric 14-3-3, and (ii) proteins preferentially bound to monomeric 14-3-3. The results show that monomeric 14-3-3, unlike its dimeric counterpart, binds to cereblon, a crucial E3 ubiquitin-ligase adaptor protein of pharmacological interest.

The combination of CRISPR technology and isothermal amplification methods for nucleic acid detection offers substantial promise in the realm of point-of-care diagnostics. However, the majority of present-day methods depend on fluorescent or lateral flow assay readout, necessitating external activation or the subsequent transfer of the amplified reaction. By employing a pair of dCas9-based probes that facilitate split NanoLuc luciferase complementation, a novel bioluminescent nucleic acid sensor (LUNAS) platform was developed to enable sequence-specific detection of target dsDNA. Recombinase polymerase amplification (RPA), readily integrated with LUNAS, enables attomolar sensitivity in a quick, one-step assay. Real-time monitoring of the RPA reaction is empowered by a luciferase calibrator for a robust ratiometric outcome, all achievable through a simple digital camera. We devised an RT-RPA-LUNAS assay capable of SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation protocols, demonstrating its diagnostic efficacy on nasopharyngeal swab samples from COVID-19 patients. Point-of-care infectious disease testing gains a promising new tool in RPA-LUNAS, as it detects SARS-CoV-2 with viral RNA loads of 200 cp/L in a swift 20 minutes.

The spike protein of SARS-CoV-2, with DC-SIGN, a C-type lectin receptor, as its coreceptor, has been extensively studied. A finding in research is that Polyman26, a multivalent glycomimetic ligand, successfully inhibits SARS-CoV-2’s trans-infection process, which relies on DC-SIGN. The molecular components and interactions responsible for avidity development in these systems are inadequately characterized. Utilizing dendrimer constructs based on Polyman26, we sought to delineate the multifaceted role of chelation, clustering, and rebinding in multivalency. A specifically designed rod-like core was used to target two binding sites on the tetrameric DC-SIGN simultaneously. A range of biophysical techniques, including the recently developed surface plasmon resonance oriented-surface methodology, have been used to examine the binding properties of these compounds. We utilized molecular modeling to analyze, for the first time, the contribution of carbohydrate recognition domains’ flexibility within the DC-SIGN tetramer to the compounds’ avidity. A deeper comprehension of the various binding modes revealed how a construct, despite its limited valency, manages to achieve nanomolar affinity. The multi-pronged experimental and theoretical approach yields a detailed understanding of multivalent ligand/multimeric protein interactions, potentially guiding future predictions. New virus attachment blockers, adaptable to the different C-type lectin receptors found on viruses, are made possible by this research.

A pressing need exists for small-molecule prodrug approaches capable of selectively activating cancer treatments within the confines of tumors. In this research, the first antitumor prodrugs, activated by thiol-manifold oxidoreductases and directed at the thioredoxin (Trx) system, were produced. The Trx system, a pivotal cellular redox axis linked to dysregulated redox/metabolic states in cancer, is currently neglected by bioreductive prodrugs, primarily those centered on oxidized nitrogen species. By employing Trx/TrxR-specific artificial dichalcogenides, we were able to manipulate the bioactivity of ten duocarmycin prodrugs that change from inactive to active states upon reduction. In 177 cell lines, the cell-free and cellular reductase-dependent activity of the prodrugs were evaluated to understand the broad redox-based cellular bioactivity trends displayed by the dichalcogenides. In vivo mouse studies displayed excellent tolerance to the compounds, indicating a low level of systemic duocarmycin release; the in vivo anti-tumor efficacy studies in mouse models of breast and pancreatic cancer revealed positive signs, suggesting the targeted drug delivery mechanism operates effectively through in situ bioreductive activation. This investigation unveils a chemically unique class of bioreductive prodrugs, specifically designed for a previously uncharacterized reductase chemotype. It validates the capacity to synthesize in vivo-compatible small molecule prodrugs, even those derived from powerfully cumulative toxins. Importantly, it introduces a carefully tailored approach using dichalcogenides as a platform for specific bioreduction-based release.

Brain cancer, neurodegeneration, and neuropsychiatric disorders are connected to the problematic activity of kinases, despite the challenge of identifying kinase inhibitors that operate efficiently within the brain’s complex environment. The inability of blood drug levels to predict brain drug efficacy stems from the blood-brain barrier’s exclusion of the majority of compounds. Evaluating kinase inhibition within the cerebral cortex mandates the physical separation of brain tissue and the subsequent execution of biochemical assays, a process that is both time-consuming and resource-intensive. Longitudinal, noninvasive imaging of drug activity within the brain is achieved via kinase-modulated bioluminescent indicators (KiMBIs), relying on a newly optimized luciferase-luciferin system. To address the lack of bioluminescent indicators for inhibitors of the Ras-Raf-MEK-ERK pathway, we developed an ERK KiMBI. ERK KiMBI analysis discriminates between brain-penetrant and non-brain-penetrant MEK inhibitors, indicating blood-tumor barrier leakage in xenograft models, and documenting MEK inhibitor pharmacodynamic responses in native brain tissue and intracranial xenografts. Employing ERK KiMBI, we screen ERK inhibitors for their impact on the brain, ultimately revealing temuterkib as a promising brain-active ERK inhibitor, a result not anticipated from its chemical profile. In summary, KiMBIs allow for the rapid identification and detailed evaluation of the pharmacodynamic properties of kinase inhibitors, positioning them as potential treatments for brain diseases.

Despite their widespread use in clinical and industrial settings, protein-polymer conjugates face a significant hurdle in rational design, stemming from the lack of experimental data correlating protein-polymer interactions with improved protein stability. Significant progress in synthetic chemistry has led to enhanced polymer designs, featuring non-linear frameworks, novel monomer architectures, and controlled hydrophobicity; nonetheless, more experimental validation is necessary to effectively translate these chemical improvements into the next generation of conjugates. An integrative biophysical analysis was conducted to uncover the molecular mechanisms responsible for the thermal stabilization of the human galectin protein Gal3C, which was conjugated to polymers exhibiting a range of linear and nonlinear architectures, degrees of polymerization, and hydrophobicities. The ability to independently modify the polymerization degree and polymer structure permitted the identification of specific polymer characteristics that aided protein stability. Thermally stable conjugates of polymer-conjugated Gal3C backbones exhibited shared patterns of protein-polymer interactions across linear and nonlinear architectures, according to NMR spectroscopic data.