Final Report Summary - THINC (Targeting HIV integration cofactors, targeting cellular proteins during nuclear import or integration of HIV) Executive summary: The insight grew that HIV requires multiple cellular proteins to serve as cofactors for viral replication. The overall objective was to develop novel treatment strategies by targeting cellular proteins required for human immunodeficiency virus (HIV) replication. Focus was laid on the cellular proteins that mediate HIV trafficking, nuclear import and integration. The scientific and valorisation highlights were the following: 1. A novel cofactor of HIV nuclear import was identified and validated as a novel target for anti-HIV therapy: transportin-SR2 2. First-in-class drugs against the recently validated cellular target LEDGF / p75 were developed. 3. A novel cofactor of nuclear import, with code name PC2, was identified and validated. Depletion of PC2 resulted in reduced HIV replication. Q-PCR analysis indicated that PC2 is involved in nuclear import of HIV. 4. KAP1 was identified as a restriction factor of HIV integration and elucidated the mechanism in a high-impact paper. 5. Multiple Protein-protein interaction (PPI) assays were established (AlphaScreen in vitro, FCCS and MAPPIT in vivo). The AlphaScreen assay was instrumental in identifying LEDGINs and would be used to discover inhibitors of IN/TRN-SR2 interaction. Multiple drug discovery projects between medicinal chemists and biologists would continue. Assays and technology platforms were also offered to industry at fee-for-service (economic valorisation). Part of the research done was of generic value (inhibitors of protein protein interactions). 6. Novel technologies were implemented to follow enhanced green fluorescent protein (eGFP) labelled HIV Pre-integration complexes (PICs) during their journey to the nucleus. This technology was instrumental in validating TRN-SR2 as an HIV nuclear import factor. 7. The genetic variability of LEDGF / p75 (Single-nucleotide polymorphisms or SNPs and one mutation at position 472) was determined and a correlation in African populations with susceptibility to infection and / or disease progression was found. This work was published in AIDS. 8. The work was performed in the perspective of those who would benefit most: the HIV infected people all over the world by accomplishing a licence agreement in 2010 with Pfizer for further clinical development of LEDGINs. If successful, these drugs would be of major benefit for people in resource poor countries. To evaluate the global use of LEDGINs we evaluated the sensitivity of different HIV clades. No major differences were seen in between clades. Project context and objectives: Standard therapy of infection with the HIV type 1 (HIV-1) is based on potent cocktails of drugs targeting viral proteins. This treatment was associated with severe side effects and was almost unaffordable for the patients living in sub-Saharan Africa. Moreover, incomplete suppression of HIV replication results in emergence of drug-resistant strains. Therefore, a continued research effort is required to develop more potent, cheaper and less toxic antivirals. The initial steps of target validation and hit identification should be taken by academia supported by public funding. However, further optimisation and (pre)-clinical development of drugs are ideally undertaken in close collaboration with industry. The insight grew that HIV requires multiple cellular proteins to serve as co-factors for viral replication. The overall objective of the THINC consortium was to develop novel treatment strategies by targeting cellular proteins required for HIV replication. The virus may find more difficulties in developing antiviral resistance against drugs targeting interaction between invariable cellular proteins and conserved viral protein domains. Focus was laid on the cellular proteins that mediate HIV trafficking, nuclear import and integration. The multidisciplinary consortium was composed of three biologists / virologists (one partner is from South Africa), two medicinal chemists, one structural biologist and 1 SME. The tight collaboration between the partners significantly increased the basic understanding of PPIs during HIV nuclear import and integration. Project results: 1. A high throughput co-immunoprecipitation experiment was conducted for the identification of HIV integrase cofactors by nanoLC and MS (WP1). From this experiment Dr. Debyser's group identified and validated a novel cofactor of nuclear import, with code name PC2 at month 48. Depletion of PC2 resulted in reduced HIV replication. Quantitative real-time polymerase chain reaction (Q-PCR) analysis indicated that PC2 is involved in nuclear import of HIV. In a follow-up project it would be investigated whether PC2 could be exploited as novel HIV drug target. 2. The project aimed to validate a novel cofactor of trafficking, nuclear import and integration as antiviral target (WP1). This objective was reached ahead of time since Transportin-SR2 had been validated as novel cofactor of nuclear import (month 12). The finding that interaction between HIV integrase and an import factor, TRN-SR2, may mediate nuclear import stirred the field. The cofactor was identified in two out of four large-scale small interfering RNA (siRNA) screens for HIV cofactors. Although not all molecular details of this process had been revealed, this THINC finding was considered as a major scientific breakthrough that eventually could clarify the nuclear import step. It also opened new perspectives for anti-HIV therapy. This was explored during THINC and would be further elaborated in a follow-up project. The group of Dr Cereseto identified KAP1 as a restriction factor of HIV integration and elucidated the mechanism in a high impact-paper. 3. Novel technologies for the analysis of virus-host protein-protein interactions in vitro and in the living cell were developed (WP2). This objective was reached since multiple PPI assays had been operational since month 24. The AlphaScreen assay was instrumental in identifying LEDGINs and would be used to discover inhibitors of IN-TRN-SR2 interaction. 4. The project aimed to develop novel technologies for tracking of HIV particles in the cell (WP2). This objective was reached since HIV tracking technology had been operational since month 12; technology to visualise HIV integration in the cell was operational at month 30. Dr Cereseto's group pioneered on novel technologies to follow eGFP labelled HIV PICs during their journey to the nucleus. This technology was instrumental in validating TRN-SR2 as a HIV nuclear import factor and would be used to validate inhibitors of HIV nuclear import in a follow up project. Dr Cereseto recently optimised revolutionary technology to visualise HIV integration in the infected cell. 5. The project sought to develop inhibitors of integrase-LEDGF / p75 interaction (WP3). This objective was reached ahead of time (month 12 instead of 36) by rationally designing small molecule inhibitors (LEDGINs) as first-in-class allosteric inhibitors of HIV integration at KU Leuven. Co-crystals with integrase and thorough antiviral profiling of the compounds confirmed the mechanism of action. LEDGINs were licenced to Pfizer in United Kingdom (UK) for further clinical development. The laboratory of Dr Chimirri also explored the LEDGF / p75binding site in HIV-1 for drug discovery and developed inhibitors based on novel chemical scaffolds. These inhibit the LEDGF / p75-IN interaction in vitro and have demonstrated moderate antiviral activity in vivo. 6. THINC aimed to develop inhibitors of integrase-DNA interaction (WP3). This objective was reached by Dr Botta by identifying novel small molecules that block the dimerisation of HIV integrase required for interaction with DNA using a novel dimerisation assay established in the laboratory of Dr Debyser 7. Resistance development against inhibitors of integrase-DNA and integrase-LEDGF / p75 interaction was thoroughly studied (WP4). This objective was reached for inhibitors targeting IN-LEDGF interaction (month 30) and extensive resistance selection studies revealed a mutation (A128T) in the LEDGF / p75 binding pocket of HIV-1 IN. New LEDGIN congeners could circumvent this mutation. 8. The cell biology of LEDGF / p75 and its impact on potential toxicity of specific inhibitors was thoroughly studied (WP4). This objective was reached since two toxicity counterscreens (JPO2 and pogZ) had been established using AlphaScreen and had been used in developing LEDGINs. Most potent LEDGINs reached high selectivity (above 10 000) in cell culture without toxicity. Advanced toxicological studies with preclinical candidates were ongoing. Dr Rezacova performed successful structural biology studies on JPO2 and pogZ to study potential toxicity. 9. The genetic variability of validated cofactors in different populations was determined and the genotype was correlated with disease progression and susceptibility to infection (WP5). This objective was reached ahead of schedule since the genetic variability of LEDGF / p75 (SNPs and one mutation at position 472) was determined and a correlation in African populations with susceptibility to infection and / or disease progression was found. This work by Dr Ndungu had been published in AIDS. A similar work was done with the newly validated cofactor transportin-SR2 (WP1). 10. At each decision level the final goal to develop new drugs that could become available to patients in resource poor settings was considered. In 2010 a licence agreement was signed between KU Leuven and Pfizer for further clinical development of LEDGINs. If successful, these drugs would be of major benefit for people in resource poor countries. To evaluate the global use of LEDGINs the sensitivity of different HIV clades was evaluated. LEDGINs are active against a broad spectrum of clinical isolates and clades, including those prevalent in sub-Saharan Africa. Potential impact: Apart from the results already obtained (publications, valorisation, dissemination) following lines of research would be continued in collaborations between Member States. WP1 - New cofactors of HIV integration and replication A drug discovery project targeting IN / transportin-SR2 interaction and / or nuclear import was ongoing in a collaboration between the laboratories of Dr Debyser (KU Leuven) and Dr Cereseto (U Trento). Identification of novel inhibitors of nuclear import would signify a scientific breakthrough with a huge potential impact on future treatment of HIV infection. Follow-up projects were applied for in collaboration with European Small and medium-sized enterprises (SMEs). For PC2, a novel cofactor of nuclear import, a target validation project was initiated in the laboratory of Dr Debyser in collaboration with Dr Cereseto. Dr Cereseto would further study the mechanism of KAP1 restriction with a potential translational impact on elite controllers / HIV latency. WP2 - Novel assays for protein-protein interactions Multiple assays developed during THINC were operational in the partner laboratories. Multiple drug discovery projects between medicinal chemists (Dr Botta, Dr Chimirri) and biologists (Dr Debyser, Dr Cereseto) would continue. Assays and technology platforms were also offered to industry at fee-for-service (economic valorisation). Since part of the research done is of generic value (inhibitors of protein protein interactions) new research projects and valorisation outside the HIV field will be done (eg in the leukemia and neurodegeneration fields). WP3 - Drug discovery The discovery of LEGDINs as first-in-class inhibitors of LEDGF / p75-IN interaction and the license agreement with Pfizer underlined the huge socioeconomic impact of the THINC project, demonstrating that - academic drug discovery supported with public money can indeed reveal new targets and deliver new drugs; - deliverables from a European consortium had the potential generating a financial return through out-licensing to European industry; - results of basic research had a huge societal impact. In a research collaboration between KU Leuven and Pfizer / ViiV Healthcare chemical biology projects and (pre)clinical development projects had been initiated and would continue in the next years. WP4 - Cell biology of HIV cofactors The different partners (Dr Cereseto, Dr Debyser, Dr Luban, Dr Rezacova) would continue to study the molecular virology and cell biology of HIV cofactors and restriction factors identified by THINC in bilateral and multilateral projects. This work would continue to feed the HIV molecular virology field but would have also impact on other research fields (e.g. cancer). High impact papers, training of students and socioeconomic valorisation are likely to result from this effort. WP5- Genetic variability of HIV cofactors. The established collaborations between Dr Ndungu in South-Africa on the one hand, and Dr Debyser and Dr Luban in Europe on the other hand, on the impact of genetic variability of HIV cofactors on disease progression will continue. One of the south African docs would perform a postdoc in Leuven to strengthen the collaboration. The patient cohorts provided a strong asset for Dr Ndungu. The results obtained in the collaboration were highly relevant for his country hit hard by the pandemic.
