Barbosa Pereira, P. J. | Search bioRxiv

bioRxiv2023

Development of supramolecular anticoagulants with on-demand reversibility

Drugs are administered at a dosing schedule set by their therapeutic index and termination of action is achieved by clearance and metabolism of the drug (hours to days for small molecules, weeks to months for biologics). In some cases, it is important to achieve a fast reversal of the drugs action to overcome dangerous side effects or in response to unforeseen events. A case in point is for anticoagulant drugs. Here we report a general strategy to achieve on-demand reversibility by leveraging supramolecular assembly of drug fragments and showcase the approach with thrombin-inhibiting anticoagulants. In our supramolecular drug design, the action of the drug is reinforced by a dynamic hybridisation of peptide nucleic acids (PNAs) between drug fragments. We show that this design enables the generation of very potent bivalent direct thrombin inhibitors (Ki 74 pM) and this inhibition can be reversed through the use of a PNA antidote. We demonstrate that these supramolecular inhibitors exhibit potent anticoagulant activity in vitro and in vivo and that this activity can also be reversed on demand.

biochemistry↗

bioRxiv2020

A secreted NlpC/P60 endopeptidase from Photobacterium damselae subsp. piscicida cleaves the peptidoglycan of potentially competing bacteria

Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. Here, it is described the structural and functional characterization of an NlpC/P60-containing peptidase from Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes high mortality of warm-water marine fish with great impact for the aquaculture industry. PnpA (Photobacterium NlpC-like Protein A) has a four-domain structure with a hydrophobic and narrow access to the catalytic center and specificity for the {gamma}-D-glutamyl-meso-diaminopimelic acid bond. However, PnpA does not cleave the PG of Phdp and neither PG of several Gram-negative and Gram-positive bacterial species. Interestingly, it is secreted by the Phdp type II secretion system and degrades the PG of Vibrio anguillarum and V. vulnificus. This suggests that PnpA is used by Phdp to gain an advantage over bacteria that compete for the same resources or to obtain nutrients in nutrient-scarce environments. Comparison of the muropeptide composition of PG susceptible and resistant to the catalytic activity of PnpA, showed that the global content of muropeptides is similar, suggesting that susceptibility to PnpA is determined by the three-dimensional organization of the muropeptides in the PG. IMPORTANCEPeptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, originating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or joint its components. Here, it is shown that Photobacterium damselae subsp. piscicida, a bacterium that causes high mortality in warm-water marine fish, produces PnpA, an enzyme that is secreted into the environment and is able to cleave the PG of potentially competing bacteria, either for gaining competitive advantages and/or to get nutrients. The specificity of PnpA to the PG of some bacteria and its inability to cleave others may be explained by differences in the structure of the PG mesh and not by different muropeptide composition.

microbiology↗