Biology subjects

Whittaker, M. R.

Publications and source records attributed to Whittaker, M. R..

4 recordsLinked to original sources

bioRxiv2026

Harnessing Diacylglycerol-Terminated Cationic Oligomers for Next-Generation Antibacterial Therapeutics

Cationic polymers, which mimic the structure of antimicrobial peptides (AMPs), are increasingly recognized as promising antimicrobial materials. Here, we report the synthesis and evaluation of a new class of cationic lipid-terminated oligomers (CLOs), comprised of 2C18-hydrophobic lipid tails, and short oligomeric cationic chains synthesised via Cu(0)-mediated reversible-deactivation radical polymerization (RDRP). Two 2-vinyl-4,4-dimethyl-5-oxazolone (VDM) oligomers with degrees of polymerization (DP) of 20 or 50 were synthesized using the lipid functional initiator (R)-3-((2-bromo-2-methylpropanoyl) oxy)propane-1,2-diyl dioctadecanoate (2C18-Br). Post-polymerization modification of the pendant oxazolone moieties was carried out using reactive amines, including N-Boc-ethylenediamine (BEDA) and N,N-dimethylethylenediamine (DMEN). Subsequent deprotection of the BEDA groups and quaternization of DMEN groups enabled the synthesis of six functional CLOs exhibiting distinct cationic functionalities. Antimicrobial assays against a panel of WHO bacterial and fungal priority pathogens (methicillin-resistant Staphylococcus aureus [MRSA], Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Candida albicans, and Cryptococcus neoformans) revealed that these CLOs exhibited potent and selective structure-dependent antibacterial activity, particularly against MRSA, with minimum inhibitory concentrations (MICs) in the clinically relevant range, below 4 {micro}g mL-1, comparable to antibiotics vancomycin and colistin. Among these, BEDA-functionalized CLOs demonstrated the strongest antimicrobial profile, which was significantly increased by increasing DP, as evidenced by a reduction in MIC values from 64 {micro}g mL-1 (for DP20) to [≤] 4 {micro}g mL-1 (for DP50) against A. baumannii. Biocompatibility assays against red blood cells and HEK293 cells indicated negligible toxicity, with haemolytic (HC50) and cytotoxic (CC50) values exceeding 512 {micro}g mL-1 across all CLOs. All CLOs displayed minimal activity against C. albicans (MIC [≥] 512 {micro}g mL-1). In contrast, activity against C. neoformans was influenced by both cationic functionality and DP, with DMEN-based CLOs exhibited superior antifungal activity at higher DP relative to their BEDA-based counterparts. Most CLOs displayed high selectivity (SI) toward MRSA (SI >128), while 2C18-O(BEDA)50 exhibited the broadest spectrum, showing potent antimicrobial activity and high selectivity against E. coli (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), A. baumannii (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), and MRSA (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), along with moderate activity against P. aeruginosa (MIC = 32 {micro}g mL-1, SI > 16). Taken together, these findings elucidate the combined influence of end-group lipidation, cationic functionality, and polymer length in modulating antimicrobial activity, thereby establishing 2C18-terminated CLOs as a rationally tunable and biocompatible platform for antimicrobial material development.

microbiology↗

bioRxiv2026

Targeting of subcellular metabotropic glutamate receptor 5 signaling to modulate pain transmission

Metabotropic glutamate receptor 5 (mGlu5) is a class C GPCR crucial for neuronal development and synaptic transmission. mGlu5 is a potential therapeutic target in pain management and modulates pain-associated gene expression and signaling pathways. Although mGlu5 inhibitors have shown promise in treating pain, none have translated to the clinic. Up to 90% of neuronal mGlu5 expression is intracellular, although the precise locations and function of different mGlu5 intracellular pools remains unclear. Building on recent evidence showing the importance of endosome-mediated nociceptive signaling by other GPCRs, we hypothesized that endosomal pools of mGlu5 contribute to pain transmission, and that targeted inhibition of intracellular mGlu5 signaling results in superior analgesia. Using calcium mobilization assays and genetically encoded resonance energy transfer biosensors, we report that upon its activation mGlu5 recruits Gq/11 and Gs to the plasma membrane. Conversely, internalized mGlu5 in endosomes recruits only Gq/11 proteins. mGlu5 signaling is highly dependent on receptor trafficking to endosomes, with sustained nuclear ERK1/2 signaling requiring both receptor internalization and active glutamate transport into the cell. We generated pH responsive nanoparticles loaded with the mGlu5 negative allosteric modulator VU0366058 (DIPMA-VU058), enabling endosome-targeted inhibition of mGlu5. Nanoparticle encapsulation of VU0366058 enhanced inhibition of both acute and sustained nuclear ERK1/2 signaling, and significantly reduced neuronal excitability in nociceptive circuits in spinal cord slices from rats with neuropathic pain. Intrathecal administration of DIPMA-VU058 achieved superior analgesia in both inflammatory and neuropathic models of pain in mice compared to free VU0366058 and the reference compound fenobam. These studies demonstrate the importance of endosome-associated receptors for the complete mGlu5 signaling response. Furthermore, we show that manipulating the cellular distribution of an allosteric modulator can engender location-biased pharmacological effects. Together, we have revealed new and unappreciated roles for endosome-specific mGlu5 signaling and demonstrate that endosome-selective targeting may offer an alternative therapeutic approach for modulating mGlu5 activity.

pharmacology and toxicology↗

bioRxiv2025

Bioactive Cationic Lipidated Oligomers (CLOs) as Antimicrobial Materials: Metabolomic Insights into MRSA Membrane Disruption

The escalating incidence of antimicrobial resistance in Staphylococcus aureus, particularly the methicillin-resistant strain (MRSA), necessitates the development of novel therapeutic strategies. Cationic lipidated oligomers (CLOs) have emerged as promising membrane-active antimicrobial agents; however, their mechanisms of action remain insufficiently understood. In this study, untargeted metabolomics was employed to systematically profile the temporal metabolic perturbations induced by two structurally distinct CLOs, C12-o-DMEN-10 and C12-o-BEDA-10, in MRSA across four defined time points (0.25, 0.5, 1, and 3 hours). These CLOs previously demonstrated differential antibacterial activity, as evidenced by dose-dependent propidium iodide (PI) uptake and growth inhibition assays. Metabolomic analysis revealed pronounced and sustained disruptions in bacterial membrane lipid metabolism, including significant depletion of phosphatidylglycerols ([≥] -2.5 log2FC, p < 0.05), alongside elevated levels of phosphatidylethanolamines, lysophospholipids, and fatty acid-derived metabolites indicative of membrane destabilization and lipid remodelling. Although both CLOs affected overlapping metabolic pathways, they differed in the extent and temporal dynamics of their effects. These findings provide mechanistic insights into CLO-mediated antibacterial activity and highlight the value of metabolomics in elucidating both direct and downstream cellular responses, which extend beyond the scope of conventional membrane integrity assays, such as PI fluorescence.

systems biology↗

bioRxiv2024

Providing insight into the mechanism of action of Cationic Lipidated Oligomers (CLOs) using metabolomics

The increasing resistance of clinically relevant microbes against current commercially available antimicrobials underpins the urgent need for alternative and novel treatment strategies. Cationic lipidated oligomers (CLOs) are innovative alternatives to antimicrobial peptides, and have reported antimicrobial potential. An understanding of their antimicrobial mechanism of action is required to rationally design future treatment strategies for CLOs, either in monotherapy or synergistic combinations. In the present study, metabolomics was used to investigate the potential metabolic pathways involved in the mechanisms of antibacterial activity of one CLO, C12-o-(BG-D)-10, which we have previously shown to be effective against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. The metabolomes of MRSA ATCC 43300 at 1, 3 and 6 h following treatment with C12-o-(BG-D)-10 (48 {micro}g/mL i.e., 3x MIC) were compared to those of the untreated controls. Our findings reveal that the studied CLO, C12-o-(BG-D)-10, disorganized the bacterial membrane as the first step towards its antimicrobial effect, as evidenced by marked perturbations in the bacterial membrane lipids and peptidoglycan biosynthesis observed at early time points i.e., 1, and 3 h. Central carbon metabolism, and biosynthesis of DNA, RNA, and arginine were also vigorously perturbed, mainly at early time points. Moreover, bacterial cells were under osmotic and oxidative stress across all time points, evident by perturbations of trehalose biosynthesis and pentose phosphate shunt. Overall, this metabolomics study has, for the first time, revealed that the antimicrobial action of C12-o-(BG-D)-10 may potentially stem from the dysregulation of multiple metabolic pathways. ImportanceAntimicrobial resistance poses a significant challenge to healthcare systems worldwide. Novel anti-infective therapeutics are urgently needed to combat drug-resistant microorganisms. Cationic lipidated oligomers (CLOs) show promise as new antibacterial agents against Gram-positive pathogens like Staphylococcus aureus (MRSA). Understanding their molecular mechanism(s) of antimicrobial action may help design synergistic CLO treatments along with monotherapy. Here, we describe the first metabolomics study to investigate the killing mechanism(s) of CLOs against MRSA. The results of our study indicate that the CLO, C12-o-(BG-D)-10, had a notable impact on the biosynthesis and organization of the bacterial cell envelope. C12-o-(BG-D)-10 also inhibits arginine, histidine, central carbon metabolism, and trehalose production, adding to its antibacterial characteristics. This work illuminates the unique mechanism of action of C12-o-(BG-D)-10 and opens an avenue to design innovative antibacterial oligomers/polymers for future clinical applications.

microbiology↗