Biology subjects

Cowan, A.

Publications and source records attributed to Cowan, A..

3 recordsLinked to original sources

bioRxiv2025

Tuning the open-close equilibrium of Cereblon with small molecules influences protein degradation

Most PROTACs and molecular glue degraders currently approved or in clinical trials recruit Cereblon (CRBN) as the ubiquitin E3 ligase. Upon binding ligands and molecular glues, CRBN undergoes a significant structural rearrangement from an open to closed state, defined by the positioning of the thalidomide-binding domain (TBD) with respect to the Lon domain. However, the exact molecular basis for this ligand-induced conformational change and its implication to neo-substrate degradation remain elusive. During our campaign to discover novel CRBN binders, we found hits exhibiting distinct biophysical behaviour from classical thalidomide-based ligands. By combining orthogonal biophysical methods of differential scanning fluorimetry, isothermal titration calorimetry, and small-angle X-ray scattering, supported by X-ray crystallography and cryo-EM structures of ligand-bound complexes, we classify CRBN binders between those that can induce CRBN closure, and those that cannot. Mutational studies identify key residues in the CRBN ligand binding pocket and N-terminal belt that are essential for the Lon and TBD domains to come together in the closed state. Finally, we show that the probability to yield active degrader molecules is greatly influenced by whether binders can or cannot induce CRBN closure. Together, our study reveals new molecular insights into the structural basis for how CRBN open-closed equilibrium is directly modulated by compound binding and impact target degradability by CRBN, with important implications to the design of PROTACs and molecular glue degraders.

biochemistry↗

bioRxiv2018

Vector-Mediated Transport Producing Drug-like Peptides

Drugs that are structural mimetics of peptides (e.g. small molecules) have been plagued by problems associated with oral availability and transcellular movement. Vector-mediated transport, where a potentially therapeutic drug is covalently linked to another molecule that is a ligand for an active transport or transcytosis system, was developed as an approach for moving a drug across the blood-brain-barrier. We now report a vector approach that produced peptides with oral activity, blood-brain-barrier transport, and extended in vivo half-life. Generating these properties requires secondary structure stabilization into a {beta} hairpin, and the addition of a C-terminal dipeptide sequence composed of non-polar residues. Peptides with biological activity incompatible with these derivatizations were covalently linked to a model transport vector, producing a chimera with the therapeutic activity of the peptide and the transport properties of the vector. Our platform technology may be a general approach for the design of drug-like peptides.

pharmacology and toxicology↗

bioRxiv2018

Autoregulation of the S. mutans SloR metalloregulator is constitutive and driven by an independent promoter

Streptococcus mutans, one of [~]600 bacterial species in the human oral cavity, is among the most acidogenic constituents of the plaque biofilm. Considered to be the primary causative agent of dental caries, S. mutans harbors a 25kDa SloR metalloregulatory protein which controls metal ion transport across the bacterial cell membrane to maintain essential metal ion homeostasis. The expression of SloR derives, in part, from transcriptional readthrough of the sloABC operon which encodes a Mn2+/Fe2+ ABC transport system. Herein, we describe the details of the sloABC promoter that drives this transcription, as well as a novel independent promoter in an intergenic region (IGR) that contributes to downstream sloR expression. RT-PCR studies support sloR transcription that is independent of sloABC expression, and the results of 5' RACE revealed a sloR transcription start site in the IGR from which the -10 and -35 promoter regions were predicted. The results of gel mobility shift assays support direct SloR binding to the IGR, albeit with lower affinity than SloR binding to the sloABCR promoter. Function of the sloR promoter was validated in qRT-PCR experiments. Interestingly, sloR expression was not significantly impacted when grown in the presence of high manganese, whereas expression of the sloABC operon was repressed under these conditions. The results of in vitro transcription studies support SloR-mediated transcriptional-activation of sloR and -repression of sloABC. Taken together, these findings implicate SloR as a bifunctional regulator that represses sloABC promoter activity and encourages sloR transcription from an independent promoter.\n\nImportanceTooth decay is a ubiquitous infectious disease that is especially pervasive in underserved communities worldwide. S. mutans-induced carious lesions cause functional, physical, and/or aesthetic impairment in the vast majority of adults, and in 60-90% of schoolchildren in industrialized countries. Billions of dollars are spent annually on caries treatment, and productivity losses due to absenteeism from the workplace are significant. Research aimed at alleviating S. mutans-induced tooth decay is important because it can address the socioeconomic disparity that is associated with dental cavities and improve overall general health which is inextricably linked to oral health. Research focused on the S. mutans SloR metalloregulatory protein can guide the development of novel therapeutics and so alleviate the burden of dental cavities.

microbiology↗