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Novel strategies for anti-HIV-1 therapy: Small molecules targeting RNA partners of the nucleocapsid protein

Periodic Reporting for period 2 - BICEPSvsHIV (Novel strategies for anti-HIV-1 therapy: Small molecules targeting RNA partners of the nucleocapsid protein)

Reporting period: 2018-07-01 to 2019-06-30

Human Immunodeficiency Virus (HIV) infection remains of major public health importance. 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” proposed a new anti-HIV strategy focused on RNA.
Specific RNA sequences of the viral genome are substrates of proteins, such as the HIV-1 nucleocapsid (NC), a highly conserved protein known for promoting remodeling of nucleic acid structures acting in essential steps of the virus replication cycle. Our working hypothesis was that the employment of bis-3-chloropiperidines (BICEPS) as RNA targeting agents could represent a novel pharmacological treatment to impair NC-mediated processes, while overcoming drug resistance. Small molecules able to bind to the RNA substrates of NC and freeze their three-dimensional configurations were already shown to block NC-mediated remodelling of nucleic acids secondary structures.

The Experienced Researcher (ER) worked at The RNA Institute (SUNY Albany, NY, USA) in the research laboratory of Prof. Dan Fabris during the outgoing phase, whereas she performed her research at the Department of Pharmaceutical and Pharmacological Sciences (University of Padova, Padova, Italy) in the research laboratory of Prof. Barbara Gatto during the incoming phase.

During these two years, all the proposed research objectives of the project were met.
The conclusions of the action are: 1) we identified positive hits targeting selectively the RNA substrates of NC, with different reactivity and selectivity, and we described their detailed molecular mechanism of reactions towards RNA, including the fast and specific cross-linking of different RNA structures; 2) in the in vitro biological analysis, we established how the exquisite reactivity with RNA led to the stabilization of their dynamical conformation leading to the inhibition of NC-mediated remodelling of the nucleic acids structures; 3) we established the structure-activity relationships of BICEPS, useful to optimize RNA specific cross-linking agents for the development of anti-NC lead compounds.
From the beginning of the project to the end of the period covered by the report the main results achieved are:

In the first year of the “BICEPSvsHIV” project, research activities were mainly focused on the investigation of bis-3-chloropiperidines (BICEPS) reactivity towards RNA by means of unique and innovative mass spectrometric techniques. A wide library of compounds was examined for the direct interaction with RNA constructs. The obtained results allowed the identification of the most promising BICEPS targeting RNA.

In the second year of the project, the research focused on the biological evaluation of the in vitro NC inhibition by BICEPS and on hits validation. We optimized the experimental platform to specifically test BICEPS derivatives and identified potent in vitro inhibitors of NC activities as hit compounds.

Hit compounds were further characterized in cell systems by analyzing their cytotoxicity. In particular, the group of BICEPS bearing the flexible alkyl- linker, which resulted to be the most highly reactive RNA cross-linkers, exhibited poor cytotoxicity. We believe that they fast react with targets in the cytoplasmic environment, therefore not damaging the genomic DNA of the host, hence representing optimal candidates for the development of RNA-targeting antiviral agents. In addition, the unexpected result of the biological analyses showed us how the “useless” compounds, meaning those poorly reactive with RNA and uninteresting as antivirals, were instead promising candidates for further development as anticancer agents, and, most specifically, for pancreatic tumors.

Scientific outcomes achieved during the project were published in high-impact peer-reviewed journals and generated interest to the Italian and European Chemistry Society and to their Medicinal Chemistry Divisions as demonstrated by the success obtained at several important international conferences where the ER was selected for oral communications.
Progress beyond the state of the art relates to i) the discovery of new molecules with an exquisite, fast and selective reactivity towards RNA, and ii) the development of innovative mass spectrometry-based tools for the analysis of RNA structures, structure-function relationships of RNA-ligands and RNA-protein complexes. The obtained results are highly informative in order to identify the most promising BICEPS targeting RNA, to evaluate their in vitro NC inhibition and to elucidate their mechanisms of NC inhibition, and we successfully identified effective NC inhibitors targeting its RNA partners.

The progress, while applicable to a project dedicated to potential drugs targeting important steps in the lifecycle of viruses, have indeed a wider perspective to the applied research in the fast-growing field of RNA biology: results of “BICEPSvsHIV” will have a durable impact in future research aimed at understanding the role of the newly discovered RNAs with essential biological functions.

The impact of the “BICEPSvsHIV” project has achieved significant benefits. The ER developed outstanding knowledge in mass spectrometry for RNA science needed in Europe to improve research excellence and to increase the number of women working in the field. Moreover, this project represented a great opportunity for the European Institution involved in the action to improve academic collaborations and long-standing relationships not only with SUNY Albany (USA) but also with other European Universities, namely JLU in Giessen (Germany) and Charles University in Prague (Czech Republic).
Schematic representation of the strategy to inhibit NC by BICEPS