RSC Chemical Biology最新文献

The self-exchange of subunits by protein homodimers is a common protein–protein interaction in vivo. In heterozygous genetic disorders involving homodimeric gene products, both mutant and WT proteins can exchange subunits (heterodimerize). This form of heterodimerization can be analytically challenging to study. In this paper, we used capillary electrophoresis to investigate how deamidation of multiple asparagine residues (to aspartate) in homodimeric Cu, Zn superoxide dismutase-1 (SOD1) affected the rate and free energy of heterodimerization between WT and mutant SOD1 that cause amyotrophic lateral sclerosis (ALS). To model asparagine deamidation, Asn to Asp substitutions were introduced at five Asn residues predicted to undergo the most rapid deamidation in SOD1 (N26D, N131D, N139D, N65D, N19D). This model of penta-deamidated SOD1 did not heterodimerize with WT SOD1 or E100K SOD1 (linked to ALS). In contrast, the quad-variant N26D/N131D/N139D/N19D SOD1 did heterodimerize. These results suggest that the WT SOD1 protein has an intrinsic “timer” or “molecular clock” (as spontaneous Asn deamidation has been described) that effectively stops its heterodimerization after the SOD1 protein has existed in solution for ∼3 months.

Development of chemically modified oligonucleotides, nucleic acid mimics, protein-based constructs, and other ligands – capable of sequence-unrestricted recognition of specific double-stranded (ds) DNA regions – is an area of research that continues to attract considerable attention. Efforts are fueled by the need for diagnostic agents, modulators of gene expression, and novel therapeutic modalities against genetic diseases. While pioneering approaches focused on accessing nucleotide-specific features from the grooves of DNA duplexes, recent developments have entailed strand-invading probes, i.e., probes capable of binding to DNA duplexes by breaking existing Watson–Crick base pairs and forming new, more stable base pairs. For the past twenty years, our laboratory has pursued the development of a type of dsDNA-targeting strand-invading probes, which we have named Invader probes. These double-stranded oligonucleotide probes feature intercalator-functionalized nucleotides that are specifically arranged to promote destabilization of the probe duplex, whereas individual strands exhibit very high affinity towards complementary DNA. This account details the discovery, principles, and applications of Invader probes.

Peptide nucleic acid (PNA) oligomers have tremendous potential as therapeutics; however, their delivery is challenging and has limited their development as therapeutics. In recent years, new strategies for delivering water-soluble backbone-modified PNA oligomers into cells for antisense and gene-editing applications have attracted significant attention. This review critically examines earlier delivery approaches and their limitations, highlights recent advances in PNA engineering and nanocarrier design, and discusses the future directions necessary to advance PNA-based therapeutics. By integrating these innovations, PNAs hold the potential to transform biomedical applications and contribute to the next generation of medicine.

A variety of oligonucleotide-based probes have been developed for specific and selective sensing of RNA and DNA. Among these, FIT-PNAs (forced intercalation-peptide nucleic acids) and FIT probes (DNA- and RNA-based sensors) have been studied for a variety of RNA biomarkers in cell culture and tissues, and in vivo. FIT-PNAs and FIT probes are RNA/DNA sensors that exhibit fluorescence upon sequence-specific RNA/DNA hybridization. Several synthetic approaches have been successfully applied to increase the brightness and selectivity of these molecules, including the introduction of cyclopentane (cp) modified PNA monomers (cpPNA) as well as locked nucleic acids (LNAs—for FIT probes). In this report, we have explored the biophysical properties of FIT-PNAs that are modified with gamma-L-serine PNAs (γPNAs). We found that introducing a single γ-PNA flanking the fluorophore (BisQ) in the FIT-PNA sequence is sufficient to achieve a 46-fold increase in fluorescence for the PNA:RNA duplex, similarly to cpPNA. Interestingly, when two γ-PNAs flank BisQ on both sides, a significant increase in RNA affinity is observed (over an 8 °C increase in melting temperature, T_m). Altogether, γ-PNAs are a beneficial chemical modification that leads to brighter FIT-PNAs with improved binding affinities to targeted RNA.

Chemical conjugation to carrier proteins has been traditionally used to improve polysaccharides immunogenicity and to overcome the limitations of T-independent antigens, including lack of immunological memory and efficacy in infants. A double-hit approach, meaning that both polysaccharide and carrier protein belong to the same pathogen, may be particularly useful for targeting bacterial species with large glycan variability. Recently, bacterial protein glycosylation has been exploited to obtain glycosylated proteins in E. coli cytoplasm. In our work we have combined cytoplasmic glycoengineering and chemical conjugation for the development of novel selective glycoconjugates, with the aim to preserve the immunogenicity of the protein chosen as carrier. The potential protective protein MrkA, the major component of Klebsiella pneumoniae type 3 fimbriae, was successfully modified with a lactose moiety in E. coli. K. pneumoniae K2 K-antigen and O1v1 O-antigen were then covalently linked to MrkA at the level of this unique sugar handle and tested in vivo. Immune response against MrkA and sugars was evaluated in animal models. This work contributes to expand the application of the glycoengineering technology for the development of effective glycoconjugate vaccines.

RNA-based vaccines have emerged as a highly effective delivery platform. However, this approach eliminates the possibility for immunogen purification, common in manufacturing of recombinant immunogens. In HIV-1 vaccine design, this is of particular importance because non-native epitopes can compromise the desired immune response, and native immunogen assembly is important for presentation of glycan-based epitopes targeted by broadly neutralizing antibodies. Here, we investigate the assembly and glycosylation of the archetypal trimeric HIV-1 immunogen, BG505, in the soluble single-chain format (NFL.664) that bypasses the need of maturation by furin cleavage. We have investigated the presence of the trimer-associated mannose-patch as oligomannose-type structures at these N-linked glycosylation sites are indicative of native-like glycoprotein structure. Despite the presence of features of native-like glycosylation, both electron microscopy and glycopeptide analysis indicated the presence of a sub-population of non-native material. We also investigated the glycosylation of material derived from cell-types that likely produce immunogens near the site of intramuscular RNA injection. We show that replicon-transformed dendritic and muscle cell lines generate immunogens displaying similar oligomannose-type glycan content, whereas sites presenting complex-type glycosylation differed substantially in the levels of glycan processing. Overall, the control of the immunogen assembly by protein engineering is sufficient to drive native-like glycosylation at the majority of glycosylation sites independent of producer cells. Furthermore, we explored the engineering of RNA immunogens to improve glycan site occupancy. Controlling immunogen assembly at the nucleotide level offers a route to enhanced RNA-based immunogens.

Proteins form complex networks critical to various biological processes; many become involved in disease-related pathologies – only a subset of these proteins are considered to be druggable by conventional, non-covalent small-molecule therapeutics. Covalent drugs, which encompass irreversible inhibitors and reversible covalent inhibitors, are small-molecule modalities that chemically conjugate with their therapeutic targets and have emerged as a strategy to more effectively target these proteins, with structure-based approaches guiding their design, and achieving an improved therapeutic effect, predominantly through sustained inhibitions. In this review, we focus on the impact of covalent bond formation on protein structural dynamics, such as the generation/trapping of cryptic pockets, and how these phenomena may be leveraged in orthosteric and allosteric drug design. Further, while irrreversible inhibitors result in longer residence times with permanent changes of target proteins that will require protein re-synthesis, reversible covalent inhibitors enjoy the benefit of samplng different adducts, wherein one particular conjugate may be favoured through stabilizing structural reogranizations; this may prove significant when a protein presents multiple nucleophilic residues, and selectivity is a concern. Herein, we explore selected case studies that examine the mechanistic consequences of protein–drug conjugations, recommending a more dynamic structural perspective in rational drug development.

Correction for ‘Proteolysis targeting chimeras (PROTACs) come of age: entering the third decade of targeted protein degradation’ by Michael J. Bond et al., RSC Chem. Biol., 2021, 2, 725–742, https://doi.org/10.1039/D1CB00011J.

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.