RSC Chemical Biology最新文献

We describe rational chemical engineering to enhance the proteolytic stability of a chimeric peptide using a combination of unique strategies that involve the incorporation of a series of d-amino acids into the parent l-peptide sequence and restricting the conformational freedom of the peptide by covalent stitching. We hypothesize that replacing a stretch of sequence of an unstructured peptide motif with d-amino acids would increase its proteolytic stability without significantly affecting its affinity to the target protein. Also, considering the C_β-C_β distances, replacing an appropriate pair of residues with cysteine to form an additional disulfide bond in the molecule would provide additional stability to the engineered peptide. To verify this hypothesis, we have implemented these strategies to a previously reported peptidic inhibitor RR, against P. falciparum invasion into red blood cells (RBCs) and designed two novel heterochiral chimeric peptides, RR-I and RR-II. We have demonstrated that these peptides exhibit remarkable inhibitory activity with dramatically enhanced proteolytic stability. Finally, we have designed a cyclic analog, RR-III, to enhance the stability of the peptide against endopeptidases. The RR-III peptide exhibits the same inhibitory activity as RR-II while demonstrating impressive resistance to enzymatic degradation and prolonged stability in human plasma. These developments hold promise for a new generation of peptide-based therapeutics, showcasing the potential of residue selection for tailored modifications, as demonstrated in this work.

Aberrant and dysregulated protein-protein interactions (PPIs) drive a significant number of human diseases, which is why they represent a major class of targets in drug discovery. Although a number of high-affinity antibody-based drugs have emerged in this therapeutic space, the discovery of smaller PPI inhibitors is lagging far behind, underscoring the need for novel scaffold modalities. To bridge this gap, we introduce a biomimetic platform technology - adaptive design of antibody paratopes into therapeutics (ADAPT) - that enables the paratope-forming binding loops of antibodies to be crafted into large β-hairpin scaffolds (ADAPTins). In this study, we describe a novel strategy for engineering native CDR-H3 "hot loops" with varying sequences, lengths, and rigidity into ADAPTins, ultimately transforming these compounds into irreversible covalent inhibitors. A proof-of-concept was established by creating a series of ADAPTin blockers of the PD1:PDL1 immune checkpoint PPI (blocking activity EC₅₀ < 0.3 μM) which were subsequently modified into potent covalent PD1 inhibitors. The compelling rate of stable and folded ADAPTins above physiological temperature (21 out of 29) obtained across six different scaffolds suggests that the platform technology could provide a novel opportunity for high-quality peptide display and biological screening.

Nitroreductases are important enzymes for a variety of applications, including cancer therapy and bioremediation. They often require encapsulation to improve stability and activity. We focus on genetically encoded encapsulation of nitroreductases within protein capsids, like encapsulins. Our study showcases the encapsulation of nitroreductase NfsB as functional dimers within encapsulins, which enhances protein activity and stability in diverse conditions. Mutations within the pore region are beneficial for activity of the encapsulated enzyme, potentially by increasing diffusion rates. Cryogenic electron microscopy reveals the overall architecture of the encapsulated dimeric NfsB within the nanoreactor environment and identifies multiple pore states in the shell. These findings highlight the potential of encapsulins as versatile tools for enhancing enzyme performance across various fields.

Solubility and aggregation of proteins are crucial factors for their functional and further biological roles. Aggregation of proteins in vivo, such as the amyloid beta (Aβ_1-40) peptide into fibrils, is significantly modulated by membrane lipids, abundantly present in cells. We developed a model membrane system, composed of lipid hybrid-vesicles bearing embedded hydrophilic polymers to in vitro study the aggregation of the Aβ_1-40 peptide. Focus is to understand and inhibit the primordial, nucleation stages of their fibrillation by added hybrid-vesicles, composed of a natural lipid and amphiphilic polymers. These designed hybrid-vesicles are based on 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), displaying embedded hydrophilic (EO) _m P _n A_EG polymers (m = 2 or 3; P _n = 10 to 52 with M _n = 2800-9950 gmol^-1) in amounts ranging from 5-20 mol%, anchored to the POPC vesicles via hydrophobic hexadecyl-, glyceryl- and cholesteryl-moieties, affixed to the polymers as end-groups. All investigated hybrid-vesicles significantly delay fibrillation of the Aβ_1-40 peptide as determined by thioflavin T (ThT) assays. We observed that the hybrid-vesicles interacted with early aggregating species of Aβ_1-40 peptide, irrespective of their composition or size. A substantial perturbation of both primary (k ₊ k _n ) and secondary (k ₊ k ₂) nucleation rates of Aβ_1-40 by the POPC-polymer vesicles compared to POPC vesicles was observed, particularly for the cholesteryl-anchored polymers, interfering with the fragmentation and elongation steps of Aβ_1-40. Furthermore, morphological differences of the aggregates were revealed by transmission electron microscopy (TEM) images supported the inhibitory kinetic signatures.

Radical SAM cyclophane synthases catalyze C-C, C-N, and C-O crosslinking reactions in the biosynthesis of bioactive peptide natural products. Here, we studied an uncharacterized rSAM enzyme, HtkB from Pandoraea sp., and found this enzyme to catalyze the formation of a HisC2-to-LysCβ crosslink. We used a combination of ColabFold and mutagenesis studies to show that residues D214 in HtkB and H204 in HaaB (another cyclophane synthase) are important for substrate specificity. Mutation of these residues changes the specificity and lowers substrate recognition on the wild-type motifs. This result opens opportunities to alter the specificity and promiscuity for rSAM peptide modifying enzymes.

Self-labeling protein tags are an efficient means to visualize, manipulate, and isolate engineered fusion proteins with suitable chemical probes. The SNAP-tag, which covalently conjugates to benzyl-guanine and -chloropyrimidine derivatives is used extensively in fluorescence microscopy, given the availability of suitable SNAP-ligand-based probes. Here, we extend the applicability of the SNAP-tag to targeted protein degradation. We developed a set of SNAP PROteolysis TArgeting Chimeras (SNAP-PROTACs), which recruit the VHL or CRBN-ubiquitin E3 ligases to induce the degradation of SNAP-fusion proteins. Endogenous tagging enabled the visualization and the selective depletion of a SNAP-clathrin light chain fusion protein using SNAP-PROTACs. The addition of PROTACs to the SNAP-tag reagent toolbox facilitates the comprehensive analysis of protein function with a single gene tagging event.

Bioorthogonal reactions are extremely useful for the chemical modification of biomolecules, and are already well studied in mammalian cells. In contrast, very little attention has been given to the feasibility of such reactions in bacteria. Herein we report modified coumarin dyes for monitoring the internalisation and activity of bioorthogonal catalysts in the Gram-positive bacterial species Bacillus subtilis. Two fluorophores based on 7-aminocoumarin were synthesised and characterised to establish their luminescence properties. The introduction of an allyl carbamate (R₂N-COOR') group onto the nitrogen atom of two 7-aminocoumarin derivatives with different solubility led to decreased fluorescence emission intensities and remarkable blue-shifts of the emission maxima. Importantly, this allyl carbamate group could be uncaged by the bioorthogonal, organometallic ruthenium catalyst investigated in this work, to yield the fluorescent product under biologically-relevant conditions. The internalisation of this catalyst was confirmed and quantified by ICP-OES analysis. Investigation of the bacterial cytoplasm and extracellular fractions separately, following incubation of the bacteria with the two caged dyes, facilitated their localisation, as well as that of their uncaged form by catalyst addition. In fact, significant differences were observed, as only the more lipophilic dye was located inside the cells and importantly remained there, seemingly avoiding efflux mechanisms. However, the uncaged form of this dye is not retained, and was found predominantly in the extracellular space. Finally, a range of siderophore-conjugated derivatives of the catalyst were investigated for the same transformations. Even though uptake was observed, albeit less significant than for the non-conjugated version, the fact that similar intracellular reaction rates were observed regardless of the iron content of the medium supports the notion that their uptake is independent of the iron transporters utilised by Gram-positive Bacillus subtilis cells.

The CDK12 inhibitor SR-4835 promotes the proteasomal degradation of cyclin K, contingent on the presence of CDK12 and the CUL4-RBX1-DDB1 E3 ligase complex. The inhibitor displays molecular glue activity, which correlates with its enhanced ability to inhibit cell growth. This effect is achieved by facilitating the formation of a ternary complex that requires the small molecule SR-4835, CDK12, and the adaptor protein DDB1, leading to the subsequent ubiquitination and degradation of cyclin K. We have successfully solved the structure of the ternary complex, enabling the de novo design of molecular glues that transform four different CDK12 scaffold inhibitors, including the clinical pan-CDK inhibitor dinaciclib, into cyclin K degraders. These results not only deepen our understanding of CDK12's role in cell regulation but also underscore significant progress in designing molecular glues for targeted protein degradation in cancers associated with dysregulated cyclin K activity.

Tryptophan plays a critical role in proteins by contributing to stability, allostery, and catalysis. Using fluorine (¹⁹F) nuclear magnetic resonance (NMR), protein conformational dynamics and structure-activity relationships (SARs) can be studied via fluorotryptophan reporters. Tryptophan analogs such as 4-, 5-, 6-, or 7-fluorotryptophan can be routinely incorporated into proteins during heterologous expression by arresting endogenous tryptophan biosynthesis. Building upon the large ¹⁹F chemical shift dispersion associated with 5-fluorotryptophan, we introduce an approach to the incorporation of ¹³C-enriched 5-fluorotryptophan using a direct biosynthetic precursor, 5-fluoroanthranilic acid-(phenyl-¹³C₆). The homodimeric enzyme fluoroacetate dehalogenase (FAcD), a thermophilic alpha/beta hydrolase responsible for the hydrolysis of a C-F bond in fluoroacetate, was expressed and biosynthetically labeled with (phenyl-¹³C₆) 5-fluorotryptophan. The resulting two-dimensional ¹⁹F-¹³C (transverse relaxation optimized spectroscopy) TROSY heteronuclear correlation spectra provide complete resolution of all 9 tryptophan residues in the apo enzyme and FAcD saturated with the substrate analog bromoacetate. The (¹⁹F,¹³C) correlation spectra also reveal a multitude of minor resonances in the apo sample. The role of each tryptophan residue in allosteric communication was validated with computational rigidity transmission allostery analysis, which in this case explores the relative interprotomer communication between all possible tryptophan pairs.

Malignant primary brain tumors are a group of highly aggressive and often infiltrating tumors that lack adequate therapeutic treatments to achieve long time survival. Complete tumor removal is one precondition to reach this goal. A promising approach to optimize resection margins and eliminate remaining infiltrative so-called guerilla cells is photodynamic therapy (PDT) using organic photosensitizers that can pass the disrupted blood-brain-barrier and selectively accumulate in tumor tissue. Hypericin fulfills these conditions and additionally offers outstanding photophysical properties, making it an excellent choice as a photosensitizing molecule for PDT. However, the actual hypericin-induced PDT cell death mechanism is still under debate. In this work, hypericin-induced PDT was investigated by employing the three distinct fluorescent probes hypericin, resorufin and propidium iodide (PI) in fluorescence-lifetime imaging microscopy (FLIM). This approach enables visualizing the PDT-induced photodamaging and dying of single, living glioma cells, as an in vitro tumor model for glioblastoma. Hypericin PDT and FLIM image acquisition were simultaneously induced by 405 nm laser irradiation and sequences of FLIM images and fluorescence spectra were recorded to analyze the PDT progression. The reproducibly observed cellular changes provide insight into the mechanism of cell death during PDT and suggest that apoptosis is the initial mechanism followed by necrosis after continued irradiation. These new insights into the mechanism of hypericin PDT of single glioma cells may help to adjust irradiation doses and improve the implementation as a therapy for primary brain tumors.