Building a Molecular Trap for a Serine Protease from Aptamer and Peptide Modules

Abstract

In drug development, molecular intervention strategies are usually based on interference with a single protein function, such as enzyme activity or receptor binding. However, in many cases, protein drug targets are multifunctional, with several molecular functions contributing to their pathophysiological actions. Aptamers and peptides are interesting synthetic building blocks for the design of multivalent molecules capable of modulating multiple functions of a target protein. Here, we report a molecular trap with the ability to interfere with the activation, catalytic activity, receptor binding, etc. of the serine protease urokinase-type plasminogen activator (uPA) by a rational combination of two RNA aptamers and a peptide with different inhibitory properties. The assembly of these artificial inhibitors into one molecule enhanced the inhibitory activity between 10- and 10,000-fold toward several functions of uPA. The study highlights the potential of multivalent designs and illustrates how they can easily be constructed from aptamers and peptides using nucleic acid engineering, chemical synthesis, and bioconjugation chemistry. By aptamer to aptamer and aptamer to peptide conjugation, we created, to the best of our knowledge, the first trivalent molecule which combines three artificial inhibitors binding to three different sites in a protein target. We hypothesize that by simultaneously preventing all of the functional interactions and activities of the target protein, this approach may represent an alternative to siRNA technology for a functional knockout.

Aptamers in HIV research diagnosis and therapy

“…Dupont et al, has presented detailed review on biochemical mechanisms of aptamer selection, protein-aptamer recognition, protease inhibition, and advantages of aptamers for pharmacological intervention with pathophysiological functions of proteases [141]. Several studies have reported proteaseinhibiting aptamers directed against blood coagulation factors, which are currently undergoing clinical trials as anticoagulant drugs [141][142][143]. Duclair et al, isolated high-affinity RNA Aptamers against the HIV-1 protease to inhibit protease activity [144].…”

Synthetic Nucleic Acid Analogues in Gene Therapy: An Update for Peptide–Oligonucleotide Conjugates

“…Such a multivalent molecule was synthesized by combining three artificial inhibitors, two aptamers, and a peptide. The conjugated moieties bind the serine protease urokinase‐type plasminogen activator at three different sites and simultaneously prevent all functional interaction of the same …”

“…[64] Given the ability of oligonucleotidest oc ontrol peptide conformation and regulate protein activity,t he combination of peptides and oligonucleotides as POCs is ah ighly effective approach when the two parts of the POC work together. [64] In addition, the potential of multivalent designs that are capable of modulating more functions of at arget protein has been highlighted by Dupont et al [65] This approachi sagood alternative to functional knockout. Such amultivalent molecule was synthesized by combining three artificial inhibitors, two aptamers, and ap eptide.…”

“…The conjugated moieties bind the serine protease urokinase-typep lasminogen activatora tt hree different sites and simultaneously prevent all functional interaction of the same. [65] Ta king into account all of the aforementioned results, CPPs and their conjugates are promising candidates for conjugation to NATs .H owever,d espite knowledge gained of CPPs in recent decades, there is still no unified CPP design that can internalize variousc argos into all cell types. This obstacle creates a demand for efficient methods to developn ew CPPs.…”

Multivalent Aptamers: Versatile Tools for Diagnostic and Therapeutic Applications

“…The concrete method of connection is determined by peculiarities of aptamer-target interaction and by the desired functions of a multi-aptamer construct. In principle, aptamer domains can be covalently fused in an end-to-end manner, i.e., with zero linkers, but this approach is quite rarely used [19,20,21,22]. As a rule, linkers of different length and nature are required for proper functioning of multivalent constructs.…”