Researchers have developed a system for designing protein inhibitors with reversible activity. The method is based on a supramolecular drug that results from the interaction of two or more molecules through non-covalent bonds. The formation and dissociation of the supramolecular drug complex are controlled by the binding of synthetic peptide nucleic acids (PNAs).

Researchers from the Universities of Geneva and Sydney utilized this approach to create supramolecular drugs with binding sites for two distinct PNAs: an activator and an inhibitor. These drugs exhibit remarkable efficacy and can reverse their anticoagulant effects. This design strategy can be applied to therapeutic targets with two identifiable binding sites.

The research article, “Development of supramolecular anticoagulants with on-demand reversibility,” was published in Nature Biotechnology

The anticoagulant risk

Anticoagulants can cause bleeding in cases of adverse events or injury, with bleeding and side effects related to anticoagulants accounting for approximately 15% of all emergency department visits caused by negative drug effects.

An often employed strategy to mitigate the impact of anticoagulants is to administer nonspecific reversal agents. This process entails administering coagulation factors specifically engineered to counteract the effects of anticoagulants in the bloodstream. Monoclonal antibodies with a high affinity for specific small-molecule anticoagulants have recently been developed. This binding facilitates the reversal of the inhibition of coagulation factor activity. While these approaches can be effective, they have limitations and are costly.

Supramolecular drugs and PNAs

Supramolecular entities depend on unstable noncovalent interactions and, due to their inherent characteristics, are dynamic and can be reversed in response to particular environmental cues or stimuli by altering the balance in the system. These characteristics of supramolecular systems have been skillfully utilized in molecular recognition, catalysis, molecular motors, stimuli-responsive polymers, and drug discovery and delivery. However, they have not been implemented in medicinal chemistry and pharmacology. 

PNAs are known for their high metabolic stability and cannot traverse cell membranes unless deliberately modified. These characteristics make PNAs an optimal selection for directing the action of supramolecular drugs toward extracellular targets, thereby minimizing unintended effects on nontarget areas and any inherent pharmacological activity for the antidote. The utilization of different sequences of cost-effective PNAs to encode assembly allows for the possibility of multiplexing programmable supramolecular drug candidates. 

Anticoagulants with reversible activity

Millicent Dockerill, a PhD student at the University of Geneva, and her colleagues have developed a method to create powerful anticoagulants that inhibit thrombin and can be reversed using programmed supramolecular assembly. The strategy relies on the capacity to connect two fragments through a reversible supramolecular interaction. The two fragments can synergistically interact with the target at two separate locations, forming the active inhibitor as directed by the target. Interrupting the supramolecular interaction connecting the two fragments leads to decreased cooperative behavior and, consequently, a loss of inhibitory function.

The supramolecular assembly was designed based on natural thrombin inhibitors in hematophagous organisms like leeches, ticks, mosquitoes, and flies. These organisms produce small protein thrombin inhibitors in their salivary glands to aid in acquiring and digesting a blood meal. These salivary proteins effectively inhibit thrombin by interacting with two binding sites on the enzyme. However, their strong affinity for thrombin makes it difficult to reverse this inhibition. 

The designed supramolecular anticoagulants exhibited strong inhibitory effects on thrombin and demonstrated anticoagulation activities in laboratory conditions. These effects could be promptly reversed by employing small antidotes based on PNAs. The strong ability to prevent blood clotting, which can be reversed when needed, was also shown in a live model of blood clot formation. This finding serves as a foundation for the potential future use of this treatment method for potential anticoagulant drugs. 

In this case, the strategy offers a comprehensive approach to rapidly initiating or halting therapeutic activity and is thus not limited to its use in thrombosis applications. For instance, the application of the supramolecular concept could prove advantageous in immunotherapy scenarios where there is a need for an antidote to counteract the response of chimeric antigen receptor T cells or to reverse the effects of immunomodulators in cases of severe infection.

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