RESUMO
The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15-dependent chemotherapeutics. Lead compound TH1760 demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.
Assuntos
Pirofosfatases/antagonistas & inibidores , Pirofosfatases/metabolismo , Sítios de Ligação , Linhagem Celular , Desenho de Fármacos , Desenvolvimento de Medicamentos , Escherichia coli , Humanos , Pirofosfatase Inorgânica/antagonistas & inibidores , Pirofosfatase Inorgânica/genética , Pirofosfatase Inorgânica/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Pirofosfatases/química , Pirofosfatases/genética , Relação Estrutura-AtividadeRESUMO
In zebrafish, cilia movement within the Kupffer's vesicle (KV) generates a fluid flow responsible for accumulating nodal signals exclusively in the left lateral plate mesoderm, thereby initiating left-right patterning (LRP). Defects in LRP cause devastating congenital disorders including congenital heart malformations due to organ mis-positioning. We identified the miR-103/107 family to be involved in regulating LRP. Depletion of miR-103/107 in zebrafish embryos resulted in malpositioned and malformed visceral organs and hearts due to disturbed LRP gene expression, indicating early defects in LRP. Additionally, loss of miR-103/107 affected KV morphogenesis and cilia formation without disturbing endoderm development. Human fibroblasts depleted of miR-103a/107 often failed to extend cilia or developed shorter cilia, indicating functional conservation between species. We identified arl6, araf and foxH1 as direct targets of miR-103/107 providing a mechanistic link to cilia development and nodal signal titration. We describe a new microRNA family controlling KV development and hence influencing establishment of internal organ asymmetry.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Peixe-Zebra/genética , Animais , Padronização Corporal , Linhagem Celular , Cílios/genética , Embrião não Mamífero/anormalidades , Embrião não Mamífero/metabolismo , Coração/embriologia , Humanos , Mesoderma/embriologia , Mesoderma/metabolismo , Peixe-Zebra/embriologiaRESUMO
The release of porcine endogenous retrovirus (PERV) particles from pig cells is a potential risk factor during xenotransplantation by way of productively infecting the human transplant recipient. Potential countermeasures against PERV replication are restriction factors that block retroviral replication. SAMHD1 is a triphosphohydrolase that depletes the cellular pool of dNTPs in non-cycling cells starving retroviral reverse transcription. We investigated the antiviral activity of human SAMHD1 against PERV and found that SAMHD1 potently restricts its reverse transcription in human monocytes, monocyte-derived dendritic cells (MDDC), or macrophages (MDM) and in monocytic THP-1 cells. Degradation of SAMHD1 by SIVmac Vpx or CRISPR/Cas9 knock-out of SAMHD1 allowed for PERV reverse transcription. Addition of deoxynucleosides alleviated the SAMHD1-mediated restriction suggesting that SAMHD1-mediated degradation of dNTPs restricts PERV replication in these human immune cells. In conclusion, our findings highlight SAMHD1 as a potential barrier to PERV transmission from pig transplants to human recipients during xenotransplantation.
Assuntos
Retrovirus Endógenos/fisiologia , Xenoenxertos/metabolismo , Xenoenxertos/virologia , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Animais , Sistemas CRISPR-Cas/fisiologia , Linhagem Celular , Células HEK293 , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Monócitos/metabolismo , Monócitos/virologia , Transcrição Reversa/fisiologia , Suínos , Células THP-1 , Transplante Heterólogo/métodos , Replicação Viral/fisiologiaRESUMO
Human immunodeficiency virus type 1 (HIV-1) infection of dividing and nondividing cells involves regulatory interactions with the nuclear pore complex (NPC), followed by translocation to the nucleus and preferential integration into genomic areas in proximity to the inner nuclear membrane (INM). To identify host proteins that may contribute to these processes, we performed an overexpression screen of known membrane-associated NE proteins. We found that the integral transmembrane proteins SUN1/UNC84A and SUN2/UNC84B are potent or modest inhibitors of HIV-1 infection, respectively, and that suppression corresponds to defects in the accumulation of viral cDNA in the nucleus. While laboratory strains (HIV-1NL4.3 and HIV-1IIIB) are sensitive to SUN1-mediated inhibition, the transmitted founder viruses RHPA and ZM247 are largely resistant. Using chimeric viruses, we identified the HIV-1 capsid (CA) protein as a major determinant of sensitivity to SUN1, and in vitro-assembled capsid-nucleocapsid (CANC) nanotubes captured SUN1 and SUN2 from cell lysates. Finally, we generated SUN1-/- and SUN2-/- cells by using CRISPR/Cas9 and found that the loss of SUN1 had no effect on HIV-1 infectivity, whereas the loss of SUN2 had a modest suppressive effect. Taken together, these observations suggest that SUN1 and SUN2 may function redundantly to modulate postentry, nuclear-associated steps of HIV-1 infection.IMPORTANCE HIV-1 causes more than 1 million deaths per year. The life cycle of HIV-1 has been studied extensively, yet important steps that occur between viral capsid release into the cytoplasm and the expression of viral genes remain elusive. We propose here that the INM components SUN1 and SUN2, two members of the linker of nucleoskeleton and cytoskeleton (LINC) complex, may interact with incoming HIV-1 replication complexes and affect key steps of infection. While overexpression of these proteins reduces HIV-1 infection, disruption of the individual SUN2 and SUN1 genes leads to a mild reduction or no effect on infectivity, respectively. We speculate that SUN1/SUN2 may function redundantly in early HIV-1 infection steps and therefore influence HIV-1 replication and pathogenesis.
Assuntos
Proteínas do Capsídeo/genética , Infecções por HIV/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Sistemas CRISPR-Cas/genética , Linhagem Celular , DNA Viral/genética , Inativação Gênica , Células HEK293 , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/patogenicidade , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Poro Nuclear/metabolismo , Proteínas Nucleares/genéticaRESUMO
UNLABELLED: Type I interferons (IFNs), including IFN-α, upregulate an array of IFN-stimulated genes (ISGs) and potently suppress Human immunodeficiency virus type 1 (HIV-1) infectivity in CD4(+) T cells, monocyte-derived macrophages, and dendritic cells. Recently, we and others identified ISG myxovirus resistance 2 (MX2) as an inhibitor of HIV-1 nuclear entry. However, additional antiviral blocks exist upstream of nuclear import, but the ISGs that suppress infection, e.g., prior to (or during) reverse transcription, remain to be defined. We show here that the HIV-1 CA mutations N74D and A105T, both of which allow escape from inhibition by MX2 and the truncated version of cleavage and polyadenylation specific factor 6 (CPSF6), as well as the cyclophilin A (CypA)-binding loop mutation P90A, all increase sensitivity to IFN-α-mediated inhibition. Using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology, we demonstrate that the IFN-α hypersensitivity of these mutants in THP-1 cells is independent of MX2 or CPSF6. As expected, CypA depletion had no additional effect on the behavior of the P90A mutant but modestly increased the IFN-α sensitivity of wild-type virus. Interestingly, the infectivity of wild-type or P90A virus could be rescued from the MX2-independent IFN-α-induced blocks in THP-1 cells by treatment with cyclosporine (Cs) or its nonimmunosuppressive analogue SDZ-NIM811, indicating that Cs-sensitive host cell cyclophilins other than CypA contribute to the activity of IFN-α-induced blocks. We propose that cellular interactions with incoming HIV-1 capsids help shield the virus from recognition by antiviral effector mechanisms. Thus, the CA protein is a fulcrum for the dynamic interplay between cell-encoded functions that inhibit or promote HIV-1 infection. IMPORTANCE: HIV-1 is the causative agent of AIDS. During acute HIV-1 infection, numerous proinflammatory cytokines are produced, including type I interferons (IFNs). IFNs can limit HIV-1 replication by inducing the expression of a set of antiviral genes that inhibit HIV-1 at multiple steps in its life cycle, including the postentry steps of reverse transcription and nuclear import. This is observed in cultured cell systems, as well as in clinical trials in HIV-1-infected patients. The identities of the cellular antiviral factors, their viral targets, and the underpinning mechanisms are largely unknown. We show here that the HIV-1 Capsid protein plays a central role in protecting the virus from IFN-induced inhibitors that block early postentry steps of infection. We further show that host cell cyclophilins play an important role in regulating these processes, thus highlighting the complex interplay between antiviral effector mechanisms and viral survival.
Assuntos
Antivirais/metabolismo , Proteína do Núcleo p24 do HIV/metabolismo , HIV-1/imunologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Fatores Imunológicos/metabolismo , Linhagem Celular , Proteína do Núcleo p24 do HIV/genética , Humanos , Imunidade Inata , Interferon-alfa/imunologia , Monócitos/virologia , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismoRESUMO
The deoxycytidine analogue cytarabine (ara-C) remains the backbone treatment of acute myeloid leukaemia (AML) as well as other haematological and lymphoid malignancies, but must be combined with other chemotherapeutics to achieve cure. Yet, the underlying mechanism dictating synergistic efficacy of combination chemotherapy remains largely unknown. The dNTPase SAMHD1, which regulates dNTP homoeostasis antagonistically to ribonucleotide reductase (RNR), limits ara-C efficacy by hydrolysing the active triphosphate metabolite ara-CTP. Here, we report that clinically used inhibitors of RNR, such as gemcitabine and hydroxyurea, overcome the SAMHD1-mediated barrier to ara-C efficacy in primary blasts and mouse models of AML, displaying SAMHD1-dependent synergy with ara-C. We present evidence that this is mediated by dNTP pool imbalances leading to allosteric reduction of SAMHD1 ara-CTPase activity. Thus, SAMHD1 constitutes a novel biomarker for combination therapies of ara-C and RNR inhibitors with immediate consequences for clinical practice to improve treatment of AML.
Assuntos
Citarabina/farmacologia , Leucemia Mieloide Aguda , Pirofosfatases/metabolismo , Ribonucleotídeo Redutases/antagonistas & inibidores , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Animais , Arabinofuranosilcitosina Trifosfato/metabolismo , CamundongosRESUMO
SAMHD1 is a human restriction factor known to prevent infection of macrophages, resting CD4+ T cells, and dendritic cells by HIV-1. To test the contribution of MxB to the ability of SAMHD1 to block HIV-1 infection, we created human THP-1 cell lines that were knocked out for expression of MxB, SAMHD1, or both. Interestingly, MxB depletion renders SAMHD1 ineffective against HIV-1 but not SIVmac. We observed similar results in human primary macrophages that were knockdown for the expression of MxB. To understand how MxB assists SAMHD1 restriction of HIV-1, we examined direct interaction between SAMHD1 and MxB in pull-down experiments. In addition, we investigated several properties of SAMHD1 in the absence of MxB expression, including subcellular localization, phosphorylation of the SAMHD1 residue T592, and dNTPs levels. These experiments showed that SAMHD1 restriction of HIV-1 requires expression of MxB.
Assuntos
Infecções por HIV/metabolismo , HIV-1/fisiologia , Proteínas de Resistência a Myxovirus/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Vírus da Imunodeficiência Símia/fisiologia , Motivos de Aminoácidos , Infecções por HIV/genética , Infecções por HIV/virologia , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas de Resistência a Myxovirus/genética , Fosforilação , Ligação Proteica , Proteína 1 com Domínio SAM e Domínio HD/química , Proteína 1 com Domínio SAM e Domínio HD/genética , Especificidade da EspécieRESUMO
Sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1) is a (deoxy)guanosine triphosphate (dGTP/GTP)-activated deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase involved in cellular dNTP homoeostasis. Mutations in SAMHD1 have been associated with the hyperinflammatory disease Aicardi-Goutières syndrome (AGS). SAMHD1 also limits cells' permissiveness to infection with diverse viruses, including human immunodeficiency virus (HIV-1), and controls endogenous retroviruses. Increasing evidence supports the role of SAMHD1 as a tumor suppressor. However, SAMHD1 also can act as a resistance factor to nucleoside-based chemotherapies by hydrolyzing their active triphosphate metabolites, thereby reducing response of various malignancies to these anticancer drugs. Hence, informed cancer therapies must take into account the ambiguous properties of SAMHD1 as both an inhibitor of uncontrolled proliferation and a resistance factor limiting the efficacy of anticancer treatments. Here, we provide evidence that SAMHD1 is a double-edged sword for patients with acute myelogenous leukemia (AML). Our time-dependent analyses of The Cancer Genome Atlas (TCGA) AML cohort indicate that high expression of SAMHD1, even though it critically limits the efficacy of high-dose ara-C therapy, might be associated with more favorable disease progression.
Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , Resistência a Medicamentos/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Malformações do Sistema Nervoso/genética , Proteínas Supressoras de Tumor/genética , Doença Aguda , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Doenças Autoimunes do Sistema Nervoso/metabolismo , Azacitidina/análogos & derivados , Azacitidina/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Citarabina/uso terapêutico , Decitabina , Humanos , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Malformações do Sistema Nervoso/metabolismo , Proteína 1 com Domínio SAM e Domínio HD , Análise de Sobrevida , Resultado do Tratamento , Proteínas Supressoras de Tumor/metabolismoRESUMO
Recently, we demonstrated that sterile α motif and HD domain containing protein 1 (SAMHD1) is a major barrier in acute myelogenous leukemia (AML) cells to the cytotoxicity of cytarabine (ara-C), the most important drug in AML treatment. Ara-C is intracellularly converted by the canonical dNTP synthesis pathway to ara-CTP, which serves as a substrate but not an allosteric activator of SAMHD1. Using an AML mouse model, we show here that wild type but not catalytically inactive SAMHD1 reduces ara-C treatment efficacy in vivo. Expanding the clinically relevant substrates of SAMHD1, we demonstrate that THP-1 CRISPR/Cas9 cells lacking a functional SAMHD1 gene showed increased sensitivity to the antimetabolites nelarabine, fludarabine, decitabine, vidarabine, clofarabine, and trifluridine. Within this Extra View, we discuss and build upon both these and our previously reported findings, and propose SAMHD1 is likely active against a variety of nucleoside analog antimetabolites present in anti-cancer chemotherapies. Thus, SAMHD1 may constitute a promising target to improve a wide range of therapies for both hematological and non-haematological malignancies.
Assuntos
Antimetabólitos/efeitos adversos , Neoplasias/tratamento farmacológico , Nucleosídeos/efeitos adversos , Substâncias Protetoras/farmacologia , Substâncias Protetoras/uso terapêutico , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Modelos Biológicos , Neoplasias/patologia , Nucleosídeos/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/genéticaRESUMO
The cytostatic deoxycytidine analog cytarabine (ara-C) is the most active agent available against acute myelogenous leukemia (AML). Together with anthracyclines, ara-C forms the backbone of AML treatment for children and adults. In AML, both the cytotoxicity of ara-C in vitro and the clinical response to ara-C therapy are correlated with the ability of AML blasts to accumulate the active metabolite ara-C triphosphate (ara-CTP), which causes DNA damage through perturbation of DNA synthesis. Differences in expression levels of known transporters or metabolic enzymes relevant to ara-C only partially account for patient-specific differential ara-CTP accumulation in AML blasts and response to ara-C treatment. Here we demonstrate that the deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) promotes the detoxification of intracellular ara-CTP pools. Recombinant SAMHD1 exhibited ara-CTPase activity in vitro, and cells in which SAMHD1 expression was transiently reduced by treatment with the simian immunodeficiency virus (SIV) protein Vpx were dramatically more sensitive to ara-C-induced cytotoxicity. CRISPR-Cas9-mediated disruption of the gene encoding SAMHD1 sensitized cells to ara-C, and this sensitivity could be abrogated by ectopic expression of wild-type (WT), but not dNTPase-deficient, SAMHD1. Mouse models of AML lacking SAMHD1 were hypersensitive to ara-C, and treatment ex vivo with Vpx sensitized primary patient-derived AML blasts to ara-C. Finally, we identified SAMHD1 as a risk factor in cohorts of both pediatric and adult patients with de novo AML who received ara-C treatment. Thus, SAMHD1 expression levels dictate patient sensitivity to ara-C, providing proof-of-concept that the targeting of SAMHD1 by Vpx could be an attractive therapeutic strategy for potentiating ara-C efficacy in hematological malignancies.