Final Report Summary - HIVINNOV (Generation of a new class of antiretrovirals targeting HIV-cellular cofactors interactions) Executive Summary:1- Executive summaryHighly active antiretroviral therapy has reduced HIV-associated mortality and morbidity. However the AIDS pandemic remains a major public-health threat and with transmission of drug-resistant viruses that is increasing, it is essential that new antiretroviral (ARV) agents become available. In the HIVINNOV consortium, we aim to discover new targets and develop novel classes of ARVs targeting specific virus-host interactions, such as integrase (IN)-LEDGF involved in the HIV integration sites selection, and the Transportin-SR2 pathway that interferes with Capsid during viral import in the nucleus. HIVINNOV started October 1st 2012 and ended September 30th 2015. It was composed of 6 partners from 4 EU countries: one SME, BIODIM (FR) as coordinator, and 5 outstanding academic laboratories well recognized in the HIV research field, A. Fassati (UCL (UK)), P. Cherepanov (CRUK and F. Crick Inst. (UK)), A. Zamborlini and S. Emiliani (INSERM & CNRS (FR)), Ben Berkhout (AMC (NL)), and J. Gatell (FCRB (SP)). HIVINNOV was able to achieve remarkable discoveries:- Identification by BIODIM of a drug candidate, BDM2, belonging to a new class of ARV, Allosteric Integrase Inhibitor (ALLINI) targeting IN-LEDGF interaction. BDM2 is a very potent ARV, equivalent to the best anti-HIV drugs currently on the market, with full activity against viruses resistant to all current drugs.- Elucidation of its unique dual mode of action at two different steps of the HIV replication cycle (weak inhibition at integration and strong antiviral effect at maturation during virus production). - Preclinical in vivo validation for the first time ever of this new class of ARV in the humanized mouse model of HIV infection (BIODIM and Ben Berkhout). Since no compound of the ALLINI class has been tested in phase I/IIa Proof of concept (POC) clinical trial, this achievement makes BIODIM world leader in the ALLINI field.- Achievement of BDM2 non-regulatory preclinical pharmacology and toxicity with no no-go signals that could impair its future development, and initiation of the synthesis of an 8 kg GMP batch required for next preclinical and clinical studies. Although phase I and IIa POC clinical trials could not be completed before the end of HIVINNOV, BDM2 clinical validation will be completed in two years after the end of HIVINNOV. - Validation of the NGS-HIS mice model of HIV infection as suitable for anti-HIV drug efficiency studies by oral drug administration (Ben Berkhout) - Elucidation of the first crystallographic 3D structure of the Transportin-SR2 protein and of the structural basis for retroviral integration into nucleosomes by Single-particle cryo-electron microscopy. (P. Cherepanov)- Identification of the first small chemical molecules (hits) targeting the Transportin-SR2 pathway (A. Fassati), and the interaction between Transportin-SR2 and one of its cargo proteins (CPSF6) that interferes with Capsid and is involved in the HIV nuclear import (A. Fassati, P. Cherepanov & BIODIM)- Discovery of a new role of Hsp90 in the control of HIV reactivation from latency (A. Fassati) and of the complex LEDGF/p75:Iws1:Spt6 in interaction with the PTW/PP1 complex, in the establishment of HIV latency (S. Emiliani). Thus, very exciting findings on the control of HIV latency by Hsp90 and LEDGF/p75 have emerged. - Discovery of the interaction between IN of a model retro-element (Ty1) and a chromatin-bound cellular protein, AC40, involved in site-specific integration of retro-elements; identification of a splice variant of the integrase SUMO E3 ligase, PIAS3, in quiescent T cells that are resistant to HIV infection (A. Zamborlini). In terms of dissemination, HIVINNOV has been very productive with 39 peer-reviewed scientific publications in first class international journals or in the best reviews of Retrovirology, with 48 dissemination activities (oral presentations, posters, press releases…) in international scientific meetings ; the organization of a satellite meeting of the EACS meeting in Barcelona 2015 together with two other EU consortia working on HIV, Hit Hidden HIV and Thinpad. In terms of exploitation: i) seven patent families have been submitted by BIODIM on its ALLINIs and discussions have been initiated with big pharmaceutical companies for partnership in the development of its drug candidate BDM2; ii) UCL is looking for partnership and new sources of financing to ensure the lead optimisation and development of the Transportin-SR2 hits identified by A. Fassati lab.; iii) AMC is looking for exploitation of the Ben Berkhout lab. process of oral drug administration in the humanized mice model of HIV infection; iv) INSERM and CNRS filed a patent on a new latency vector designed by S. Emiliani. The great number of patents (more than 50), deposited on ALLINIs, in particular by most of the big pharmaceutical companies involved in HIV therapies is a clear sign of extreme interest for this new class of drugs and besides for the new targets against HIV discovered in HIVINNOV. The social and economic impacts of HIVINNOV are already important and the interest for its products will be increasing when BDM2 will be clinically validated, and the other lead compounds or new targets discovered by the consortium will be fully developed. HIVINNOV partners agreed that the work and collaborations that have been established during HIVINNOV will continue through different forms after the end of the consortium. Project Context and Objectives:The AIDS pandemic remains a major global public-health threat with an estimated 33 million people infected with HIV, mainly in Sub-Saharan Africa. Highly active antiretroviral therapy, i.e. combination therapy of three or more drugs with different mechanism of action, is effective at controlling HIV-1 replication, and has reduced both HIV-associated mortality and morbidity. HIV infection has become a chronic disease with life-long anti-retroviral treatment (ART). Life expectancy of patients has increased and approaches that of uninfected individuals. The tendency to start treatment earlier and the lack of adherence due to long-term toxic side effects, are factors that increase the risk of spread of drug resistance. Transmission of drug-resistant viruses is increasing, including viruses resistant to the newly developed integrase strand transfer inhibitors. With no effective vaccine currently available, and the inability to eradicate and cure HIV, prevention and anti-retroviral (ARV) drugs remain today the only means to reduce virus transmission and replication. With patients failing current ARV treatments and with expansion of ARV therapy programs, the emergence and transmission of drug-resistant viruses has become a new serious public health challenge. It is therefore essential that new ARV agents become available. There are currently 27 licensed drugs falling into 6 different classes: nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), fusion inhibitor (FI), co-receptor inhibitor (CRI) and integrase strand transfer inhibitors (INSTIs). Altogether, these drugs target only 4 or 5 different steps out of the estimated dozen or so needed by the virus to complete its replication cycle in infected cells. The development of new drugs with a completely different mechanism of action that will be active on viruses resistant to the present drugs remains an important issue. Since the introduction of INSTIs late 2007, no novel class of drugs have been introduced in the anti-HIV drug market. HIV requires numerous interactions with cellular proteins and other cellular components, to complete its viral replication cycle and escape the intracellular antiviral innate restriction mechanisms. More recently, major discoveries, to which the partners of the consortium have actively participated, led to the identification of important cellular cofactors for HIV replication. In the HIVINNOV consortium, we aim to develop novel classes of ARVs inhibiting specific virus-host interactions. The consortium selected two cellular targets, LEDGF/p75 and Transportin-SR2 (Trn-SR2), cofactors of Integrase (IN) and Capsid (CA) respectively, important for viral integration and nuclear transport. There is a broad consensus on the importance of these cellular cofactors in the replication cycle of HIV: LEDGF is a chromatin-bound protein involved in tethering IN to actively transcribed genes and in the selection of HIV integration sites Transportin-SR2 or Transportin 3 (TRN-SR2 or TNPO3) is implicated in nuclear import of splicing factors namely SR proteins such as ASF/SF2 and CPSF6. It is believed that TRN-SR2 promotes HIV-1 infectivity, by facilitating HIV import to the nucleus through HIV Capsid (CA) binding to CPSF6. The elucidation of the 3D structure of the IN-LEDGF interfaces by partner 3 and colleagues led to the recent discovery of a new class of IN allosteric inhibitors that can inhibit LEDGF-IN interaction. Several acronyms that are fully equivalent are found in the literature to name this new class of ARV drugs (LEDGINs = LEDGF Integrase Inhibitor; ALLINIs = Allosteric Integrase Inhibitors; INLAIs = Integrase LEDGF Allosteric Inhibitors; NCINIs = Non Catalytic Integrase Inhibitors; MINIs = Multimerization Integrase Inhibitors). Taking into account the mode of action of these inhibitors the most appropriate name is probably ALLINI. Up to date there is no drug candidate of this class that have been validated in phases I/IIa POC clinical trials. HIVINNOV partners:The coordinator Dr. Richard Benarous, Chief Scientific Officer (CSO) of the SME BIODIM (partner 1, Romainville, France), partner 2 Dr. Ariberto Fassati (UCL, UK) partner 3 Dr. Peter Cherepanov (CRUK, UK) and partners 4 Dr. Stéphane Emiliani and Dr. Alessia Zamborlini (INSERM, Paris, France), have been instrumental in the discovery of these cellular cofactors and the elucidation of their structure and function. Partner 5 Pr. Ben Berkhout (AMC, Amsterdam, Netherlands) and partner 6 Pr. Jose Gatell (FCRB, Barcelona, Spain) bring to the consortium their fundamental expertise for in vivo drug validation in a sophisticated humanized mouse model of HIV infection (NSG-HIS mice) and in clinical trials of ARV compounds respectively.The SME BIODIM, which leads the consortium, has developed small compounds inhibitors of IN-LEDGF interaction. BIODIM compounds have a clear structure activity relationship, nanomolar ARV activity and are based on a new, structurally defined pharmacophore. The general objectives of the HIVINNOV project: 1) Optimize BIODIM IN-LEDGF advanced inhibitors up to the proof of concept (POC) in man in a phase I/IIa clinical trial with partner 6. This is the goal of the work package 1 (WP1), together with studying the activity of these compounds in the humanized mouse model of HIV infection (NSG-HIS mice) with partner 5. 2) Discover in work package 2 (WP2), small compounds that target the Trn-SR2 pathway in HIV-1 infection using a high through put screening assay validated by partner 2, determine the pharmacophore by solving the 3D structure of Trn-SR2 with partner 3, and optimize the compounds with partner 1 up to the POC in the humanized mouse model of HIV infection with partner 5. 3) Elucidate in work package 3 (WP3), the network of interactions connecting Trn-SR2 and LEDGF to CA, IN, and other cofactors, from HIV-1 uncoating to integration, with partners 1, 2, 3, 4, 5, and provide new potential ARV drug targets.4) Implement exploitation plans (WP5) for the future development of IN-LEDGF inhibitors from WP1 and of products from WP2 and WP3. WP4 was concerned with the management of the project. Project Results:3- A description of the main S&T results/ foregrounds 3.1 Summary description of the S&T results:In WP1, BIODIM developed a new series of ALLINIs with highly potent ARV activity between 3 to 20 nanomolar EC50, and elucidated the ARV mode of action of these inhibitors. Numerous BIODIM compounds were co-crystallized with IN-CCD in the LEDGF-binding pocket, facilitating the discovery of very potent ALLINIs by structure activity relationship (SAR) and promoting IN conformational change that encompasses alpha helices IN 115-122, 123-134, and 92-98. They inhibited strongly IN-LEDGF interaction and displayed partial inhibition of IN strand transfer activity and weak 3' processing inhibition. These compounds enhanced strongly IN-IN interactions and shifted the oligomerization equilibrium of IN from IN dimer toward IN tetramer. The ALLINIs are the first class of antiretrovirals that, through binding to a single target, IN, to a unique binding site on this target, have nevertheless dual anti-HIV-1 activity, at two different steps of the viral replication cycle: at integration in target cells and post-integration during production of infectious particles in producer cells. Given the function of LEDGF, the effect at integration was expected, and is linked to the inhibition of IN-LEDGF interaction. The effect at post-integration production of infectious particles was unexpected, and is linked to the dimer-tetramer IN oligomerization shift induced by compound binding to IN independently of LEDGF,Partner 1 demonstrated that both activities are linked to the compound binding to its unique target on IN, the LEDGF-binding pocket. The post-integration block of HIV-1 replication in virus-producer cells is by far much stronger than the block at integration. To explain this difference between ALLINI ARV activity at integration and post-integration stages, Partner 1 proposed the following model: LEDGF/p75 is a very abundant nuclear, chromatin-bound protein that is absent in the cytoplasm. Therefore, LEDGF/p75 can outcompete compound binding to IN in the nucleus of target cells lowering its antiretroviral activity at integration, but not in the cytoplasm where post-integration production of infectious viral particles takes place. These results illustrate the complexity of the mode of action of inhibitors targeting IN-LEDGF interaction.Partner 5 fulfilled the set-up of all preclinical tests necessary for the validation of BIODIM inhibitors in NSG-HIS mice. They demonstrated that NSG-HIS mice can be robustly and reproducibly infected with HIV virus. All pre-clinical tests, including the monitoring of CD4+/CD8+ T cell dynamics (FACS), plasma RNA viral load (qPCR) and intracellular p24 staining (FACS) have been implemented successfully as well as studies for continuous administration of drug candidates via novel MediDrop technology. BIODIM succeeded to isolate a drug candidate, BDM2 with very potent ARV activity, equivalent to that of the best ARV drugs currently on the market, with activity conserved on resistant viruses to all current drugs, full virologic validation with demonstration of its ability to be combined efficiently to other classes of drugs currently used in clinic.BIODIM achieved the synthesis of 500g pilot batch of BDM2 as a first step to scale up 8kg GMP batch required for regulatory preclinical and phase I/IIa POC clinical trials, completion of the acute toxicity studies in mice and dogs in search of the Maximum Tolerated Dose and Dose Range Finding (MTD/DRF studies) that did not show no-go signals that could preclude further development of BDM2. Partner 1 also advanced in Pharmacokinetics (PK) studies in mice rats and dogs, basis for simulations for prediction of possible BDM2 QD dosing in man. Very importantly, Partner 1 in collaboration with Partner 5 succeeded to demonstrate for the first time ever the ARV activity of these IN-LEDGF inhibitors in vivo in the NSG-HIS mice model of HIV infection. This result constitutes the first preclinical in vivo validation of the ARV efficiency of this class of drugs, which makes BIODIM a world leader in this field. Partner 6 prepared protocols and made necessary arrangements for the phase I study of the selected drug candidate of partner 1 with healthy volunteers and the phase IIa Proof of Concept study with treatment naïve HIV infected subjects.In WP2, Partner 2, looked for compounds that could interfere with the TRN-SR2 pathway required for HIV-1 infection. The rational here is the following: a compound that interferes with TRN-SR2 could impair the transport of HIV to the nucleus thus inhibiting HIV replication. Partner 2 carried out a cell-based high-content cellular screening using a sub-library of 10,000 small chemical compounds provided by Partner 1. Several hits were identified and two were validated independently by partners 1 and 2 as a new class of ARV and showed selective inhibition of HIV-1 WT (TRN-SR2 dependent) versus N74D CA mutant TRN-SR2 independent.Partner 3 in collaboration with Partner 2 elucidated the crystal structure of TRN-SR2, revealing the critical residues for binding to SR proteins ASF/SF2 (not involved in HIV replication) and CPSF6, which is implicated in HIV-1 infection by facilitating the transport of HIV-1 in the nucleus. Crystallography studies of Partner 3 established the mode of binding and release of diverse RS domain-containing cargoes by Tnpo3. Although the RS domains of ASF/SF2 and CPSF6 engage the same region of the Tnpo3 surface, the marked discordance in their Tnpo3 mutant interaction profiles suggests that TRN-SR2 may be exploitable as a target for the development of small molecules to selectively inhibit its interactions with a subset of cargoes. Given the potent antiviral properties of cytoplasmic CPSF6, redirecting even a small fraction of this protein into the by inhibiting TRN-SR2-CPSF6 interaction could be a viable strategy to block HIV-1 replication. So this structure led to the development of a new biochemical screening assay based on TRN-SR2-CPSF6 interaction that has been designed in collaboration with Partner 1. Using this assay, partner 2 was able to complete the screening of the BIODIM library of 130,000 chemical compounds and to identify several promising hits.In WP3 Partner 1 and Partner 4 identified protein-protein interactions between the capsid (CA) and integrase (IN) protein of HIV-1, that are part of the HIV-1 PIC, and cellular proteins such as HSP90, CypA, Cyp40, TNPO3, SR-25, EIF3H and the components of the cellular SUMOylation machinery (E3 SUMO ligases of the PIAS family, SUMO E2 conjugating enzyme Ubc9) that promote covalent modification of cellular and viral proteins by SUMO moieties. - Regarding the interplay between HIV proteins and SUMOylation, Partner 4 has identified a PIAS3 variant which enhances the conjugation of SUMO proteins to HIV-1 IN. This PIAS3 variant is expressed in quiescent T cells, which are resistant to HIV infection, but is undetectable in activated T cells, which are permissive to HIV. These results suggest that PIAS3 might play a role as a viral cofactor during the early stages of the HIV life cycle. - By studying the integration site choice of a model retroelement (Ty1) that is closely related to HIV Partner 4 deepened the understanding about the site-specific integration of retroelements (retrotransposons and retroviruses). This process relies on the specific interaction between the integrase protein of Ty1 and a chromatin-bound cellular protein, AC40, a polymerase III subunit.- Partner 3 elucidated by Single-particle cryo-electron microscopy the structural basis for retroviral integration into nucleosomes.- Partner 4 characterized a novel LEDGF/p75-associated complex, containing Iws1 and Spt6 proteins, and has shown that this complex participates to the establishment of HIV latency that can arise from infection of activated primary CD4+T cells, and that its recruitment to latent provirus correlates with the presence of repressive histone marks. Partner 4 characterized an interaction between Iws1 and the PTW/PP1 complex that contains PNUTS, Tox4, Wdr82 and the protein phosphatase 1 (PP1). Partner 4 also developed a Dual-color HIV vector to identify and isolate latently infected cells (patent deposited EP14305885.7).- Partner 2 identified a new role of the heat shock protein 90 Hsp90 in the control of HIV reactivation from latency. Partner 2 had previously shown that Hsp90 is required for HIV-1 gene expression and mediates greater HIV-1 replication in some conditions. Partner 4 found that specific inhibitors of Hsp90 such as 17-(N-allylamino)-17-demethoxygeldanamycin and AUY922 prevent HIV-1 reactivation in CD4+ T cells. Thus, very exciting and important findings concerning the control of HIV latency by Hsp90 and LEDGF/p75 have emerged from WP3. 3.2 Detailed description of S&T results by task:WP1 : Development of new ARVs targeting IN-LEDGF interactionSelection of the preclinical drug candidate and synthesis of back-up compounds (Partner 1)After abandoning a first drug candidate BDM1 because of its failure to conserve full activity against some polymorphic viruses, BDM2 was selected by Partner 1 as drug candidate at the end of the first 18 month period of HIVINNOV on the basis of: - excellent antiretroviral activity with EC50 in the 10nM range and PA EC90 at 60ng/mL, potency which is equivalent to that of the best anti-HIV drug currently in the market, the catalytic integrase inhibitor Dolutegravir® (VIIV). - activity well conserved against all polymorphic viruses tested and against all primary isolates from different clades tested.- physico-chemical properties which show very good solubility and membrane permeability, low MW (<400 DA)- low cytotoxicity against a panel of different human cells including primary PBMC, with high selectivity index (>12.000 with PBMC)- low or undetected potential of cardiotoxicity and genotoxicity- no significant inhibition of cytochromes and low potential for drug-drug interaction- favorable preliminary ADME profile with excellent bioavailability, PK parameters that do not preclude further development of BDM-2 or of the back-up compounds.During the second 18 month period, partner 1 successfully developed two back-up compounds of BDM2, BDM3 and BDM4; partner 5 together with partner 1 achieved major advances in dissecting the mode of action of this new class of ARVs; and partner 1 demonstrated that BDM2 and back-ups could be efficiently combined with all the other classes of drugs without antagonism, but additivity and even moderate synergy with some current drugs. i) BDM3 had even more potent ARV activity than BDM2, below 5nM EC50 for all polymorphic viruses tested with similar PA EC90, but was more difficult to synthesize than BDM2. - BDM4 had a slightly lower ARV activity than BDM2, with EC50 of 15nM, well conserved activity against all polymorphic viruses, with more favorable profile against BDM2 resistant viruses although with a lower but significant impact of the most detrimental BDM2 resistant mutations (see below details in Task 1.3).- So altogether these data confirmed the selection of BDM2 as preferred drug candidate of partner 1, but two excellent back-up compounds are available if BDM2 development will fail at some step.ii) Partner 5 with partner 1 elucidated further the mode of action of this class of inhibitors: During the first 18 month period, it was demonstrated by partner 1 that these compounds had a dual antiretroviral activity with a weak inhibitory activity at integration and a very potent activity at the post-integration production of infectious viral particles. HIV-1 produced in the presence of such compounds showed a deficiency in infectivity for the progeny virions, which strongly inhibits viral replication. The weaker inhibition of integration was linked to blocking of the IN-LEDGF interaction. And the stronger inhibition at the virus production’s stage was linked to a shift of IN toward multimerization promoted by inhibitor binding to the LEDGF binding pocket on IN (Lerouzic et al. Retrovirology 2013).During the second period, Partner 5 and Partner 1 performed mechanistic studies to accurately map the virus defect imposed by ALLINI-like inhibitors. For this, we analyzed several key components of virion particles that were produced in the presence of the drug. Quite remarkably, we measured normal levels of viral RNA, tRNA primer and Reverse Transcriptase enzyme. Combined with the published electron microscopy images of such viruses, this means that all components are properly packaged, but apparently not in the proper configuration to execute the essential process of reverse transcription (Van Bel et al. PLoS One 2014). iii) Studies of BDM2 combination with the other classes of drugs currently used in the clinic were performed in order to determine whether BDM2 did show any antagonistic behaviour with the other drug classes with which it could be combined. Also on reverse it was important to determine whether these other classes of drugs exhibited no antagonism on the ARV activity of BDM2. Taking into account that all anti-HIV treatments are combination treatments (HAART) of several drugs with different mechanism of action, these studies were important to validate the different combinations of drugs in which BDM2 could be potentially used. We found by Mac Synergy plot II analysis with combination of increasing concentrations of BDM2 with each of the other classes of drugs that BDM2 had no antagonistic effect with any of the other classes of drugs used in clinic, including the three INSTIs Raltegravir, Elvitegravir and Dolutegravir, NRTIs, NNRTIs and PIs. We found additive effects with all these drugs, and even moderate but significant synergistic effects with some of these including Elvitegravir and Lopinavir (PI).Preclinical test of the in vivo activity of lead compound in NSG-HIS mice (partner 5)During the first 18 month period Partner 5 set up the HIS mouse model, optimized drug delivery by MediDrop and performed pilot tests with the well-known anti-HIV drug Raltegravir. In period 2 partner 5 focused exclusively on the in vivo testing of the previous BDM1 drug candidate developed by partner 1, as reference for all compounds of this class including BDM2, as explained in the report of the first 18 month period, since BDM1 had full conserved ARV activity against the HIV-1 virus used in these in vivo experiments of HIV-1 infection in NSG-HIS mice.- Partner 5 in collaboration with partner 1 obtained the first in vivo proof of principle for the ARV activity of the BDM1 drug compound in the HIS mouse model. This is the first worldwide in vivo proof of principle of the preclinical validation of an antiretroviral activity of a molecule belonging to the Allosteric integrase Inhibitor (ALLINI) the new class of ARV drug in development. In the absence of clinical phase IIa POC trial, this result in an animal model of HIV infection in humanized mice is a major achievement that validates the entire class of ALLINIs as new class of potent ARV drug, and positions BIODIM as world leader in the development of ALLINIs. Partner 5 further optimized drug delivery using the MediDrop method (manuscript in preparation) and validated the efficacy of the lead compound BDM-1 in HIV-1 infected HIS mice. Although BDM-1 was abandoned as drug candidate for clinical testing because of resistance due to naturally occurring sequence polymorphisms in several HIV-1 viruses, the JRCSF strain is fully sensitive to BDM-1. To date, no animal experiments have been conducted with any of the new class on ALLINIs. From literature, we know that all ALLINIs act via the same molecular mechanism. Therefore, we believe that testing ALLINIs in the HIS mice for the very first time will provide critical information that can be used for the development of second generation ALLINI compounds less susceptible to drug resistance mutations.Indeed, we obtained good virus inhibition when BDM-1 was administered at 350 mg/kg/day via MediDrop. We observed an 1.2 log10 inhibition of the plasma viral load over a 1 week period in three out of five mice, which is very comparable to the 1.45log10 reduction we observed upon treatment with the validated Integrase inhibitor Raltegravir at 175mg/kg/day, taking into account that Raltegravir is 5 to 10 times more potent than BDM1.Estimation of the genetic barrier to resistance (partners 1 & 5)Partner 1 selected ALLINI-resistant virus by infecting in vitro MT4 human T lymphocytic, target cells of HIV-1, in the presence of increasing concentrations of drug candidate and other compounds of the same class. A series of resistant mutations to the BDM-2 drug candidate and other IN-LEDGF inhibitors was found, corresponding to single and multiple point mutants, in the LEDGF-binding pocket or in general close to this pocket: IN T174>I, IN A128>T/S, IN Y99>H, IN N222>K. T174I with a fold change (FC) of 264 times the EC50 for wt virus, was the most detrimental resistance mutation. The other mutations selected promoted very low resistance with FC value around 2-3. The kinetics of emergence of resistant mutants as well as the identity of these resistant mutations was equivalent for all ALLINIs, including those from competitors, which means that all these compounds, corresponding to the first generation of this inhibitor class, behave similarly. These data lead to the conclusion that the genetic barrier to resistance for the first generation of ALLINIs is not very high and seems similar to that observed in vitro for Nevirapine. In addition to these resistance mutations, we found that Integrase polymorphisms at position 124 or 125 on the left edge of the compound binding site were detrimental for some inhibitors, such as an A residue at position 125 for the previous BIODIM drug candidate BDM1. Studying further the influence of polymorphism at IN residues 124 125 on the ARV activity of ALLINIs, we found that ALLINIs from competitors were susceptible to an N residue at position 124, GS13 from Gilead (WO2012/003497) with fold shift of 5 and Cpd I-82 from Shionogi (WO 2013/062028) with Fold shift of 10. All BIODIM compounds including BDM1 were not susceptible to this IN polymorphism.During the second period, virus escape from the candidate BDM1 antiviral was also studied in vivo. Most importantly, Partner 5 used the HIS mouse model to select for BDM1-resistant viruses. To do so, we extended the treatment period to three weeks and used a sub-optimal drug concentration (100 mg/kg/day). In these conditions, we did not observe a significant reduction of the plasma viral load, suggesting that either the drug concentration was too low or indeed drug-resistant virus variants evolved rapidly. We analyzed the population sequence of the full-length Integrase gene and observed the acquisition of mutations after two weeks of treatment in all mice. Moreover, the selected mutations are identical to those observed in vitro, which thus confirms the value and reliability of the HIS mouse model.Preclinical regulatory studies and preparation of IND (Investigational New Drug) dossier (Partner 1)With several ADMETOX studies, BDM-2 was shown to have a low potential for genotoxicity, cardiotoxicity and cytochrome inhibition. BDM-2 metabolic profiles among 5 species (mouse, rat, dog, monkey, human) was examined, thus permitting rodent and non-rodent species selection for further Toxicology studies (mouse/dog). Its PK in mice, rats and dogs was also determined with good bioavailability. For the preclinical studies the chemical synthesis of the drug candidate had been optimized internally by partner 1 and transferred to a selected specialized CRO for scaling up to a pilot batch of 500 g, intermediate step required to ensure the feasibility of the 8kg GMP batch synthesis required for GLP preclinical and clinical studies. i) Completion of the synthesis of the 500g pilot batch:A first CRO was unable to scale up step 6 of the synthesis process. An alternative solution was looked for with a second CRO selected on the basis of its ability to achieve the scaling up of this 6th step and finish the synthesis. This second CRO succeeded in delivering the pilot batch in April 2015 after the delivery of a batch that was contaminated at an unacceptable level. ii) BDM-2 human dose simulation: taking into account that most of the ARV drugs currently used in clinic are once day (QD) dosing, it was of critical importance for the future development of BDM2 to assess by simulation studies from animal data whether the PK of BDM2 could be compatible with QD dosing in human. Several PK studies in mice, rats and dogs and consultancies with recognized international PK simulation experts, the conclusion was that QD regimen scenarios for BDM-2 could be predicted in human. This was a very important step, as the HIV market today requires QD drugs.iii) BDM-2 in vivo toxicology studies (MTD/DRF) with the 500g pilot batch: Following pilot batches delivery in April 2015, the MTD/DRF (non-regulatory toxicology) studies were started in May 2015 in dog and mice in order to see if BDM2 did not induce in vivo unacceptable toxicity, at what dose the first signs of toxicity could appear (determination of Non Observed Adverse Effect level (NOAEL), and consequently assess the security window between toxicity and activity Macroscopic and microscopic observations undertaken on dogs led to the conclusion that at high dose (400mg/kg/day), no adverse effects were observed in the liver. Adverse observations were made in the kidneys & gall bladder. In mice the NOAEL could be estimated at the highest dose of 1000mg/kg/day. The toxicity data was in a range found in preclinical studies of various ARV used currently in clinic and partner 1 decided to go forward in the development of BDM-2 up to and including the clinical phases I/IIa. iv) Synthesis of the 8kg GMP batch: Currently the selection process of a CRO will be achieved soon allowing the start of the synthesis of the 8kg GMP batch of BDM2.v) Completion of the GLP regulatory preclinical studies and submission of the IND dossier: These tasks require the completion of the 8 kg of the BDM2 GMP batch synthesis. Due to the fact that BDM-2 synthesis requires 13 different steps, some that might be challenging at scaling up to kg quantities, it is difficult to predict accurately when the GMP batch will become available. Partner 1 anticipates that these GLP regulatory preclinical tasks will be initiated around the end of 2016 early 2017.Status of this task: delayedFirst in man phase I clinical trial (Partner 1 & 6) and Proof of concept Phase IIa clinical trial (Partner 1 & 6)These tasks will be performed after the delivery of the BDM2 GMP batch and the submission of the IND after completion of the regulatory preclinical pharmacology and toxicity studies. Partner 1 anticipates that these tasks will be initiated within two years after the end of HIVINNOV. Partner 1 underlines that it is fully committed to continue the development of BDM2 up to its clinical validation through phase I and phase IIa POC clinical trials. Partner 6 completed very detailed protocols for these trials, both for the two phase I and the phase IIa POC clinical trials. These protocols are provided in annex of this report:- Phase I Randomized, Double Blind, Placebo Controlled “first-in-human” Study To Assess the Safety and Tolerability of Single Ascending Oral Doses of BDM-2 in Male Healthy Volunteers (78 pages) - Phase I Randomized, double blind, placebo controlled, parallel groups study to assess the safety, tolerability and pharmacokinetic profile of BDM-2 after multiple oral doses, in healthy volunteers (68 pages)- Phase IIa: A pilot, double-blind, randomized, placebo-controlled, repeated dose clinical trial to assess the safety and antiviral activity of BDM-2 in naive HIV-1 infected patients (38 pages).Status of these tasks: Postponed within about two years after the end of HIVINNOV. WP2 : New ARV compounds targeting the Trn-SR2 pathway in HIV-1 infectionThe overall WP2’s objective is to develop a new generation of ARVs targeting the Trn-SR2 pathway in HIV-1 infection and to lead the optimization up to the POC in the NSG-HIS mice.High content cellular screening campaign (partner 2)During the first period, Partner 2 carried out a cell-based cellular screening using a sub-library of 10,000 small chemical compounds provided by partner 1. Status of this task: Completed.Hit selection (partners 1, 2, 4).Several hits were identified; three hits showed selective inhibition of HIV-1 WT (TRN-SR2 dependent) versus N74D CA mutant TRN-SR2 independent). Partners 1 and 2 proceeded to validate the hits independently. Two hits have been selected for further tests for final selection.Target identification (partners 1, 2, 4).The first step for target identification was to demonstrate that the selected hits were a novel class of ARV thus not concerned with the different targets that correspond to the class of ARV already identified. To do so, hits were further validated against HIV-1 mutants resistant to known classes of ARVs including ALLINIs. All compounds tested fully retained their antiviral activity, including against a panel of 5 ALLINI-resistant viruses, indicating that they had a new mechanism of action.The best hits, according to several independent assays had an EC50 below 5 μM and a CC50 > 50 μM. We therefore re-synthesized them and tested them in infection assays. Both hits confirmed their antiretroviral activity. Hits 25 and 53 were selected for further studies to determine if the compounds depend on the TRN-SR2 pathway.Hit to lead optimization by medicinal chemistry (partner 1).Chembridge has several derivatives of hit 25 and 53 available hence we plan to perform an initial SAR with such compounds.Crystallographic support to the medicinal chemistry efforts (partner 3).Partner 3 (CRUK) in collaboration with Partner 2 (UCL) elucidated the crystal structure of TRN-SR2 (Maertens et al. 2014 PNAS 111: 2728), revealing the critical residues for binding to SR proteins ASF/SF2 and CPSF6. CPSF6 like ASF/SF2 is transported to the nucleus by interaction with TRN-SR2. CPSF6 is implicated in HIV-1 infection presumably by interaction with capsid which could play a role in the import of HIV-1 to the nucleus In contrast to CPSF6, ASF/SF2 also transported to the nucleus by TRN-SR2 is not involved in the HIV-1 replication cycle. Interestingly, CPSF6 and ASF/SF2 appear to bind to a few different residues in the arginine rich helix ofTRN-SR2, which suggests that it may be possible to selectively perturb by small molecules TRN-SR2 interaction with CPSF6 without affecting ASF/SF2 binding. This may have therapeutic implications to inhibit HIV-1 infection and reduce possible cytotoxicity due to loss of nuclear ASF/SF2, a critical splicing factor.To this end, in collaboration with Partner 1 and Partner 3, we have set up and validated a biochemical high through put screening assay based on FRET to identify small molecules that selectively inhibit the interaction between CPSF6 and TRN-SR2. We were able to develop a robust screening assay. BIODIM has provided to UCL a 130,000 compounds preformatted library into 408x 384 well plates. - 130,560 compounds were screened for TNPO3:CPSF6 inhibitors in 8 sets. - 659 hits were selected in total using 5 Medium Average Deviations cut-off, and 33 were confirmed in secondary screen.BIODIM has provided UCL with a sub-library of cherry picked compounds, which is currently being counter screened against CPSF6 and ASF/SF2 (from Partner3).POC in NSG-HIS mice to test efficacy of validated compounds in vivo and preliminary toxicity studies (partner 5). This task has not started yet because the two hits were validated recently and we did not have enough compound to start the experiments. WP3 : From uncoating to nuclear translocation and integration: identification of an interaction network involving PIC components and Trn-SR2, TRIM 5 α and the Hsp90 chaperone complexThe overall objective for WP3 is - to elucidate the function and activities of the network of interaction in HIV-1 infection from uncoating to integration (comprising among other the PIC components, Trn-SR2, TRIM 5α and the Hsp90 chaperone complex), - to discover and validate novel anti-HIV-1 drug targets, and to set up a High Throughput Screening on the most promising validated target to identify new inhibitors. Molecular characterization of the PIC interactome (partners 1, 2, 3, 4).- Partner 1 and Partner 4 identified protein-protein interactions between the capsid (CA) and integrase (IN) protein of HIV-1, that are part of the HIV-1 PIC, and cellular proteins such as HSP90, CypA, Cyp40, TNPO3, SR-25 and the components of the cellular SUMOylation machinery (E3 SUMO ligases of the PIAS family, SUMO E2 conjugating enzyme Ubc9) by yeast two hybrid screening (Y2HS). These studies also revealed interesting potential interactions between some cellular proteins involved in the replication cycle of HIV-1, specifically Trim5α, and components of the cellular SUMOylation machinery.- Partner 4 has extended the network of interaction between cellular and viral proteins that was initially described in the proposal, with a particular focus on HIV-1 IN. First, a new cofactor of HIV-1 IN, EIF3H a subunit from the EIF3 complex involved in the control of translation, was identified by Y2HS (collaboration with Partner 1). The interaction between IN and EIF3H was confirmed and the functional validation of this interaction during HIV replication is in progress. Functional validation of the interactome: Impact of the Hsp90 complex on HIV-1 replication. (partners 2 and 4) - Partner 2 identified a new role of the heat shock protein 90 Hsp90 in the control of HIV reactivation from latency (Anderson et al., Proc Natl Acad Sci U S A. 2014). Partner 2 had previously shown that Hsp90 is required for HIV-1 gene expression and mediates greater HIV-1 replication in some conditions. Partner 4 found that specific inhibitors of Hsp90 such as 17-(N-allylamino)-17-demethoxygeldanamycin and AUY922 prevent HIV-1 reactivation in CD4+ T cells.- Using proteomic approaches, Partner 4 characterized also a novel LEDGF/p75-associated complex, containing Iws1 and Spt6, and has shown that this complex regulates HIV-1 replication at a post integration step and participates to silencing of the HIV-1 provirus and to the establishment and maintenance of HIV latency (Gerard et al. Cell Host Microbe, 2015). The recruitment of this complex to latent provirus correlates with the presence of repressive histone marks recognized by LEDGF. Thus, very exciting and important findings concerning the control of HIV latency by Hsp90 and LEDGF/p75 have emerged from WP3.Partner 4 also developed a Dualcolour HIV vector to identify and isolate latently infected cells (patent deposed EP14305885.7). - Partner 4 also characterized an interaction between Iws1 and the PTW/PP1 complex that contains PNUTS, Tox4, Wdr82 and the protein phosphatase 1 (PP1). The effect of this complex on HIV latency revealed an opposite role of some the subunits of the PTW/PP1 complex and indicates that PP1 could play a role in The work of Partner 4 also deepened the understanding about the process of site-specific integration of retroelements (retrotransposons and retroviruses) by studying the integration site choice of a model retroelement (Ty1) that is closely related to HIV. This process relies on the specific interaction between the IN protein and a chromatin-bound cellular protein, AC40, a polymerase III subunit (Bridier-Nahmias et al. Science, 2015). These results also emphasize the role of retroelements in the evolution and adaptation of organisms, and their potential value for gene therapy.Role of SUMOylation pathway in early steps of HIV replication (partner 4)- Regarding the interplay between HIV proteins and SUMOylation, Partner 4 has identified and characterized the physical and functional interactions between HIV-1 IN and the SUMO E2 conjugation enzyme Ubc9 and SUMO E3 ligases PIAS family members. A splice variant of the SUMO E3 ligase PIAS3 which enhances the conjugation of SUMO proteins to HIV-1 IN was also characterized. This PIAS3 variant is expressed in quiescent T cells, which are resistant to HIV infection, but is undetectable in activated T cells, which are permissive to HIV. Overexpression of either the long or the short PIAS3 isoform modestly increased HIV infectivity, while siRNA-mediated depletion of endogenous PIAS3 slightly reduced HIV infectivity. These results suggest that PIAS3 plays a role as a viral cofactor during the early stages of the HIV life cycle. - Partner 4 has also confirmed that the HIV restriction factor Trim5alpha is modified by SUMO and is currently addressing the functional role of this modification. We also established that the interaction between Trim5alpha and SUMO occurs mainly in the nucleus, by performing immunofluorescence and Proximity ligation assay. This result is intriguing, because overexpressed Trim5alpha is distributed in the cytoplasm, while the proteins of the SUMOylation machinery are enriched in the nucleus. However, Trim5alpha accumulates in nuclear foci in cells treated with LMB, an inhibitor of Crm-1-mediated nuclear export (Diaz-Griffero F., Retrovirology 2011). Our goal now is to identify the determinants of Trim5alpha nucleo-cytoplasmic trafficking and establish whether a correlation exists with conjugation of SUMO to Trim5alpha. HTS assay on the most promising new target and screening campaign to identify new inhibitors. (partner 1).Postponed because validation of the new targets was completed at the end of the second period and the choice of the most promising target from the point of view of best pharmacological target for identification of a novel class of ARV putatively interfering with the HIV latency process is still in debate.Crystallographic support to the medicinal chemistry efforts (partner 3).Partner 3 elucidated by Single-particle cryo-electron microscopy the structural basis for retroviral integration into nucleosomes (Maskell et al. Nature, 2015). Retroviral integration is catalyzed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Since the complete 3D structure of HIV-1 IN is still missing, Partner 3 used for this study that of a close homolog, the IN of the prototype foamy virus (PFV) in stable complex with viral DNA ends as intasome, he elucidated previously (Maertens et al., Nature, 2010). Partner 3 showed that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy revealed a multivalent intasome–nucleosome interface involving both gyres of nucleosomal DNA and one H2A–H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A–H2B heterodimer to allow integration at strongly preferred superhelix location ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Partner 3 findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration. This work provides further structural support for the isolation of novel inhibitors of integration. Overall conclusion of the work progress and achievements during the entire 3 years period of HIVINNOV.All partners actively participated to the advances achieved during the three years period of the HIVINNOV project. Remarkably most of the results obtained stem from very efficient, fruitful and smooth collaborative works between partners in all WPs of the project. The information and reagents have been circulating without restrictions, and each consortium meeting has been an opportunity for very productive discussions.Although some deliverables were not reached within the three years period of the consortium, such as phases I and IIa POC clinical trials for the partner 1 BDM2 drug candidate, the identification of BDM2 as a very potent ARV new class of drug, its full characterization as a very good drug candidate through deep preclinical investigation, including acute toxicity studies in several animal models, its in vivo preclinical validation of efficient ARV in the NSG-HIS mice model of HIV infection, first ever in vivo validation of this new class of drugs, all these important advances are very encouraging for the final success of this drug candidate in the clinical trials that partner 1 is fully committed to achieve with a delay of about two years following the three year period of HIVINNOV. All WPs and all partners have also succeeded to realize remarkable achievements such as the validation of the NSG_HIS mice model suitable for oral administration studies of anti-HIV drugs, the identification of new compounds and new targets very promising for the development of new class of ARV, and the important elucidation of 3D structures of new proteins involved in pathway implicated in HIV infection or new complexes of intasome with nucleosomes at integration sites. All these remarkable realizations are attested by a quite brilliant list of publications resulting from the work of the HIVINNOV partners.Potential Impact:4- The potential impact In the submitted HIVINNOV proposal, various important impacts, medical, scientific and socio-economic were expected from the completion of this project. These impacts were the followings:i) Impact of HIVINNOV compounds on anti-retroviral treatments with:a) IN-LEDGF inhibitors as salvage therapy for patients in virological failure due to viruses resistant to INSTIsb) IN-LEDGF inhibitors in association to INSTIsc) Inhibitors of the Trn-SR2 dependent HIV replication pathwayii) Access to new treatments in developing countriesiii) Scientific impact of HIVINNOVa) Protein-protein interactions as druggable targetsb) Impact on HIV molecular virology: toward the elucidation of crucial pathways in the HIV replication cycle and the identification of new targets for ARV developmentc) Impact on the development of drugs in other microbial diseasesiv) EU HIV R&D area, Biotech sector and market size v) Impact on the development of the partner 1 SME BIODIMIn this report, we will examine what are the potential impacts of the HIVINNOV project that could be expected in respect to the results that have been reached at the end of this 3 years period of intense work. i) Impact on ARV treatments:As indicated in the description of the main S&T results of HIVINNOV, some important deliverables that could not be reached within the three years period of HIVINNOV, such as phase I/IIa POC trials as clinical validation of the partner 1 drug candidate BDM2, are delayed in the two years following the end of HIVINNOV. As a result of this delay, the full consequence of the project, on the:- Impact of HIVINNOV compounds on anti retroviral treatments and on - the Access to new treatments in developing countries will also be delayed of two years provided that the clinical trials planed will be successful. Here again it should be recalled that partner 1 is fully committed to achieve these clinical trials once the GMP batch will be delivered and the regulatory preclinical studies completed. However, even in the field of the ARV treatments, the results already achieved with the partner 1 drug candidate BDM2 are expected to be highly significant in respect ofa) IN-LEDGF inhibitors as salvage therapy for patients in virological failure due to viruses resistant to INSTIs: In fact we know already from the work achieved in WP1, on resistance studies in vitro on MT4 cells infected with HIV-1 and on similar studies achieved in vivo on NSG-HIS mice model of HIV infection, that BDM2 and all the ARV class of ALLINIs are fully active on all viruses resistant to all anti-HIV drugs currently on the market. This is the condition required to validate future ALLINI treatments as salvage therapy for patients in virological failure. b) IN-LEDGF inhibitors in association to INSTIs: From the results obtained with combination studies performed using BDM2 in association with all classes of present anti-HIV drugs we have established that BDM2 can be combined with all other classes of drugs including INSTIs without any antagonistic effect detectable. We even found some synergistic effect withan INSTI, Elvitegravir, and a protease inhibitor Lopinavir. Also from resistance in vitro studies performed with viruses resistant to INSTIs and with viruses resistant to ALLINIs, we established that ALLINIs are fully active on viruses resistant to INSTIs, and on reverse that INSTIs were fully active on ALLINI-resistant viruses. So here also the expected impact on the association of BDM2 with INSTIs is clearly anticipated from the results already obtained. c) Inhibitors of the Trn-SR2 dependent HIV replication pathway: we identified in this pathway by using two different screening assays: i) the high content differential cellular screening assay with wt virus, replication-dependent, and mutant virus replication-independent of the TRN-SR2 pathway; ii) biochemical screening of inhibitors of the TRN-SR2/CPSF6 interaction, interaction believed to be required for the import of HIV-1 to the nucleus. These compounds were validated as a new class of ARV with respect to all drug classes currently on the market, but also with respect of ALLINIs. In this part of the project, although we could not complete full validation of these compounds in vivo in the NSG-HIS mice model of HIV infection, their identification in vitro in MT4 cells infected by HIV-1, as a new class of ARV, different from all current drug classes including ALLINIs, will impact very positively the search for alternative new treatments. Our results already demonstrated that the TRN-SR2 pathway could be considered as an effective new target allowing this new class of anti-HIV drugs to be identified thanks to the screening assays we developed. Another interesting characteristic of these new compounds is that they are directed against cellular targets implicated in HIV replication and not against viral targets. Such characteristics presumably will make these compounds less susceptible to drug resistance because host factors encoding by cellular genes mutate rarely compared to viral genes. iii)- Scientific impact of HIVINNOV:Since with the results already achieved during the 3 years period of HIVINNOV, we were able to:- Identify very potent new class of ALLINI drug candidates of partner 1 including back-up compounds, - Elucidate their mode of action, characterize their preclinical pharmacology and toxicity profiles, - Achieve for the first time their full preclinical validation as potent new class of ARV in the NSG-HIS mice model of HIV infection,- Validate the NSG-HIS mice model for continuous oral drug administration via novel MediDrop technology.- Identify new targets involved in the HIV replication cycle and in the establishment of HIV latency- Elucidate the tri-dimensional structure of TRN-SR2 and characterize its interaction with two cargo proteins that are transported to the nucleus by TRN-SR2, one of which, CPSF6 being involved in the import of HIV to the nucleus- Elucidate by a superb up-to-date technology, single-particle cryo-electron microscopy, the structural basis for retroviral integration into nucleosomes.One can consider that the scientific impact of HIVINNOV will be considerable and will even overcome our best expectations:a) In the field of Protein-protein interactions as druggable targets, our results demonstrate that i) it is feasible to identify extremely potent protein-protein interaction inhibitors (PPII): as a striking example we found that the back-up compound, BDM3 a small chemical compound of less than 400 MW, was able to disrupt the complex IN-LEDGF/p75 of more than 250,000 MW with an IC50 as low as 12nM! This one of the most potent PPII ever identified; ii) these PPII display pharmacology profile and antiviral activity that makes them very promising drugs. So our results fully validate the search of PPII as a very promising pathway in drug discovery for the identification of a new generation of drugs, in particular for the development of drugs in other microbial diseases. It is useful to recall that up to date there is still no drug on the market in any pathological field that have been discovered on the basis of PPII. We are proud that HIVINNOV is one of the few actors paving the way toward this new pathway in drug discovery.b) Impact on HIV molecular virology: toward the elucidation of crucial pathways in the HIV replication cycle and the identification of new targets for ARV developmentThe elucidation of the peculiar mode of action of the ALLINIs by partner 1 and partner 5 revealed an unexpected function of IN at the late stage of the HIV replication cycle, during maturation and production of infectious viral particles. Also, besides their use as potent new class of ARV drug, BDM2 and all ALLINIs developed by partner 1 can be considered as very interesting reagent that can be used for fundamental research in virology on the HIV replication cycle. Same is true for the compounds identified by partner 2 in the TRN-SR2 pathway.The identification of new targets, namely those involved in the establishment of HIV latency, the integration choice of retroelements, along the SUMOylation pathway, and the elucidation of the tri-D structure of TRN-SR2 and of the basis of retroviral integration into nucleosomes, will have a considerable influence in the HIV research field.The importance of the scientific impact of HIVINNOV is fully attested by the brilliant list of papers, patents and scientific communications already published by the HIVINNOV partners.iv) EU HIV R&D area, Biotech sector The impact of HIVINNOV is already important in the EU HIV R&D area and in the EU biotech sector involved in HIV drug discovery.The HIVINNOV consortium took the initiative for the organization of a public meeting satellite of the European AIDS Clinical Society (EACS) congress in Barcelona 2015 October 20th. We contacted our colleagues of two other EU consortia working on HIV, Hit Hidden HIV and Thinpad that immediately agreed to hold this joint public meeting. The scientific program of this meeting is provided in annex. The meeting was a great success and more than 100 scientists from all over Europe attended the meeting to which the EU officer in charge of HIVINNOV, Dr. Alessandra Martini participated. EACS president Manuel Battegay and the organizer of the EACS congress in Barcelona, our Partner 6 colleague, Pr. Jose Gatell were very pleased with this satellite meeting that offered to many European clinician scientists the opportunity to hear about progress achieved by the outstanding fundamental and pharmacology European scientists members of HIVINNOV and of the two other EU consortia working on HIV, Hit Hidden HIV and Thinpad. Interestingly, the EACS president decided to renew this experience of satellite meeting with EU consortia, for the future EACS congress. In addition all the SME partners of these three consortia actively participated to the meeting and initiated useful contacts between them and with the partners of the other consortia.v) Impact on the development of the partner 1 SME BIODIM Based on the results obtained so far by partner 1 on the identification and characterization of the drug candidate BDM2 and back-up compounds, as very potent new ARV drugs from the class of ALLINIs, partner 1 could established good contacts with major pharmaceutical companies active in the anti-HIV drug research. The goal of partner 1 is to look for a partnership with one of these major pharmaceutical companies to ensure the development of BDM2 up to phase II and phase III trials up to its access to the market. Indeed, very importantly, one of these pharmaceutical companies which wanted to validate our drug candidate found very identical results to those found by partner 1, further confirming the attractive properties of BDM2 as potent ARV. The full impact of HIVINNOV as anticipated since the beginning of HIVINNOV will be reached provided in about two years from the end of HIVINNOV, BDM2 will be successfully validated in phase I/phase IIa POC clinical trials. The contacts already established by partner 1 with some pharmaceutical companies are very encouraging for the future development of partner 1 once BDM2 will be clinically validated. Partner 1 which is in excellent financial situation is fully committed to develop BDM2 on its own resources within the coming two years following the end of HIVINNOV, up to the clinical validation in phase I/IIa POC trials. Main dissemination activities and exploitation of results:Objectives and main achievements: The overall objectives of this Work Package are to:Look for partnership with a larger pharmaceutical laboratory to further develop the BIODIM drug candidate towards the clinical phase IIb and the regulatory authorization.Keep a watch on the patentability of the project’s results, especially the optimized molecules originating from WP2 screening, and the new targets discovered in WP3.To disseminate the knowledge generated within HIVINNOV.To organise at the end of the project a public scientific workshop on new ARV compounds and new targets.Exploitation plan for future development of WP1 validated drugsA “teaser” has been prepared by partner 1 in the first months of the project to establish contact with potential partners in the pharmaceutical industry. Concerning the BDM-2 lead compound, BIODIM has initiated contacts with 3 major pharmaceutical companies from the HIV field in order to explore a potential partnership. BDM2 samples were sent to some of these companies as mean of further validation of preclinical ARV activity by these companies. The results of these tests were very encouraging for the future development of BDM2 since these tests fully confirm the high potential of BDM2 as most advanced drug candidate of the family of this new ARV class ALLINIs. These contacts will be continued during the future preclinical and clinical development of BDM2 in the two years to come after the end of HIVINNOV. However Partner 1 is facing some complicated problems due to the fact that some of these major players in the HIV field announced recently their decision to stop their drug discovery research efforts in the HIV and HBV fields. BIODIM has implemented a development plan on its own resources, to pursue the work of HIVINNOV WP1 in the two-three coming years for the 8 kg BDM2 GMP batch synthesis and completion of BDM2 regulatory preclinal and phases I/IIa POC clinical trials. The financial situation of BIODIM is excellent, thus ensuring that the internal financing of BDM2 development plan for the coming tears, as previously planned in HIVINNOV, is granted. Clinical validation of BDM2 will facilitate certainly the conclusion of partnership with pharmaceutical companies for the further development of BDM2 toward phase II and phase III clinical trials if the phase I/IIa POC trials are successful.- Six patents families on BIODIM BDM1, BDM2 drug candidate, BDM3 and BDM4 back-ups and other BIODIM ALLINIs have been deposited, extended worldwide and national phases initiated. It should be underlined that worldwide, more than 51 patents have been deposited to date on ALLINIs, including those of BIODIM. Most of major players in the anti-HIV drug field (BMS, GSK, VIIV, Pfizer, Shionogi, Gilead) have been very active in deposit of patents on ALLINI compounds. This is a clear sign of the great interest for this new class of drug. This is also very encouraging for the future exploitation of BDM2, if POC clinical trials are successful. Intellectual Property sharing and exploitation plan of WP2 and WP3 productsIn WP2, Partner 2 is implementing a development plan for further validation, optimization and exploitation of the hits identified in the high-content cellular screening and biochemical screening in search of compounds interfering with the TRN-SR2 pathway that is required for HIV infection.Validation of the hits resulting from the high-content cellular screening conducted in WP2 is still ongoing. To increase the chances of finding new compounds active in the TRN-SR2 pathway, a new biochemical screening assay was developed in close collaboration between partner 1 (BIODIM) partner 2 (UCL) and partner 3 (CRUK & F. CRICK Inst.). An important High-Throughput screening of 130,000 compounds from partner’s 1 library was performed successfully by Partner 2, resulting in several hits (see report on WP2). However no patentable compound has emerged so far. While discussing with the Scientific Advisory Board, Partner 2 learnt about possible UK funding schemes to support the development of these hits. This can be a way to sustain the HIVINNOV collaboration even after the end of the European support. Partner 2 and Partner 3 are looking actively toward such possibility. Partner 1 supports these efforts of Partners 2 and 3 and ensures future collaboration. - In WP3, Partner 4 deposited a patent on a Dual-colour HIV vector to identify and isolate latently infected cells (patent deposed EP14305885.7). Partner 3, with INSERM Transfer is looking for partnership with big pharmaceutical companies active in the field of HIV latency and HIV cure.Publication, communication and dissemination of resultsUpon the project’s launch we have produced a press-release in English and French (translated in Spanish by the Hospital Clinic partner). This press-release has been relayed at least 21 times in the international press (see annex). The HIVINNOV consortium took the initiative for the organization of a public meeting satellite of the EACS congress in Barcelona 2015 October 20th, together with two other EU consortia working on HIV, Hit Hidden HIV and Thinpad. The scientific program of this meeting is provided below. The meeting was a great success and more than 100 scientists from all over Europe attended the meeting to which the EU officer in charge of HIVINNOV, Dr. Alessandra Martini participated. EACS president Manuel Battegay and the organizer of the EACS congress in Barcelona, our Partner 6 colleague, Pr. Jose Gatell were very pleased with this satellite meeting that offered to many European clinician scientists the opportunity to hear about progress achieved by the outstanding fundamental and pharmacology European scientists members of HIVINNOV and of the two other EU consortia working on HIV, Hit Hidden HIV and Thinpad. Interestingly, the EACS president decided to renew this experience of satellite meeting with EU consortia, for the future EACS congress. The following deposited patents and articles related to the HIVINNOV activities have been published by the partners in the second period, illustrating with the articles published during the first period, the efficient collaborations taking place in the consortium.Talks and posters related to the project’s activities have been presented at various events of the field. The list is available on the participant portal and below.It has been decided at the last Steering Committee meeting (May 7th 2014) to assemble a public HIVINNOV newsletter summarizing the main achievements so far. This communication is intended to raise awareness about our activities towards the scientific community or patient organizations.List of Websites:http:// www.hivinnov.eu Scientific Coordinator : Dr. Richard Benarous – Chief Scientific Officer. BiodimTel: +33 1 57 14 05 23 or 20E-mail: Richard.benarous@mutabilis.fr Project Manager : Ibrahima Guillard - BiodimTel: +33 1 57 14 05 34E-mail : Ibrahima.guillard@mutabilis.fr