A highly potent and safe pyrrolopyridine-based allosteric HIV-1 integrase inhibitor targeting host LEDGF/p75-integrase interaction site.

Allosteric integrase inhibitors (ALLINIs) are a class of experimental anti-HIV agents that target the noncatalytic sites of the viral integrase (IN) and interfere with the IN-viral RNA interaction during viral maturation. Here, we report a highly potent and safe pyrrolopyridine-based ALLINI, STP0404, displaying picomolar IC50 in human PBMCs with a >24,000 therapeutic index against HIV-1. X-ray structural and biochemical analyses revealed that STP0404 binds to the host LEDGF/p75 protein binding pocket of the IN dimer, which induces aberrant IN oligomerization and blocks the IN-RNA interaction. Consequently, STP0404 inhibits proper localization of HIV-1 RNA genomes in viral particles during viral maturation. Y99H and A128T mutations at the LEDGF/p75 binding pocket render resistance to STP0404. Extensive in vivo pharmacological and toxicity investigations demonstrate that STP0404 harbors outstanding therapeutic and safety properties. Overall, STP0404 is a potent and first-in-class ALLINI that targets LEDGF/p75 binding site and has advanced to a human trial.

Mechanistic cross-talk between DNA/RNA polymerase enzyme kinetics and nucleotide substrate availability in cells: Implications for polymerase inhibitor discovery.

Enzyme kinetic analysis reveals a dynamic relationship between enzymes and their substrates. Overall enzyme activity can be controlled by both protein expression and various cellular regulatory systems. Interestingly, the availability and concentrations of intracellular substrates can constantly change, depending on conditions and cell types. Here, we review previously reported enzyme kinetic parameters of cellular and viral DNA and RNA polymerases with respect to cellular levels of their nucleotide substrates. This broad perspective exposes a remarkable co-evolution scenario of DNA polymerase enzyme kinetics with dNTP levels that can vastly change, depending on cell proliferation profiles. Similarly, RNA polymerases display much higher Km values than DNA polymerases, possibly due to millimolar range rNTP concentrations found in cells (compared with micromolar range dNTP levels). Polymerases are commonly targeted by nucleotide analog inhibitors for the treatments of various human diseases, such as cancers and viral pathogens. Because these inhibitors compete against natural cellular nucleotides, the efficacy of each inhibitor can be affected by varying cellular nucleotide levels in their target cells. Overall, both kinetic discrepancy between DNA and RNA polymerases and cellular concentration discrepancy between dNTPs and rNTPs present pharmacological and mechanistic considerations for therapeutic discovery.

The dNTPase activity of SAMHD1 is important for its suppression of innate immune responses in differentiated monocytic cells.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase (dNTPase) with a nuclear localization signal (NLS). SAMHD1 suppresses innate immune responses to viral infection and inflammatory stimuli by inhibiting the NF-κB and type I interferon (IFN-I) pathways. However, whether the dNTPase activity and nuclear localization of SAMHD1 are required for its suppression of innate immunity remains unknown. Here, we report that the dNTPase activity, but not nuclear localization of SAMHD1, is important for its suppression of innate immune responses in differentiated monocytic cells. We generated monocytic U937 cell lines stably expressing WT SAMHD1 or mutated variants defective in dNTPase activity (HD/RN) or nuclear localization (mNLS). WT SAMHD1 in differentiated U937 cells significantly inhibited lipopolysaccharide-induced expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) mRNAs, as well as IFN-α, IFN-ß, and TNF-α mRNA levels induced by Sendai virus infection. In contrast, the HD/RN mutant did not exhibit this inhibition in either U937 or THP-1 cells, indicating that the dNTPase activity of SAMHD1 is important for suppressing NF-κB activation. Of note, in lipopolysaccharide-treated or Sendai virus-infected U937 or THP-1 cells, the mNLS variant reduced TNF-α or IFN-ß mRNA expression to a similar extent as did WT SAMHD1, suggesting that SAMHD1-mediated inhibition of innate immune responses is independent of SAMHD1's nuclear localization. Moreover, WT and mutant SAMHD1 similarly interacted with key proteins in NF-κB and IFN-I pathways in cells. This study further defines the role and mechanisms of SAMHD1 in suppressing innate immunity.

Host SAMHD1 protein restricts endogenous reverse transcription of HIV-1 in nondividing macrophages.

BACKGROUND: SAM domain and HD domain containing protein 1 (SAMHD1) is a host anti-HIV-1 restriction factor known to suppress viral reverse transcription in nondividing myeloid cells by its dNTP triphosphorylase activity that depletes cellular dNTPs. However, HIV-2 and some SIV strains rapidly replicate in macrophages due to their accessory protein, viral protein X (Vpx), which proteosomally degrades SAMHD1 and elevates dNTP levels. Endogenous reverse transcription (ERT) of retroviruses is the extra-cellular reverse transcription step that partially synthesizes proviral DNAs within cell-free viral particles before the viruses infect new cells. ERT activity utilizes dNTPs co-packaged during budding from the virus-producing cells, and high ERT activity is known to enhance HIV-1 infectivity in nondividing cells. Here, since Vpx elevates cellular dNTP levels in macrophages, we hypothesize that HIV-2 should contain higher ERT activity than HIV-1 in macrophages, and that the Vpx-mediated dNTP elevation should enhance both ERT activity and infectivity of HIV-1 particles produced in macrophages. RESULTS: Here, we demonstrate that HIV-2 produced from human primary monocyte derived macrophages displays higher ERT activity than HIV-1 produced from macrophages. Also, HIV-1 particles produced from macrophages treated with virus like particles (VLPs) containing Vpx, Vpx (+), displayed large increases of ERT activity with the enhanced copy numbers of early, middle and late reverse transcription products within the viral particles, compared to the viruses produced from macrophages treated with Vpx (-) VLPs. Furthermore, upon the infection with an equal p24 amount to fresh macrophages, the viruses produced from the Vpx (+) VLP treated macrophages demonstrated higher infectivity than the viruses from the Vpx (-) VLP treated macrophages. CONCLUSIONS: This finding identifies the viral ERT step as an additional step of HIV-1 replication cycle that SAMHD1 restricts in nondividing myeloid target cells.

SAMHD1 expression contributes to doxorubicin resistance and predicts survival outcomes in diffuse large B-cell lymphoma patients.

Diffuse large B-cell lymphoma (DLBCL) is a commonly diagnosed, aggressive non-Hodgkin's lymphoma. While R-CHOP chemoimmunotherapy is potentially curative, about 40% of DLBCL patients will fail, highlighting the need to identify biomarkers to optimize management. SAMHD1 has a dNTPase-independent role in promoting resection to facilitate DNA double-strand break (DSB) repair by homologous recombination. We evaluated the relationship of SAMHD1 levels with sensitivity to DSB-sensitizing agents in DLBCL cells and the association of SAMHD1 expression with clinical outcomes in 79 DLBCL patients treated with definitive therapy and an independent cohort dataset of 234 DLBCL patients. Low SAMHD1 expression, Vpx-mediated, or siRNA-mediated degradation/depletion in DLBCL cells was associated with greater sensitivity to doxorubicin and PARP inhibitors. On Kaplan-Meier log-rank survival analysis, low SAMHD1 expression was associated with improved overall survival (OS), which on subset analysis remained significant only in patients with advanced stage (III-IV) and moderate to high risk (2-5 International Prognostic Index (IPI)). The association of low SAMHD1 expression with improved OS remained significant on multivariate analysis independent of other adverse factors, including IPI, and was validated in an independent cohort. Our findings suggest that SAMHD1 expression mediates doxorubicin resistance and may be an important prognostic biomarker in advanced, higher-risk DLBCL patients.

SUMOylation of SAMHD1 at Lysine 595 is required for HIV-1 restriction in non-cycling cells.

SAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic T592E mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells.

SAMHD1 Functions and Human Diseases.

Deoxynucleoside triphosphate (dNTP) molecules are essential for the replication and maintenance of genomic information in both cells and a variety of viral pathogens. While the process of dNTP biosynthesis by cellular enzymes, such as ribonucleotide reductase (RNR) and thymidine kinase (TK), has been extensively investigated, a negative regulatory mechanism of dNTP pools was recently found to involve sterile alpha motif (SAM) domain and histidine-aspartate (HD) domain-containing protein 1, SAMHD1. When active, dNTP triphosphohydrolase activity of SAMHD1 degrades dNTPs into their 2'-deoxynucleoside (dN) and triphosphate subparts, steadily depleting intercellular dNTP pools. The differential expression levels and activation states of SAMHD1 in various cell types contributes to unique dNTP pools that either aid (i.e., dividing T cells) or restrict (i.e., nondividing macrophages) viral replication that consumes cellular dNTPs. Genetic mutations in SAMHD1 induce a rare inflammatory encephalopathy called Aicardi-Goutières syndrome (AGS), which phenotypically resembles viral infection. Recent publications have identified diverse roles for SAMHD1 in double-stranded break repair, genome stability, and the replication stress response through interferon signaling. Finally, a series of SAMHD1 mutations were also reported in various cancer cell types while why SAMHD1 is mutated in these cancer cells remains to investigated. Here, we reviewed a series of studies that have begun illuminating the highly diverse roles of SAMHD1 in virology, immunology, and cancer biology.

Simultaneous targeting of DNA replication and homologous recombination in glioblastoma with a polyether ionophore.

BACKGROUND: Despite significant endeavor having been applied to identify effective therapies to treat glioblastoma (GBM), survival outcomes remain intractable. The greatest nonsurgical benefit arises from radiotherapy, though tumors typically recur due to robust DNA repair. Patients could therefore benefit from therapies with the potential to prevent DNA repair and synergize with radiotherapy. In this work, we investigated the potential of salinomycin to enhance radiotherapy and further uncover novel dual functions of this ionophore to induce DNA damage and prevent repair. METHODS: In vitro primary GBM models and ex vivo GBM patient explants were used to determine the mechanism of action of salinomycin by immunoblot, flow cytometry, immunofluorescence, immunohistochemistry, and mass spectrometry. In vivo efficacy studies were performed using orthotopic GBM animal xenograft models. Salinomycin derivatives were synthesized to increase drug efficacy and explore structure-activity relationships. RESULTS: Here we report novel dual functions of salinomycin. Salinomycin induces toxic DNA lesions and prevents subsequent recovery by targeting homologous recombination (HR) repair. Salinomycin appears to target the more radioresistant GBM stem cell-like population and synergizes with radiotherapy to significantly delay tumor formation in vivo. We further developed salinomycin derivatives which display greater efficacy in vivo while retaining the same beneficial mechanisms of action. CONCLUSION: Our findings highlight the potential of salinomycin to induce DNA lesions and inhibit HR to greatly enhance the effect of radiotherapy. Importantly, first-generation salinomycin derivatives display greater efficacy and may pave the way for clinical testing of these agents.

Incomplete Suppression of HIV-1 by SAMHD1 Permits Efficient Macrophage Infection.

BACKGROUND: Sterile alpha motif and histidine/aspartic acid domain-containing protein (SAMHD1) is a dNTP triphosphorylase that reduces cellular dNTP levels in non-dividing cells, such as macrophages. Since dNTPs are required for reverse transcription, HIV-2 and most SIVs encode a Vpx protein that promotes proteasomal degradation of SAMHD1. It is unclear how HIV-1, which does not appear to harbor a SAMHD1 escape mechanism, is able to infect macrophages in the face of SAMHD1 restriction. METHODS: To assess whether HIV-1 had a mechanism to negate SAMHD1 activity, we compared SAMHD1 and dNTP levels in macrophages infected by HIV-1 and SIV. We examined whether macrophages infected by HIV-1 still harbored antiviral levels of SAMHD1 by assessing their susceptibility to superinfection by vpx-deleted SIV. Finally, to assess whether HIV-1 reverse transcriptase (RT) has adapted to a low dNTP environment, we evaluated SAMHD1 sensitivity of chimeric HIV-1 and SIV variants in which the RT regions were functionally exchanged. RESULTS: Here, we demonstrate that HIV-1 efficiently infects macrophages without modulating SAMHD1 activity or cellular dNTP levels, and that macrophages permissive to HIV-1 infection remained refractory to superinfection by vpx-deleted SIV. Furthermore, through the use of chimeric HIV/SIV, we demonstrate that the differential sensitivity of HIV-1 and SIV to SAMHD1 restriction is not dictated by RT. CONCLUSIONS: Our study reveals fundamental differences between HIV-1 and SIV in the strategy used to evade restriction by SAMHD1 and suggests a degree of resistance of HIV-1 to the antiviral environment created by SAMHD1. Understanding how these cellular restrictions antagonize viral replication will be important for the design of novel antiviral strategies.

SAMHD1 is recurrently mutated in T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is an aggressive malignancy with a median survival of the patients of less than two years. Besides characteristic chromosomal translocations, frequent mutations affect the ATM gene, JAK/STAT pathway members, and epigenetic regulators. We here performed a targeted mutation analysis for 40 genes selected from a RNA sequencing of 10 T-PLL in a collection of 28 T-PLL, and an exome analysis of five further cases. Nonsynonymous mutations were identified in 30 of the 40 genes, 18 being recurrently mutated. We identified recurrently mutated genes previously unknown to be mutated in T-PLL, which are SAMHD1, HERC1, HERC2, PRDM2, PARP10, PTPRC, and FOXP1. SAMHD1 regulates cellular deoxynucleotide levels and acts as a potential tumor suppressor in other leukemias. We observed destructive mutations in 18% of cases as well as deletions in two further cases. Taken together, we identified additional genes involved in JAK/STAT signaling (PTPRC), epigenetic regulation (PRDM2), or DNA damage repair (SAMHD1, PARP10, HERC1, and HERC2) as being recurrently mutated in T-PLL. Thus, our study considerably extends the picture of pathways involved in molecular pathogenesis of T-PLL and identifies the tumor suppressor gene SAMHD1 with ~20% of T-PLL affected by destructive lesions likely as major player in T-PLL pathogenesis.

Dephosphorylation of the HIV-1 restriction factor SAMHD1 is mediated by PP2A-B55α holoenzymes during mitotic exit.

SAMHD1 is a critical restriction factor for HIV-1 in non-cycling cells and its antiviral activity is regulated by T592 phosphorylation. Here, we show that SAMHD1 dephosphorylation at T592 is controlled during the cell cycle, occurring during M/G1 transition in proliferating cells. Using several complementary proteomics and biochemical approaches, we identify the phosphatase PP2A-B55α responsible for rendering SAMHD1 antivirally active. SAMHD1 is specifically targeted by PP2A-B55α holoenzymes during mitotic exit, in line with observations that PP2A-B55α is a key mitotic exit phosphatase in mammalian cells. Strikingly, as HeLa or activated primary CD4+ T cells enter the G1 phase, pronounced reduction of RT products is observed upon HIV-1 infection dependent on the presence of dephosphorylated SAMHD1. Moreover, PP2A controls SAMHD1 pT592 level in non-cycling monocyte-derived macrophages (MDMs). Thus, the PP2A-B55α holoenzyme is a key regulator to switch on the antiviral activity of SAMHD1.