Title: Novel HIV inhibitors targeting the interaction between integrase and LEDGF/p75
Other Titles: Nieuwe HIV inhibitoren gericht tegen de interactie van integrase met LEDGF/p75
Authors: Desimmie, Belete; S0203159
Issue Date: 12-Dec-2012
Abstract: Three decades after its discovery, the human immunodeficiency virus (HIV), the etiologic agent of the acquired immunodeficiency syndrome (AIDS), continues to wreak havoc on a global scale. However, thanks to the significant scientific achievements, combination antiretroviral therapy (ART) better known as highly active antiretroviral therapy (HAART) has significantly improved the prognosis of the disease. There is a continuous search for better drugs targeting alternative steps of the replication cycle with a higher genetic barrier and tolerability aiming at complementing the existing repertoire of drugs and reducing thelatent reservoir, a major obstacle of an HIV cure. Moreover, their application can help us to further understand the ‘knowable unknowns’ of thebiology of the HIV. Although most clinical anti-HIV drugs target reverse transcriptase and protease, integrase is also a prime target for anti-HIV therapy. Accordingly, several classes of integrase (IN) inhibitors have been discovered including the first IN inhibitor approved for patient treatment, raltegravir. To efficiently insert the viral DNA, IN is dependent on cellular proteins called cofactors. One such protein is lens epithelium-derived growth factor (LEDGF)/p75. The interaction between LEDGF/p75 and IN is a potential therapeutic target thatcan accommodate small molecule inhibitors. At the start of this doctoral study, through multidisciplinary efforts, our research group identified three novel classes of integration inhibitors: (i) IN catalytic site inhibitors, (ii) LEDGINs and (iii) LEDGF/p75-binding peptides identified by phage display. The present study focuses on the virological characterization and the elucidation of the mechanism of action of these inhibitors. Moreover, using some of these inhibitors we investigated the role ofLEDGF/p75 in late stage HIV replication. The first part of the study aimed at the characterization of the antiviral activityof the IN catalytic site inhibitor MB-76, a lead compound of the 2-hydroxyisoquinoline-1,3(2H, 4H)diones (HQD) series . In a series of experiments, we showed that MB-76 displays a broad-spectrum antiviral activity with low to submicromolar 50% effective concentrations (EC50) with aselectivity index (SI) of >53. MB-76 exhibits no cross-resistance with other classes of antiretrovirals (ARVs) including integrase strand transfer inhibitors (INSTIs). Using quantitative PCR and time-of-addition experiments, we demonstrated that MB-76, similar to raltegravir, solely inhibits the integration step. We could not select resistant variants afterpassaging the virus for 15 months in cell culture, indicating that the HQD compounds have a superior genetic barrier to resistance development compared to the clinical INSTIs supporting their potential for further development. In search of a novel class of allosteric IN inhibitors, we exploited the interaction between LEDGF/p75 and HIV-1 IN. This protein-protein interaction (PPI) displays a distinct ‘key and lock-like’ interaction, amenable for small molecule PPI inhibitor design. A rational drug design effort has led to the identification of small molecule allosteric IN inhibitors targeting the LEDGF/p75 binding pocket in integrase – termed LEDGINs. Here, I characterized the antiviralactivity and pinpointed the mechanism of action of LEDGINs. LEDGINs prevent the interaction between LEDGF/p75 and HIV-1 IN, and indirectly interfere with the catalytic activity of IN. LEDGINs inhibit HIV-1 replication at low micromolar EC50 by blocking the integration step. Furthermore,a resistance selection experiment revealed a single amino acid substitution, A128T, in the IN-coding region confirming the molecular target. LEDGINs lack cross-resistance with other ARVs including INSTIs. Moreover, we observed a significant effect on the infectivity of progeny virions produced in the presence of LEDGINs, suggesting that LEDGINs inhibit bothintegration and late stages of HIV. In conclusion, our work demonstrates the feasibility of rational design of small molecules inhibiting PPI between a viral protein and a cellular factor underscoring their potential for further (clinical) development. While LEDGINs inhibit HIV-1 replication by occupying the LEDGF/p75 pocket in integrase,it remained unknown whether LEDGF/p75 by itself can be targeted. Therefore, using cyclic peptides (CPs) identified by a carefully designed phage display strategy, we demonstrated for the first time that LEDGF/p75-binding ligands can inhibit HIV replication by blocking the interaction between LEDGF/p75 and HIV-1 IN. Here, we showed that the CPs inhibit HIV replication without overt toxicity. In accord with the role of LEDGF/p75 in HIV integration, CP64 and CP65 block HIV replication by inhibiting integration. Moreover, the CPs retained activity against HIV-1 strains resistant to raltegravir or LEDGINs and HIV-2, indicating their differing mechanism of action compared to LEDGINs or INSTIs. We also observed a strong effect on the infectivity of progeny virions generated in CP65 expressing cells, suggesting a yet unidentified role of LEDGF/p75 in late stage HIV replication. Serial passaging of virus in the presence of CPs did not yield resistant strains. Our work provides proof-of-concept for direct targeting of LEDGF/p75 as a novel therapeutic strategy. Moreover,the CPs may serve as a scaffold for future development of new HIV therapeutics. In the final part of this doctoral study,we elucidated the molecular mechanism of the late effect of LEDGINs, which can also explain the late effect of the LEDGF/p75-binding CPs. Through parallel approaches using LEDGINs, LEDGF/p75-depleted cells, and interaction mutants of LEDGF/p75 (D366N) and HIV-1 IN (W131A), we unveil a hitherto unknown role of LEDGF/p75 in HIV biology by demonstrating its requirement for the generation of fully infectious HIV particles. Virions produced in the presence of LEDGINs display a 2- to 4-log reduction in infectivity. Detailed analysis excluded defects in genomic RNA packaging or proteolytic maturation of the virions produced in the presence of LEDGINs; instead, our findings reveal a post-entry defect during early replication steps including reverse transcription, nuclear import, and integration in the target cells. Experiments to explain the mechanism of the late effect of LEDGINs indicated that LEDGF/p75 is incorporated in the viral particles and is inhibited by LEDGINs. We then showed that LEDGF/p75 is recruited through its direct interaction with IN and/or Pol polyprotein into nascent virions. In summary, we have identified an unanticipated role of LEDGF/p75 in HIV-1 replication and corroborated a unique mechanism of action of LEDGINs inhibiting both early and late steps of HIV replication. These novel classes of inhibitors, results of multidisciplinary efforts, represent some of the most attractive ARVs for further pharmacological development to complement the existing drugs. The inhibitors described in this study have proven multimodal anti-HIV activity and a moderate to very high genetic barrier to resistance development. Particularly, LEDGINs - allosteric IN inhibitors in advancedpreclinical development - are highly potent and have compatible pharmacokinetic profiles to advance further. Notwithstanding, their ultimate success will critically depend on further clinical research and development.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Biochemistry, Kulak (-)
Faculty of Medicine, Campus Kulak Kortrijk
Molecular Virology and Gene Therapy

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