Protection of HIV neutralizing aptamers against rectal and vaginal nucleases: implications for RNA-based therapeutics

RNA-based drugs are an emerging class of therapeutics. They have the potential to regulate proteins, chromatin, as well as bind to specific proteins of interest in the form of aptamers. These aptamers are protected from nuclease attack by chemical modifications that enhance their stability for in vivo usage. However, nucleases are ubiquitous, and as we have yet to characterize the entire human microbiome it is likely that many nucleases are yet to be identified. Any novel, unusual enzymes present in vivo might reduce the efficacy of RNA-based therapeutics, even when they are chemically modified. We have previously identified an RNA-based aptamer capable of neutralizing a broad spectrum of clinical HIV-1 isolates and are developing it as a vaginal and rectal microbicide candidate. As a first step we addressed aptamer stability in the milieu of proteins present in these environments. Here we uncover a number of different nucleases that are able to rapidly degrade 2'-F-modified RNA. We demonstrate that the aptamer can be protected from the nuclease(s) present in the vaginal setting, without affecting its antiviral activity, by replacement of key positions with 2'-O-Me-modified nucleotides. Finally, we show that the aptamer can be protected from all nucleases present in both vaginal and rectal compartments using Zn(2+) cations. In conclusion we have derived a stable, antiviral RNA-based aptamer that could form the basis of a pre-exposure microbicide or be a valuable addition to the current tenofovir-based microbicide candidate undergoing clinical trials.     

Allostery and Missense Mutations as Intermittently Linked Promising Aspects of Modern Computational Drug Discovery

Drug research and development is a multidisciplinary field with its own successes. Yet, given the complexity of the process, it also faces challenges over the long development stages and even includes those that develop once a drug is marketed, i.e. drug toxicity and drug resistance. Better success can be achieved via well designed criteria in the early drug development stages. Here, we introduce the concepts of allostery and missense mutations, and argue that incorporation of these two intermittently linked biological phenomena into the early computational drug discovery stages would help to reduce the attrition risk in later stages of the process. We discuss the individual or in concert mechanisms of actions of mutations in allostery. Design of allosteric drugs is challenging compared to orthosteric drugs, yet they have been gaining popularity in recent years as alternative systems for the therapeutic regulation of proteins with an action-at-a-distance mode and non-invasive mechanisms. We propose an easy-to-apply computational allosteric drug discovery protocol which considers the mutation effect, and detail it with three case studies focusing on (1) analysis of effect of an allosteric mutation related to isoniazid drug resistance in tuberculosis; (2) identification of a cryptic pocket in the presence of an allosteric mutation of falcipain-2 as a malarial drug target; and (3) deciphering the effects of SARS-CoV-2 evolutionary mutations on a potential allosteric modulator with changes to allosteric communication paths.