RSC Chemical Biology最新文献

5'-(E)- and 5'-(Z)-vinylphosphonate carbocyclic DNA and 5'-(E)-vinylphosphonate 2'- and 3'-O-methyl carbocyclic RNAs were incorporated at 5' termini of antisense strands of small interfering RNAs. All but the 3'-O-methyl carbocyclic analogue resulted in gene silencing activity better than the siRNA lacking a 5' phosphate in cells and in mice.

Covalent modification can enable understanding and modulation of protein function, and the identification of new therapeutic opportunities. A "direct to biology" workflow was developed that harnesses sulfonylation as a connective reaction for the synthesis of diverse sets of reactive fragments. The workflow expanded the diversity of accessible reactive fragment sets, and facilitated the discovery of pentafluorobenzene sulfonamides that modify Aurora A kinase, NEK7 kinase, and UbcH5B. Characterisation of several of the Aurora A-modifying reactive fragments revealed both their modification rates and sites. Furthermore, Cys247, a residue typically buried in Aurora A crystal structures, was identifed as a modifable residue. These findings underscore the importance of protein dynamics in determining cysteine reactivity and highlight the utility of reactive fragment sets for identifying cryptic pockets. Sulfonylation is therefore a useful complement to amide formation in "direct to biology" workflows aimed at identifying novel opportunities for targeted protein modification.

Post-translational modifications (PTMs) endow silk proteins with chemical diversity that governs their higher-order assembly, hydration, and covalent connectivity. This review highlights the principal PTMs that define silk protein function, including hydroxylation, glycosylation, phosphorylation, and covalent crosslinking. We also describe their contributions to protein structural stability and mechanical properties. Recent advances in proteomics have begun to reveal low-abundance PTMs, whereas synthetic biology and bioorthogonal chemistry enable the programmed installation of modifications to tune physicochemical properties. Understanding and harnessing these chemistries provides a foundation for the predictive design of next-generation protein-based materials at the interface of chemical biology and materials science.

Understanding small molecule-RNA interactions is a crucial part in drug development and fundamental biology. Chemotranscriptomic profiling is emerging as a powerful platform to interrogate interactions of small molecules with entire transcriptomes. This technique relies on photoaffinity probes that covalently capture small molecule RNA interactions. Most photoaffinity probes bear an alkyne handle that requires additional inefficient functionalization and purification steps after RNA capture. We sought to improve the workflow by directly desthiobiotinylating a photoaffinity probe, omitting these additional alkyne functionalization steps. Here, we compare the suitability of desthiobiotin and alkyne modified Ribocil-derived photoaffinity probes for chemotranscriptomic profiling. Our results demonstrate binding of both photoaffinity probes to their specific target, the FMN riboswitch, using in vitro transcription/translation and RT-qPCR. We also observed high unspecific interactions due to proposed weak and non-specific binding of the desthiobiotin moiety to RNA analyzed by dot blots and RT-qPCR. Finally, transcriptome-wide sequencing confirmed the unselective interaction of desthiobiotin. These findings suggest that desthiobiotin is an inefficient enrichment handle for the design of photoaffinity probes, resulting in many off-target interactions.

Affinity screenings with encoded libraries are transformative tools for rapid hit discovery from vast compound collections. Yet the adaptation of established chemical reactions to DNA-encoded libraries (DELs) remains challenging due to DNA-compatibility constraints and mismatches between barcode and chemical structure in case of incomplete reactions or side product formation. Recently, we introduced self-encoded libraries (SELs) as a barcode-free alternative to DELs. The SEL platform offers unmatched flexibility in reaction conditions and decodes screening hits directly from their chemical structure, avoiding the problem of mismatched barcode-compound pairs. Here, we expand the SEL platform to Buchwald-Hartwig aminations, enabling the construction of new high diversity SELs. We performed a thorough reaction condition optimization and tested a scope of >170 different building blocks. We adapted our automated MS/MS-based decoding methodology SIRIUS-COMET to the resulting scaffolds, enabling accurate compound decoding from complex mixtures. A 25 725-member library was synthesized and screened all at once against carbonic anhydrase IX (CAIX), resulting in robust enrichment of hits with specific building block patterns and yielding several nanomolar-affinity binders. This work showcases the seamless integration of palladium-catalyzed cross-couplings into SELs, expanding the chemical space of this technology and accelerating hit discovery with high synthetic versatility.

Precise spatiotemporal control over fluorescence labeling is a powerful approach for selective marking and tracking of proteins of interest within living systems. Here, we report a photoclickable labeling platform based on the 2,3-diaryl-indanone epoxide (DIO) photoswitch scaffold and the self-labeling protein HaloTag. Upon illumination, the protein-bound DIO undergoes reversible photoisomerization to form a metastable oxidopyrylium ylide (PY) that reacts with ring-strained dipolarophiles via [5 + 2] cycloaddition, enabling covalent spatiotemporal labeling. We synthesize and characterize a library of DIO-HaloTag and DIO-SNAP-tag ligands, systematically examining the effects of linker architecture and scaffold substitution on the photoswitching and photoclick reactivity in vitro and on living cells. We identify a naphthyl-substituted DIO ligand exhibiting superior photoswitching and photoclick efficiency, allowing fast, selective labeling of HaloTagged proteins on the surface of living cells using visible light activation (405 nm). Using this system, we achieve two- and three-color labeling of defined cell surface regions with excellent spatial and temporal precision, additionally allowing combinatorial labeling. Together, this work establishes a versatile framework for multiplexed, light-directed protein labeling compatible with living systems, with promising future applications including multiplexed long-term tracking and cellular barcoding.

Broad spectrum antivirals are critical to respond rapidly to the threat posed by newly emerging RNA viruses. One potential candidate is the natural compound thapsigargin (Tg). Tg potently induces endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). Recent studies have demonstrated that Tg has robust antiviral activity against several human coronaviruses (CoVs), including SARS-CoV-2, although the specific antiviral mechanism(s) have remained unclear. Here, we aimed to characterize the role of the UPR in the antiviral activity of Tg against HCoV-229E, a model common cold CoV. Consistent with previous findings, we show that a short 30-minute priming of A549 cells with Tg potently inhibits HCoV-229E infection. Time-of-addition assays showed that Tg is most effective when added up to 8 hours post-infection. Furthermore, Tg inhibits the accumulation of double-stranded RNA in infected cells, suggesting that Tg inhibits early stages of viral RNA replication. Using selective UPR pathway inhibitors to narrow down the role of these pathways in mediating the antiviral effect of Tg, we show that the inhibition of IRE1 or ATF6 does not impair the ability of Tg to inhibit HCoV-229E infection. The use of stable knockdown A549 cells in which IRE1, PERK, or ATF6 expression was silenced further revealed that the antiviral activity of Tg is not dependent on the expression of any of the three UPR sensors individually. However, HCoV-229E replication is inhibited in A549-shIRE1 cells, or in cells treated with the IRE1 inhibitor (KIRA6), suggesting that IRE1 activation may play a pro-viral role during HCoV-229E infection. Selective UPR pathway activators were used to further probe down the role of each pathway during HCoV-229E infection. Activation of the PERK pathway, but not IRE1 or ATF6 pathways, inhibits HCoV-229E infection. Lastly, to more broadly test the antiviral role of PERK against CoV RNA replication, we used BHK-21 cells that stably express a SARS-CoV-2 replicon. We show that PERK activation inhibits SARS-CoV-2 replication similarly to Tg. Overall, these findings provide insight into the antiviral mechanism(s) of Tg against CoV infection and demonstrate that modulation of the UPR may be exploited as an antiviral strategy.

Natural products derived from biosynthetically apt microorganisms represent an important source for chemotherapeutic agents founded first via non-specific, cytotoxicity-guided isolation, then investigated for translational potential. Analyzing biosynthetic gene clusters within microbes predicts significant untapped potential of undiscovered therapeutically relevant natural products; however, stimulating biosynthesis of such compounds in sufficient quantities for isolation, structure determination, and biological assessment remains a gap. We address this with a bioactivity discovery pipeline that first utilizes Multiplexed Activity Profiling to identify stimulus-dependent induction of bioactive secondary metabolites via functional responses in human leukemia cell lines measured with fluorescence flow cytometry. Next, active extracts are analyzed via Multiplexed Activity Metabolomics, which correlates single cell assays with spectrally defined chromatographic arrays to identify bioactive metabolomic features prior to isolation. Two experimental case studies using this workflow with cave-microbe extracts identified new molecular phenotypes of the pyridine-pyrrolidine alkaloid siderochelin and the pyrrolopyrrole-functionalized anthracycline isoquinocycline B. Finally, we investigated the effects of anthracycline functionalization on human primary cells using single cell mass cytometry to understand if changes in anthracycline aglycon structure and glycosylation impacted cell selectivity. Despite sharing an anthracyclinone pharmacophore core, variants differentially impacted responses of leukemia cell populations within and among acute myeloid leukemia (AML) patient samples. This suggests pharmacophore assumptions may not guide assessment of therapeutic potential for anthracyclines and that modest structural differences can elicit marked changes in cellular function in different patients. Taken together, the depth of information afforded by single cell molecular phenotype-based discovery and mass cytometry deep cell profiling provides new patient-level insight into biological mechanisms of new and previously discovered molecules that may find expanded use in the clinic.

Sialic acids - 9-carbon ulosonic acids - are implicated in many cell-cell and host-pathogen interactions due to their prevalent location at the non-reducing end of glycoconjugates. Sialic acids have recently been observed in microalgae, including the toxic bloom-forming Prymnesium parvum, which produces the deaminated sialic acid, ketodeoxynonulosonic acid (Kdn), through de novo biosynthesis. Here we report on the key CMP-sialic acid synthetase enzyme (CMAS), PpNeuA, which activates Kdn to its sugar nucleotide congener, CMP-Kdn. In the present study, the X-ray crystal structure of PpNeuA was determined to 1.8 Å resolution and shows that it adopts a similar overall fold to that of other sialic acid synthetase enzymes, with which it shares ca 30% amino acid sequence identity. PpNeuA specificity for Kdn is dependent upon Arg196, a hydrophilic residue that is only found in Kdn-specific sialic acid synthetases. R196L mutation switches the substrate preference of PpNeuA from Kdn to N-acetylneuraminic acid (Neu5Ac). Kinetic analysis shows that Arg196 plays both a role in substrate binding (impact on K _M) and catalysis (impact on k _cat). In the context of generating metabolic probes to identify the location and context (glycolipid vs glycoprotein) of Kdn in P. parvum, we also report on the ability of PpNeuA to accept both 5Az-Kdn and 9Az-Kdn as substrates.

Peptide stapling has emerged as a powerful strategy for stabilizing protein conformation, improving proteolytic resistance, and enhancing biomolecular recognition. Yet design principles for selecting staple sites remain elusive, so advances in stapling have depended largely on trial and error. Here we establish quantitative guidelines for staple placement by exploiting the well-defined geometry of an α-helical coiled coil to compare alternative staple sites in a controlled way. Using both experimental measurements and molecular simulations, we find that (1) staples that link residue pairs that normally form interhelical salt bridges yield greater stabilization than those linking non-salt-bridged pairs; (2) N-terminal staples are more stabilizing than C-terminal staples, where an existing interhelical disulfide constraint reduces their impact; and (3) mismatches between the staple length and site spacing can cause destabilization by forcing the structure into a compressed, non-native geometry. Together, these results show that staple-based stabilization depends on two underlying factors: unfolded-state constraint (the entropic advantage gained when the staple limits how far apart the linked residues can separate in the unfolded ensemble) and folded-state compatibility (how well the staple's maximum accessible span matches the native separation of those residues in the folded structure). These principles provide a predictive framework for rational stapled peptide design, offering a path beyond empirical screening toward principle-guided development of stabilized peptide therapeutics.