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1.
Crit Rev Biochem Mol Biol ; 54(1): 41-60, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30773935

RESUMO

Dysregulation of isoprenoid biosynthesis is implicated in numerous biochemical disorders that play a role in the onset and/or progression of age-related diseases, such as hypercholesterolemia, osteoporosis, various cancers, and neurodegeneration. The mevalonate metabolic pathway is responsible for the biosynthesis of the two key isoprenoid metabolites, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Post-translational prenylation of various proteins, including the small GTP-binding proteins (GTPases), with either FPP or GGPP is vital for proper localization and activation of these proteins. Prenylated GTPases play a critical role in cell signaling, proliferation, cellular plasticity, oncogenesis, and cancer metastasis. Pre-clinical and clinical studies strongly suggest that inhibition of protein prenylation can be an effective treatment for non-skeletal cancers. In this review, we summarize the most recent drug discovery efforts focusing on blocking protein farnesylation and/or geranylgeranylation and the biochemical and structural data available in guiding the current on-going studies in drug discovery. Furthermore, we provide a summary on the biochemical association between disruption of protein prenylation, endoplasmic reticulum (ER) stress, unfolded protein response (UPR) signaling, and cancer.


Assuntos
Vias Biossintéticas/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase/antagonistas & inibidores , Geraniltranstransferase/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Descoberta de Drogas , Inibidores Enzimáticos/uso terapêutico , Farnesiltranstransferase/metabolismo , Geraniltranstransferase/metabolismo , Humanos , Ácido Mevalônico/metabolismo , Modelos Moleculares , Neoplasias/metabolismo , Fosfatos de Poli-Isoprenil/antagonistas & inibidores , Fosfatos de Poli-Isoprenil/metabolismo , Prenilação de Proteína/efeitos dos fármacos , Sesquiterpenos/antagonistas & inibidores , Sesquiterpenos/metabolismo
2.
Chembiochem ; 17(9): 843-51, 2016 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-26792008

RESUMO

The unfolded protein response (UPR) initiated by the transmembrane kinase/ribonuclease Ire1 has been implicated in a variety of diseases. Ire1, with its unique position in the UPR, is an ideal target for the development of therapies; however, the identification of specific kinase inhibitors is challenging. Recently, the development of covalent inhibitors has gained great momentum because of the irreversible deactivation of the target. We identified and determined the mechanism of action of the Ire1-inhibitory compound UPRM8. MS analysis revealed that UPRM8 inhibition occurs by covalent adduct formation at a conserved cysteine at the regulatory DFG+2 position in the Ire1 kinase activation loop. Mutational analysis of the target cysteine residue identified both UPRM8-resistant and catalytically inactive Ire1 mutants. We describe a novel covalent inhibition mechanism of UPRM8, which can serve as a lead for the rational design and optimization of inhibitors of human Ire1.


Assuntos
Cisteína/metabolismo , Endorribonucleases/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Pirimidinonas/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Biocatálise , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/química , Endorribonucleases/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinonas/química , Pirimidinonas/farmacologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Resposta a Proteínas não Dobradas/efeitos dos fármacos
3.
J Med Chem ; 66(23): 15776-15800, 2023 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-37982711

RESUMO

Novel C6-substituted pyrazolo[3,4-d]pyrimidine- and C2-substituted purine-based bisphosphonate (C6-PyraP-BP and C2-Pur-BP, respectively) inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS) were designed and evaluated for their ability to block the proliferation of multiple myeloma (MM), pancreatic ductal adenocarcinoma (PDAC), and colorectal cancer (CRC) cells. Pyrazolo[3,4-d]pyrimidine analogs were identified that induce selective intracellular target engagement leading to apoptosis and downregulate the prenylation of Rap-1A in MM, PDAC, and CRC cells. The C6-PyraP-BP inhibitor RB-07-16 was found to exhibit antitumor efficacy in xenograft mouse models of MM and PDAC, significantly reducing tumor growth without substantially increasing liver enzymes or causing significant histopathologic damage, usually associated with hepatotoxicity. RB-07-16 is a metabolically stable compound in cross-species liver microsomes, does not inhibit key CYP 450 enzymes, and exhibits good systemic circulation in rat. Collectively, the current studies provide encouraging support for further optimization of the pyrazolo[3,4-d]pyrimidine-based GGPPS inhibitors as potential human therapeutics for various cancers.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Colorretais , Mieloma Múltiplo , Neoplasias Pancreáticas , Humanos , Camundongos , Ratos , Animais , Geranil-Geranildifosfato Geranil-Geraniltransferase , Difosfonatos/farmacologia , Difosfonatos/uso terapêutico , Neoplasias Pancreáticas/patologia , Apoptose , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Ensaios Antitumorais Modelo de Xenoenxerto
4.
J Med Chem ; 65(3): 2471-2496, 2022 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-35077178

RESUMO

Novel analogues of C-2-substituted thienopyrimidine-based bisphosphonates (C2-ThP-BPs) are described that are potent inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS). Members of this class of compounds induce target-selective apoptosis of multiple myeloma (MM) cells and exhibit antimyeloma activity in vivo. A key structural element of these inhibitors is a linker moiety that connects their (((2-phenylthieno[2,3-d]pyrimidin-4-yl)amino)methylene)bisphosphonic acid core to various side chains. The structural diversity of this linker moiety, as well as the side chains attached to it, was investigated and found to significantly impact the toxicity of these compounds in MM cells. The most potent inhibitor identified was evaluated in mouse and rat for liver toxicity and systemic exposure, respectively, providing further optimism for the potential value of such compounds as human therapeutics.


Assuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Geranil-Geranildifosfato Geranil-Geraniltransferase/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Pirimidinas/uso terapêutico , Tiofenos/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Células da Medula Óssea/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/toxicidade , Feminino , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/metabolismo , Geranil-Geranildifosfato Geranil-Geraniltransferase/metabolismo , Humanos , Fígado/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Estrutura Molecular , Ligação Proteica , Pirimidinas/síntese química , Pirimidinas/metabolismo , Pirimidinas/toxicidade , Ratos , Saccharomyces cerevisiae/enzimologia , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/metabolismo , Tiofenos/toxicidade
5.
Front Immunol ; 13: 1074740, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36601119

RESUMO

Access to commercial CD19 CAR-T cells remains limited even in wealthy countries like Canada due to clinical, logistical, and financial barriers related to centrally manufactured products. We created a non-commercial academic platform for end-to-end manufacturing of CAR-T cells within Canada's publicly funded healthcare system. We report initial results from a single-arm, open-label study to determine the safety and efficacy of in-house manufactured CD19 CAR-T cells (entitled CLIC-1901) in participants with relapsed/refractory CD19 positive hematologic malignancies. Using a GMP compliant semi-automated, closed process on the Miltenyi Prodigy, T cells were transduced with lentiviral vector bearing a 4-1BB anti-CD19 CAR transgene and expanded. Participants underwent lymphodepletion with fludarabine and cyclophosphamide, followed by infusion of non-cryopreserved CAR-T cells. Thirty participants with non-Hodgkin's lymphoma (n=25) or acute lymphoblastic leukemia (n=5) were infused with CLIC-1901: 21 males (70%), median age 66 (range 18-75). Time from enrollment to CLIC-1901 infusion was a median of 20 days (range 15-48). The median CLIC-1901 dose infused was 2.3 × 106 CAR-T cells/kg (range 0.13-3.6 × 106/kg). Toxicity included ≥ grade 3 cytokine release syndrome (n=2) and neurotoxicity (n=1). Median follow-up was 6.5 months. Overall response rate at day 28 was 76.7%. Median progression-free and overall survival was 6 months (95%CI 3-not estimable) and 11 months (95% 6.6-not estimable), respectively. This is the first trial of in-house manufactured CAR-T cells in Canada and demonstrates that administering fresh CLIC-1901 product is fast, safe, and efficacious. Our experience may provide helpful guidance for other jurisdictions seeking to create feasible and sustainable CAR-T cell programs in research-oriented yet resource-constrained settings. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT03765177, identifier NCT03765177.


Assuntos
Neoplasias Hematológicas , Linfoma não Hodgkin , Masculino , Humanos , Idoso , Linfócitos T , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Ciclofosfamida , Neoplasias Hematológicas/terapia , Recidiva , Antígenos CD19
6.
Eukaryot Cell ; 7(9): 1441-9, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18621925

RESUMO

Small GTPases of the Rho family act as molecular switches, and modulation of the GTP-bound state of Rho proteins is a well-characterized means of regulating their signaling activity in vivo. In contrast, the regulation of Rho-type GTPases by posttranslational modifications is poorly understood. Here, we present evidence of the control of the Saccharomyces cerevisiae Rho-type GTPase Rho5p by phosphorylation and ubiquitination. Rho5p binds to Ste50p, and the expression of the activated RHO5(Q91H) allele in an Deltaste50 strain is lethal under conditions of osmotic stress. An overexpression screen identified RGD2 and MSI1 as being high-copy suppressors of the osmotic sensitivity of this lethality. Rgd2p had been identified as being a possible Rho5p GTPase-activating protein based on an in vitro assay; this result supports its function as a regulator of Rho5p activity in vivo. MSI1 was previously identified as being a suppressor of hyperactive Ras/cyclic AMP signaling, where it antagonizes Npr1p kinase activity and promotes ubiquitination. Here, we show that Msi1p also acts via Npr1p to suppress activated Rho5p signaling. Rho5p is ubiquitinated, and its expression is lethal in a strain that is compromised for proteasome activity. These data identify Rho5p as being a target of Msi1p/Npr1p regulation and describe a regulatory circuit involving phosphorylation and ubiquitination.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Fator 1 de Modelagem da Cromatina , Proteínas de Ligação a DNA/genética , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Pressão Osmótica , Fosforilação , Ligação Proteica , Proteínas Quinases/genética , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Supressão Genética , Ubiquitinação , Proteínas rho de Ligação ao GTP/genética
7.
Sci Rep ; 9(1): 1265, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718665

RESUMO

Translation is a highly regulated process that is perturbed in human cancers, often through activation of the PI3K/mTOR pathway which impacts directly on the ribosome recruitment phase of translation initiation. While significant research has focused on "drugging" components of the PI3K/mTOR network, efforts have also been directed towards inhibiting eukaryotic initiation factor (eIF) 4F-dependent translation. Small molecule inhibitors of this complex have been identified, characterized, and used to validate the rationale of targeting this step to curtail tumor cell growth and modulate chemotherapy response. One such class of compounds are the rocaglates, secondary metabolites from the plant genus Aglaia, which target the RNA helicase subunit of eIF4F, eIF4A. Here we explore the ability of synthetic derivatives of aglaiastatins and an aglaroxin derivative to target the translation process in vitro and in vivo and find the synthetic derivative oxo-aglaiastatin to possess such activity. Oxo-aglaiastatin inhibited translation in vitro and in vivo and synergized with doxorubicin, ABT-199 (a Bcl-2 antagonist), and dexamethasone when tested on hematological cancer cells. The biological activity of oxo-aglaiastatin was shown to be a consequence of inhibiting eIF4A1 activity.


Assuntos
Aglaia , Antineoplásicos Fitogênicos/farmacologia , Neoplasias/tratamento farmacológico , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Aglaia/química , Animais , Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Fator de Iniciação 4A em Eucariotos/metabolismo , Feminino , Humanos , Linfoma/tratamento farmacológico , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Sulfonamidas/farmacologia
8.
Cancer Res ; 79(18): 4679-4688, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31337650

RESUMO

Overexpression of PRL-3, an oncogenic phosphatase, was identified as a novel cluster in patients with newly diagnosed multiple myeloma. However, the regulation and oncogenic activities of PRL-3 in multiple myeloma warrant further investigation. Here, we report that IL6 activates STAT3, which acts as a direct transcriptional regulator of PRL-3. Upregulation of PRL-3 increased myeloma cell viability and rephosphorylated STAT3 in a biphasic manner through direct interaction and deactivation of SHP2, thus blocking the gp130 (Y759)-mediated repression of STAT3 activity. Abrogation of PRL-3 reduced myeloma cell survival, clonogenicity, and tumorigenesis, and detailed mechanistic studies revealed "deactivation" of effector proteins such as Akt, Erk1/2, Src, STAT1, and STAT3. Furthermore, loss of PRL-3 efficiently abolished nuclear localization of STAT3 and reduced its occupancy on the promoter of target genes c-Myc and Mcl-1, and antiapoptotic genes Bcl2 and Bcl-xL. PRL-3 also played a role in the acquired resistance of myeloma cells to bortezomib, which could be overcome by PRL-3 silencing. Of clinical relevance, STAT3 and PRL-3 expression was positively correlated in five independent cohorts, and the STAT3 activation signature was significantly enriched in patients with high PRL-3 expression. Furthermore, PRL-3 could be used as a biomarker to identify high-risk patients with multiple myeloma that exhibited poor prognosis and inferior outcome even when treated with novel combinational therapeutics (proteasome inhibitors and immunomodulatory imide drugs). Conclusively, our results support a feedforward mechanism between STAT3 and PRL-3 that prolongs prosurvival signaling in multiple myeloma, and suggest targeting PRL-3 as a valid therapeutic opportunity in multiple myeloma. SIGNIFICANCE: IL6 promotes STAT3-dependent transcriptional upregulation of PRL-3, which in turn re-phosphorylates STAT3 and aberrantly activates STAT3 target genes, leading to bortezomib resistance in multiple myeloma.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interleucina-6/farmacologia , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Bortezomib/farmacologia , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/genética , Fosforilação , Prognóstico , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Tirosina Fosfatases/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Med Chem ; 61(15): 6904-6917, 2018 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-30016091

RESUMO

Post-translational prenylation of the small GTP-binding proteins (GTPases) is vital to a plethora of biological processes, including cellular proliferation. We have identified a new class of thienopyrimidine-based bisphosphonate (ThP-BP) inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS) that block protein prenylation in multiple myeloma (MM) cells leading to cellular apoptosis. These inhibitors are also effective in blocking the proliferation of other types of cancer cells. We confirmed intracellular target engagement, demonstrated the mechanism of action leading to apoptosis, and determined a direct correlation between apoptosis and intracellular inhibition of hGGPPS. Administration of a ThP-BP inhibitor to a MM mouse model confirmed in vivo downregulation of Rap1A geranylgeranylation and reduction of monoclonal immunoglobulins (M-protein, a biomarker of disease burden) in the serum. These results provide the first proof-of-principle that hGGPPS is a valuable therapeutic target in oncology and more specifically for the treatment of multiple myeloma.


Assuntos
Inibidores Enzimáticos/farmacologia , Geranil-Geranildifosfato Geranil-Geraniltransferase/antagonistas & inibidores , Mieloma Múltiplo/patologia , Prenilação de Proteína/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Domínio Catalítico , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/química , Geranil-Geranildifosfato Geranil-Geraniltransferase/química , Geranil-Geranildifosfato Geranil-Geraniltransferase/metabolismo , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Pirimidinas/química , Pirimidinas/farmacologia , Proteínas rap1 de Ligação ao GTP/metabolismo
11.
Traffic ; 8(6): 647-60, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17461799

RESUMO

Nuclear pore complexes (NPCs) form channels across the nuclear envelope and provide the sole sites of molecular exchange between the cytoplasm and nucleoplasm. The NPC is a target of a number of post-translational modifications, including phosphorylation, yet the functions of these modifications are ill defined. Here, we have investigated the mitotic specific phosphorylation of a yeast nucleoporin Nup53p. Two kinases were identified that phosphorylate Nup53p: the mitotic kinase Cdk1p/Cdc2p/Cdc28p and the casein kinase Hrr25p. Hrr25p was identified by screening 119 yeast kinases for their ability to phosphorylate Nup53p in vitro. Conditional alleles of Hrr25p support the conclusion that Hrr25p phosphorylates Nup53p in vivo. We further demonstrated using solution binding and affinity purification assays, that Hrr25p directly binds Nup53p in an interaction that is destabilized by the phosphorylation of Nup53p. Consistent with this observation, we observed that Hrr25p moves between distinct locations in the cell during the cell cycle including the nucleus, the cortex of the emerging bud and the spindle pole bodies. Cdk1p also contributes to Nup53p phosphorylation as specific inhibition of Cdk1p or mutation of Cdk1p consensus sites partially blocked its phosphorylation. The ability of nup53 alleles containing Cdk1p site mutations to complement synthetic defects of nup53 Delta nup170 Delta strains is linked to a function for Nup53p in the spindle assembly checkpoint.


Assuntos
Proteína Quinase CDC2/metabolismo , Caseína Quinase I/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alelos , Proteína Quinase CDC2/genética , Caseína Quinase I/genética , Caseína Quinase I/isolamento & purificação , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fosforilação , Plasmídeos , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Frações Subcelulares/metabolismo
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