Rational fragment-based design of compounds targeting the PWWP domain of the HRP family.

Lens epithelium-derived growth factor p75 (LEDGF/p75), member of the hepatoma-derived growth-factor-related protein (HRP) family, is a transcriptional co-activator and involved in several pathologies including HIV infection and malignancies such as MLL-rearranged leukemia. LEDGF/p75 acts by tethering proteins to the chromatin through its integrase binding domain. This chromatin interaction occurs between the PWWP domain of LEDGF/p75 and nucleosomes carrying a di- or trimethylation mark on histone H3 Lys36 (H3K36me2/3). Our aim is to rationally devise small molecule drugs capable of inhibiting such interaction. To bootstrap this development, we resorted to X-ray crystallography-based fragment screening (FBS-X). Given that the LEDGF PWWP domain crystals were not suitable for FBS-X, we employed crystals of the closely related PWWP domain of paralog HRP-2. As a result, as many as 68 diverse fragment hits were identified, providing a detailed sampling of the H3K36me2/3 pocket pharmacophore. Subsequent structure-guided fragment expansion in three directions yielded multiple compound series binding to the pocket, as verified through X-ray crystallography, nuclear magnetic resonance and differential scanning fluorimetry. Our best compounds have double-digit micromolar affinity and optimally sample the interactions available in the pocket, judging by the Kd-based ligand efficiency exceeding 0.5 kcal/mol per non-hydrogen atom. Beyond π-stacking within the aromatic cage of the pocket and hydrogen bonding, the best compounds engage in a σ-hole interaction between a halogen atom and a conserved water buried deep in the pocket. Notably, the binding pocket in LEDGF PWWP is considerably smaller compared to the related PWWP1 domains of NSD2 and NSD3 which feature an additional subpocket and for which nanomolar affinity compounds have been developed recently. The absence of this subpocket in LEDGF PWWP limits the attainable affinity. Additionally, these structural differences in the H3K36me2/3 pocket across the PWWP domain family translate into a distinct selectivity of the compounds we developed. Our top-ranked compounds are interacting with both homologous LEDGF and HRP-2 PWWP domains, yet they showed no affinity for the NSD2 PWWP1 and BRPF2 PWWP domains which belong to other PWWP domain subfamilies. Nevertheless, our developed compound series provide a strong foundation for future drug discovery targeting the LEDGF PWWP domain as they can further be explored through combinatorial chemistry. Given that the affinity of H3K36me2/3 nucleosomes to LEDGF/p75 is driven by interactions within the pocket as well as with the DNA-binding residues, we suggest that future compound development should target the latter region as well. Beyond drug discovery, our compounds can be employed to devise tool compounds to investigate the mechanism of LEDGF/p75 in epigenetic regulation.

The Impact of Lens Epithelium-Derived Growth Factor p75 Dimerization on Its Tethering Function.

The transcriptional co-activator lens epithelium-derived growth factor/p75 (LEDGF/p75) plays an important role in the biology of the cell and in several human diseases, including MLL-rearranged acute leukemia, autoimmunity, and HIV-1 infection. In both health and disease, LEDGF/p75 functions as a chromatin tether that interacts with proteins such as MLL1 and HIV-1 integrase via its integrase-binding domain (IBD) and with chromatin through its N-terminal PWWP domain. Recently, dimerization of LEDGF/p75 was shown, mediated by a network of electrostatic contacts between amino acids from the IBD and the C-terminal α6-helix. Here, we investigated the functional impact of LEDGF/p75 variants on the dimerization using biochemical and cellular interaction assays. The data demonstrate that the C-terminal α6-helix folds back in cis on the IBD of monomeric LEDGF/p75. We discovered that the presence of DNA stimulates LEDGF/p75 dimerization. LEDGF/p75 dimerization enhances binding to MLL1 but not to HIV-1 integrase, a finding that was observed in vitro and validated in cell culture. Whereas HIV-1 replication was not dependent on LEDGF/p75 dimerization, colony formation of MLLr-dependent human leukemic THP-1 cells was. In conclusion, our data indicate that intricate changes in the quaternary structure of LEDGF/p75 modulate its tethering function.

BET-Independent Murine Leukemia Virus Integration Is Retargeted In Vivo and Selects Distinct Genomic Elements for Lymphomagenesis.

Moloney murine leukemia virus (MLV) infects BALB/c mice and induces T-cell lymphoma in mice. Retroviral integration is mediated by the interaction of the MLV integrase (IN) with members of the bromodomain and extraterminal motif (BET) protein family (BRD2, BRD3, and BRD4). The introduction of the W390A mutation into MLV IN abolishes the BET interaction. Here, we compared the replication of W390A MLV to that of wild-type (WT) MLV in adult BALB/c mice to study the role of BET proteins in replication, integration, and tumorigenesis in vivo. Comparing WT and W390A MLV infections revealed similar viral loads in the blood, thymus, and spleen cells. Interestingly, W390A MLV integration was retargeted away from GC-enriched genomic regions. However, both WT MLV- and W390A MLV-infected mice developed T-cell lymphoma after similar latencies represented by an enlarged thymus and spleen and multiorgan tumor infiltration. Integration site sequencing from splenic tumor cells revealed clonal expansion in all WT MLV- and W390A MLV-infected mice. However, the integration profiles of W390A MLV and WT MLV differed significantly. Integrations were enriched in enhancers and promoters, but compared to the WT, W390A MLV integrated less frequently into enhancers and more frequently into oncogene bodies such as Notch1 and Ppp1r16b. We conclude that host factors direct MLV in vivo integration site selection. Although BET proteins target WT MLV integration preferentially toward enhancers and promoters, insertional lymphomagenesis can occur independently from BET, likely due to the intrinsically strong enhancer/promoter of the MLV long terminal repeat (LTR). IMPORTANCE In this study, we have shown that the in vivo replication of murine leukemia virus happens independently of BET proteins, which are key host determinants involved in retroviral integration site selection. This finding opens a new research line in the discovery of alternative viral or host factors that may complement the dominant host factor. In addition, our results show that BET-independent murine leukemia virus uncouples insertional mutagenesis from gene enhancers, although lymphomagenesis still occurs despite the lack of an interaction with BET proteins. Our findings also have implications for the engineering of BET-independent MLV-based vectors for gene therapy, which may not be a safe alternative.

Design, synthesis, in silico studies, and antiproliferative evaluations of novel indolin-2-one derivatives containing 3-hydroxy-4-pyridinone fragment.

Keeping in view the pharmacological properties of indolinones as promising scaffold as kinase inhibitors, herein, a novel series of 3-hydrazonoindolin-2-one derivatives bearing 3-hydroxy-4-pyridinone moiety were synthesized, studied by molecular docking, and fully characterized by spectroscopic techniques. All the prepared compounds were evaluated for their cytotoxicity attributes against a panel of tumor cell lines, including non-small cell lung cancer (A549), breast carcinoma (MCF-7), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML). They displayed moderate to promising antiproliferative effects toward A549 and MCF-7 cells but remarkable results against AML and CML. Especially, compound 10k was found to be more potent against AML (EC50 = 0.69 µM) compare to the other halogen-substituted derivatives. FMS-like tyrosine kinase 3 (FLT3) is known to be expressed in AML cancer cells. The molecular docking studies demonstrated that our prepared compounds were potentially bound to AML active site through essential H-bond and other vital interactions with critical binding residues.

Novel CFTR modulator combinations maximise rescue of G85E and N1303K in rectal organoids.

Introduction: Cystic fibrosis (CF) is a severe monogenic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Several types of CFTR modulators (correctors/potentiators) have been developed to overcome protein dysfunction associated with these mutations. CFTR modulator therapy is now available for the major CF-causing mutations; however, 10% of people with CF remain without causal treatments. By combining investigational and market-approved CFTR modulators, we aimed to maximise functional rescue of iva-, luma- and tezacaftor refractory mutants G85E and N1303K. Methods: We used the well-established forskolin-induced swelling (FIS) in primary rectal organoids to assess responses to different CFTR corrector and potentiator types. The FIS analysis was performed with brightfield microscopy, allowing both 1-h and 24-h follow-up. Corrector and potentiator activity of elexacaftor was investigated. Results: For G85E, maximal rescue was observed by a combination of elexacaftor and corr4a. For N1303K, the quadruple combination teza-elexa-ivacaftor with apigenin was required to obtain a rescue similar to that of luma-ivacaftor rescued F508del. Elexacaftor rescued G85E and N1303K by different mechanisms, with chronic corrector effects on G85E and acute potentiation of N1303K only in the presence of ivacaftor. Synergy in N1303K rescue for iva-elexacaftor and apigenin suggests at least three potentiator mechanisms for this mutant. 24-h FIS identified ivacaftor as the main CFTR modulator for N1303K and elexacaftor and apigenin as co-potentiators. Conclusions: Novel combinations of CFTR modulators can further improve functional rescue of G85E and N1303K in rectal organoids, although for N1303K, more effective CFTR modulators are still needed.

Determinants of Retroviral Integration and Implications for Gene Therapeutic MLV-Based Vectors and for a Cure for HIV-1 Infection.

To complete their replication cycle, retroviruses need to integrate a DNA copy of their RNA genome into a host chromosome. Integration site selection is not random and is driven by multiple viral and cellular host factors specific to different classes of retroviruses. Today, overwhelming evidence from cell culture, animal experiments and clinical data suggests that integration sites are important for retroviral replication, oncogenesis and/or latency. In this review, we will summarize the increasing knowledge of the mechanisms underlying the integration site selection of the gammaretrovirus MLV and the lentivirus HIV-1. We will discuss how host factors of the integration site selection of retroviruses may steer the development of safer viral vectors for gene therapy. Next, we will discuss how altering the integration site preference of HIV-1 using small molecules could lead to a cure for HIV-1 infection.

LEDGF/p75-mediated chemoresistance of mixed-lineage leukemia involves cell survival pathways and super enhancer activators.

MLL is an aggressive subtype of leukemia with a poor prognosis that mostly affects pediatric patients. MLL-rearranged fusion proteins (MLLr) induce aberrant target gene expression resulting in leukemogenesis. MLL and its fusions are tethered to chromatin by LEDGF/p75, a transcriptional co-activator that specifically recognizes H3K36me2/3. LEDGF/p75 is ubiquitously expressed and associated with regulation of gene expression, autoimmune responses, and HIV replication. LEDGF/p75 was proven to be essential for leukemogenesis in MLL. Apart from MLL, LEDGF/p75 has been linked to lung, breast, and prostate cancer. Intriguingly, LEDGF/p75 interacts with Med-1, which co-localizes with BRD4. Both are known as co-activators of super-enhancers. Here, we describe LEDGF/p75-dependent chemoresistance of MLLr cell lines. Investigation of the underlying mechanism revealed a role of LEDGF/p75 in the cell cycle and in survival pathways and showed that LEDGF/p75 protects against apoptosis during chemotherapy. Remarkably, LEDGF/p75 levels also affected expression of BRD4 and Med1. Altogether, our data suggest a role of LEDGF/p75 in cancer survival, stem cell renewal, and activation of nuclear super enhancers.

Unlike Its Paralog LEDGF/p75, HRP-2 Is Dispensable for MLL-R Leukemogenesis but Important for Leukemic Cell Survival.

HDGF-related protein 2 (HRP-2) is a member of the Hepatoma-Derived Growth Factor-related protein family that harbors the structured PWWP and Integrase Binding Domain, known to associate with methylated histone tails or cellular and viral proteins, respectively. Interestingly, HRP-2 is a paralog of Lens Epithelium Derived Growth Factor p75 (LEDGF/p75), which is essential for MLL-rearranged (MLL-r) leukemia but dispensable for hematopoiesis. Sequel to these findings, we investigated the role of HRP-2 in hematopoiesis and MLL-r leukemia. Protein interactions were investigated by co-immunoprecipitation and validated using recombinant proteins in NMR. A systemic knockout mouse model was used to study normal hematopoiesis and MLL-ENL transformation upon the different HRP-2 genotypes. The role of HRP-2 in MLL-r and other leukemic, human cell lines was evaluated by lentiviral-mediated miRNA targeting HRP-2. We demonstrate that MLL and HRP-2 interact through a conserved interface, although this interaction proved less dependent on menin than the MLL-LEDGF/p75 interaction. The systemic HRP-2 knockout mice only revealed an increase in neutrophils in the peripheral blood, whereas the depletion of HRP-2 in leukemic cell lines and transformed primary murine cells resulted in reduced colony formation independently of MLL-rearrangements. In contrast, primary murine HRP-2 knockout cells were efficiently transformed by the MLL-ENL fusion, indicating that HRP-2, unlike LEDGF/p75, is dispensable for the transformation of MLL-ENL leukemogenesis but important for leukemic cell survival.

Molecular Mechanism of LEDGF/p75 Dimerization.

Dimerization of many eukaryotic transcription regulatory factors is critical for their function. Regulatory role of an epigenetic reader lens epithelium-derived growth factor/p75 (LEDGF/p75) requires at least two copies of this protein to overcome the nucleosome-induced barrier to transcription elongation. Moreover, various LEDGF/p75 binding partners are enriched for dimeric features, further underscoring the functional regulatory role of LEDGF/p75 dimerization. Here, we dissected the minimal dimerization region in the C-terminal part of LEDGF/p75 and, using paramagnetic NMR spectroscopy, identified the key molecular contacts that helped to refine the solution structure of the dimer. The LEDGF/p75 dimeric assembly is stabilized by domain swapping within the integrase binding domain and additional electrostatic "stapling" of the negatively charged α helix formed in the intrinsically disordered C-terminal region. We validated the dimerization mechanism using structure-inspired dimerization defective LEDGF/p75 variants and chemical crosslinking coupled to mass spectrometry. We also show how dimerization might affect the LEDGF/p75 interactome.

Phenotyping of Rare CFTR Mutations Reveals Distinct Trafficking and Functional Defects.

Background. The most common CFTR mutation, F508del, presents with multiple cellular defects. However, the possible multiple defects caused by many rarer CFTR mutations are not well studied. We investigated four rare CFTR mutations E60K, G85E, E92K and A455E against well-characterized mutations, F508del and G551D, and their responses to corrector VX-809 and/or potentiator VX-770. Methods. Using complementary assays in HEK293T stable cell lines, we determined maturation by Western blotting, trafficking by flow cytometry using extracellular 3HA-tagged CFTR, and function by halide-sensitive YFP quenching. In the forskolin-induced swelling assay in intestinal organoids, we validated the effect of tagged versus endogenous CFTR. Results. Treatment with VX-809 significantly restored maturation, PM localization and function of both E60K and E92K. Mechanistically, VX-809 not only raised the total amount of CFTR, but significantly increased the traffic efficiency, which was not the case for A455E. G85E was refractory to VX-809 and VX-770 treatment. Conclusions. Since no single model or assay allows deciphering all defects at once, we propose a combination of phenotypic assays to collect rapid and early insights into the multiple defects of CFTR variants.

Affinity switching of the LEDGF/p75 IBD interactome is governed by kinase-dependent phosphorylation.

Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.

LEDGF/p75 is dispensable for hematopoiesis but essential for MLL-rearranged leukemogenesis.

Mixed lineage leukemia (MLL) represents a genetically distinct and aggressive subset of human acute leukemia carrying chromosomal translocations of the MLL gene. These translocations result in oncogenic fusions that mediate aberrant recruitment of the transcription machinery to MLL target genes. The N-terminus of MLL and MLL-fusions form a complex with lens epithelium-derived growth factor (LEDGF/p75; encoded by the PSIP1 gene) and MENIN. This complex contributes to the association of MLL and MLL-fusion multiprotein complexes with the chromatin. Several studies have shown that both MENIN and LEDGF/p75 are required for efficient MLL-fusion-mediated transformation and for the expression of downstream MLL-regulated genes such as HOXA9 and MEIS1 In light of developing a therapeutic strategy targeting this complex, understanding the function of LEDGF/p75 in normal hematopoiesis is crucial. We generated a conditional Psip1 knockout mouse model in the hematopoietic compartment and examined the effects of LEDGF/p75 depletion in postnatal hematopoiesis and the initiation of MLL leukemogenesis. Psip1 knockout mice were viable but showed several defects in hematopoiesis, reduced colony-forming activity in vitro, decreased expression of Hox genes in the hematopoietic stem cells, and decreased MLL occupancy at MLL target genes. Finally, in vitro and in vivo experiments showed that LEDGF/p75 is dispensable for steady-state hematopoiesis but essential for the initiation of MLL-mediated leukemia. These data corroborate the MLL-LEDGF/p75 interaction as novel target for the treatment of MLL-rearranged leukemia.

Protein-protein and protein-chromatin interactions of LEDGF/p75 as novel drug targets.

Lens epithelium-derived growth factor p75 (LEDGF/p75), a transcriptional co-activator, plays an important role in tethering protein complexes to the chromatin. Through this tethering function LEDGF/p75 is implicated in a diverse set of human diseases including HIV infection and mixed lineage leukemia, an aggressive form of cancer with poor prognosis. Here we provide an overview of recent progress in resolving protein-protein and protein-chromatin interaction mechanisms of LEDGF/p75. This review will focus on two well-characterized domains, the PWWP domain and the integrase binding domain (IBD). The PWWP domain interacts with methylated lysine 36 in histone H3, a marker of actively transcribed genes. The IBD interacts with the IBD binding motif, available in cellular binding partners of LEDGF/p75. Each domain forms an interesting new target for drug discovery.

Lessons Learned: HIV Points the Way Towards Precision Treatment of Mixed-Lineage Leukemia.

Protein-protein interactions are involved in most if not all pathogenic and pathophysiological processes and represent attractive therapeutic targets. Extensive biological and clinical research efforts have led to the identification and validation of several cellular hubs that are crucially involved in disease pathogenesis. An interesting example of such a hub is the lens epithelium-derived growth factor (LEDGF/p75), a protein that tethers multiple unrelated proteins and protein complexes to the chromatin. Its chromatin-tethering ability is linked to at least two unrelated diseases-HIV infection and MLL-rearranged acute leukemia. In this review we discuss recent progress in our understanding of the interaction of LEDGF/p75 with its binding partners and focus on the first steps towards therapies targeting protein-protein interactions of LEDGF/p75.

Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured consensus motif.

Lens epithelium-derived growth factor (LEDGF/p75) is an epigenetic reader and attractive therapeutic target involved in HIV integration and the development of mixed lineage leukaemia (MLL1) fusion-driven leukaemia. Besides HIV integrase and the MLL1-menin complex, LEDGF/p75 interacts with various cellular proteins via its integrase binding domain (IBD). Here we present structural characterization of IBD interactions with transcriptional repressor JPO2 and domesticated transposase PogZ, and show that the PogZ interaction is nearly identical to the interaction of LEDGF/p75 with MLL1. The interaction with the IBD is maintained by an intrinsically disordered IBD-binding motif (IBM) common to all known cellular partners of LEDGF/p75. In addition, based on IBM conservation, we identify and validate IWS1 as a novel LEDGF/p75 interaction partner. Our results also reveal how HIV integrase efficiently displaces cellular binding partners from LEDGF/p75. Finally, the similar binding modes of LEDGF/p75 interaction partners represent a new challenge for the development of selective interaction inhibitors.

Discovery of N-aryl-naphthylamines as in vitro inhibitors of the interaction between HIV integrase and the cofactor LEDGF/p75.

A series of N-aryl-naphthylamines, exemplified by the structures 11-16, were chosen for an in-house library screening to assay their ability to disrupt the interaction between the LEDGF cofactor and the HIV integrase. Structure modification led also to design and synthesize new compounds 17a-f. Compounds 11e,h,k,n, 13b, and 14 showed good activity in AlphaScreen assay. The most active compound 11e (IC50 = 2.5 µM) was selected for molecular modeling studies and showed a binding mode similar to the one of the known LEDGIN 8.

HIV-1 IN/Pol recruits LEDGF/p75 into viral particles.

BACKGROUND: The dynamic interaction between HIV and its host governs the replication of the virus and the study of the virus-host interplay is key to understand the viral lifecycle. The host factor lens epithelium-derived growth factor (LEDGF/p75) tethers the HIV preintegration complex to the chromatin through a direct interaction with integrase (IN). Small molecules that bind the LEDGF/p75 binding pocket of the HIV IN dimer (LEDGINs) block HIV replication through a multimodal mechanism impacting early and late stage replication including HIV maturation. Furthermore, LEDGF/p75 has been identified as a Pol interaction partner. This raised the question whether LEDGF/p75 besides acting as a molecular tether in the target cell, also affects late steps of HIV replication. RESULTS: LEDGF/p75 is recruited into HIV-1 particles through direct interaction with the viral IN (or Pol polyprotein) and is a substrate for HIV-1 protease. Incubation in the presence of HIV-1 protease inhibitors resulted in detection of full-length LEDGF/p75 in purified viral particles. We also demonstrate that inhibition of LEDGF/p75-IN interaction by specific mutants or LEDGINs precludes incorporation of LEDGF/p75 in virions, underscoring the specificity of the uptake. LEDGF/p75 depletion did however not result in altered LEDGIN potency. CONCLUSION: Together, these results provide evidence for an IN/Pol mediated uptake of LEDGF/p75 in viral particles and a specific cleavage by HIV protease. Understanding of the possible role of LEDGF/p75 or its cleavage fragments in the viral particle awaits further experimentation.

Host factors for retroviral integration site selection.

To achieve productive infection, retroviruses such as HIV stably integrate their reverse transcribed RNA genome into a host chromosome. Each retroviral family preferentially integrates near a unique subset of genomic features. HIV integrase (IN) is targeted to the body of active transcription units through interaction with lens epithelium-derived growth factor (LEDGF/p75). We describe the successful effort to develop inhibitors of the interaction between IN and LEDGF/p75, referred to as LEDGINs. Gammaretroviruses display a distinct integration pattern. Recently, BET (bromo- and extraterminal domain) proteins were identified as the LEDGF/p75 counterparts that target the integration of gammaretroviruses. The identification of the chromatin-readers LEDGF/p75 and BET as cellular cofactors that orchestrate lentiviral or gammaretroviral integration opens new avenues to developing safer viral vectors for gene therapy.

Design and discovery of 5-hydroxy-6-oxo-1,6-dihydropyrimidine-4-carboxamide inhibitors of HIV-1 integrase.

Raltegravir (RAL) is a first clinically approved integrase (IN) inhibitor for the treatment of HIV but rapid mutation of the virus has led to chemo-resistant strains. Therefore, there is a medical need to develop new IN inhibitors to overcome drug resistance. At present, several IN inhibitors are in different phases of clinical trials and few have been discontinued due to toxicity and lack of efficacy. The development of potent second-generation IN inhibitors with improved safety profiles is key for selecting new clinical candidates. In this article, we report the design and synthesis of potent 5-hydroxy-6-oxo-1,6-dihydropyrimidine-4-carboxamide analogues as second-generation IN inhibitors. These compounds satisfy two structural requirements known for potent inhibition of HIV-1 IN catalysis: a metal chelating moiety and a hydrophobic functionality necessary for selectivity against the strand transfer reaction. Most of the new compounds described herein are potent and selective for the strand transfer reaction and show antiviral activity in cell-based assays. Furthermore, this class of compounds are drug-like and suitable for further optimization and preclinical studies.