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BICEPSvsHIV Report Summary

Project ID: 751931
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - BICEPSvsHIV (Novel strategies for anti-HIV-1 therapy: Small molecules targeting RNA partners of the nucleocapsid protein)

Reporting period: 2017-07-01 to 2018-06-30

Summary of the context and overall objectives of the project

Human Immunodeficiency Virus (HIV) infection remains of major public health importance in Europe. Most of the commonly available drugs, although potent and selective, experienced clinical failures and severe side effects. Alternative antiretroviral drugs and novel therapeutic strategies are thus urgently needed to overcome the emergence of resistance to existing drugs.
The project “BICEPSvsHIV” proposes a new anti-HIV strategy focused on RNA. Specific RNA sequences of the viral genome are substrates of the HIV-1 nucleocapsid (NC), a highly conserved protein known for promoting remodeling of nucleic acid structures in essential steps of the virus replication cycle. Our working hypothesis is that the employment of bis-3-chloropiperidines (BICEPS) as RNA cross-linking agents could represent a novel pharmacological treatment to impair NC-mediated processes, overcoming drug resistance. Small molecules able to bind to the RNA substrates of NC and freeze their three-dimensional configurations could block their NC-mediated secondary structure remodeling.
The specific aims of the project are: 1) the identification of positive hits targeting selectively the RNA substrates of NC, the evaluation of their binding strength and selectivity, and the evaluation of their detailed molecular mechanism of reaction towards RNA; 2) the evaluation of stabilization of the dynamical conformation of RNA substrates of NC by selective RNA cross-linkers and of their strength in inhibiting in vitro NC activity; 3) the structure-activity relationship analysis of the tested compounds to optimize RNA specific cross-linking agents for the development of anti-NC lead compounds.
The reporting period corresponds to the outgoing phase of the MSCA, in which the Experienced Researcher (ER) worked at The RNA Institute (SUNY Albany, NY, USA) in the research laboratory of Prof. Daniele Fabris.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In the first year of the “BICEPSvsHIV” project, research activities were mainly focused on the investigation of BICEPS reactivity towards RNA by means of unique and innovative mass spectrometric techniques. The ER was provided with a wide library of bis-3-chloropiperidines, a new class of alkylating agents. In order to examine the wide library of BICEPS and their direct interaction with RNA constructs, the following tasks were successfully completed:
TASK 1.A: Elucidation of the detailed mechanism of BICEPS reaction towards RNA
TASK 1.B: Elucidation of the selectivity of BICEPS for viral RNAs
TASK 1.C: Investigation of BICEPS effects on NC binding to its RNA substrates
We employed direct infusion electrospray ionization (ESI) mass spectrometry (MS) to investigate the interactions between BICEPS and RNA. The direct determination of binding stoichiometry and ligands composition in complex with RNA was determined. The detailed molecular mechanism of action of BICEPS towards RNA was unambiguously resolved.
We then focused on the detection of inter-strand crosslinking adducts which are the desired ones in order to develop antiviral agents able to freeze the secondary structures of RNA sequences. Aimed at the development of a fast and reproducible tool for the unambiguous identification of inter-molecular conjugates induced by crosslinking agents, we designed and optimized a new approach based on thermal melting and ESI-MS to unambiguously discriminate between intra- and inter-molecular adducts produced by BICEPS. We screened the entire library of bis-3-chloropiperidine compounds and we identified the compounds able to induce inter-strand crosslinks on RNA. These compounds were therefore selected for the further analysis on viral RNA sequences.
In order to investigate by ESI-MS multicomponent macromolecular complexes, we developed and optimized a mass spectrometry-based assay to evaluate the effects induced by RNA ligands on NC binding to TAR and to other viral RNAs that are substrates of NC. Selected bis-3-chloropiperidine derivatives were reacted with RNA sequences replicating the NC substrates. We investigated the possible NC binding inhibition by performing ESI-MS analysis upon addition of the protein to the probed RNA substrates. We obtained clear evidences of BICEPS-induced impairment of NC binding to its RNA substrates.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Progress beyond the state of the art relates to the investigation of BICEPS reactivity towards RNA and to the development of innovative mass spectrometry-based tools for the analysis of RNA structures, structure-function relationships of RNA-ligands and RNA-protein complexes involved in the lifecycle of viruses. So far, the results obtained are highly informative in order to identify the most promising BICEPS targeting RNA and to elucidate their mechanisms of NC inhibition. We reached a valid starting point to identify effective NC inhibitors targeting its RNA partners.
In the second year of the project, during the incoming phase of the project at the Department of Pharmaceutical and Pharmacological Sciences of the University of Padova, the research will be focused on the biological evaluation of the in vitro NC inhibition by BICEPS, the results will be correlated with those obtained during the secondment year with the final aim to identify an effective anti-NC lead compound.
During the first year, the impact of the “BICEPSvsHIV” project has achieved significant benefits from the mobility of the ER. In fact, during the outgoing phase, Dr. Sosic worked in an international and multidisciplinary environment in a top-level institution. She significantly strengthened her scientific background, widening her skills in Analytical Chemistry, Molecular Biology and RNA Biology. In particular, she obtained new and profound technical competence in mass spectrometric techniques for the investigation of RNA structures and RNA-protein complexes. The new skills acquired by the fellow are attractive for both academia and pharmaceutical companies, as demonstrated by the success obtained by the ER at the “ASMS Conference on Mass Spectrometry and Allied Topics” (June 3-7, 2018, San Diego) where she was selected for an oral communication. The researcher improved her soft skills as well. She took active part in the research and financial management of the project. The ER improved her English language skills, her communication skills and attitude in networking, reinforcing and expanding international collaborations.

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