RESUMO
Renal fibrosis (RF) represents the most widespread pathological condition in chronic kidney disease (CKD). Recently, protein prenylation has been implicated in the fibrosis's progression. The research examined the renoprotective effect of zoledronic acid (ZA) (50 µg/kg/week) in a rat model of carbon tetrachloride (CCl4)-induced RF through targeting protein prenylation. Forty Wistar male rats were split up into the control group, vehicle-treated group, model-RF group, and RF-ZA group. Mean arterial blood pressure (MBP), BUN, serum creatinine, and urine albumin-creatinine ratio (uACR), protein levels of farnesyl pyrophosphate (FPP), tumour necrosis factor-alpha (TNF-α), transforming growth factor-ß (TGF-ß), and malondialdehyde (MDA), and catalase and gene expression of farnesyl pyrophosphate synthase (FPPS) and nuclear factor-kB (NF-κB) were measured. Immunohistochemical staining for renal interleukin-6 (IL-6), α-smooth muscle actin (α-SMA), and caspase-3, as well as histopathological alterations, were assessed. ZA considerably ceased the reduction in MBP, markedly reduced uACR, serum creatinine, BUN, and expression of FPPS, FPP, NF-κB, TGF-ß, TNF-α, and MDA, and significantly increased catalase levels compared to the model-RF rats. ZA ameliorated the CCl4-induced histopathological alterations and suppressed the expression of caspase-3, α-SMA, and IL-6. In conclusion, ZA preserved renal function and prevented renal fibrosis in a rat model. These were achieved through targeting protein prenylation mainly by inhibiting FPPS.
Assuntos
Fibrose , Geraniltranstransferase , Rim , Prenilação de Proteína , Ratos Wistar , Ácido Zoledrônico , Animais , Ácido Zoledrônico/farmacologia , Masculino , Ratos , Prenilação de Proteína/efeitos dos fármacos , Geraniltranstransferase/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Rim/metabolismo , Tetracloreto de Carbono , Fosfatos de Poli-Isoprenil/metabolismo , Fosfatos de Poli-Isoprenil/farmacologia , Modelos Animais de Doenças , Fator de Crescimento Transformador beta/metabolismo , Sesquiterpenos/farmacologia , Sesquiterpenos/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. As such, statin treatment not only inhibits de novo synthesis of cholesterol but also isoprenoids that are involved in prenylation, the posttranslational lipid modification of proteins. The immunomodulatory effects of statins are broad and often conflicting. Previous work demonstrated that statins increased survival and inhibited myeloid cell trafficking in a murine model of sepsis, but the exact mechanisms underlying this phenomenon were unclear. Herein, we investigated the role of prenylation in chemoattractant responses. We found that simvastatin treatment abolished chemoattractant responses induced by stimulation by C5a and FMLP. The inhibitory effect of simvastatin treatment was unaffected by the addition of either farnesyl pyrophosphate (FPP) or squalene but was reversed by restoring geranylgeranyl pyrophosphate (GGPP). Treatment with prenyltransferase inhibitors showed that the chemoattractant response to both chemoattractants was dependent on geranylgeranylation. Proteomic analysis of C15AlkOPP-prenylated proteins identified several geranylgeranylated proteins involved in chemoattractant responses, including RHOA, RAC1, CDC42, and GNG2. Chemoattractant responses in THP-1 human macrophages were also geranylgeranylation dependent. These studies provide data that help clarify paradoxical findings on the immunomodulatory effects of statins. Furthermore, they establish the role of geranylgeranylation in mediating the morphological response to chemoattractant C5a.NEW & NOTEWORTHY The immunomodulatory effect of prenylation is ill-defined. We investigated the role of prenylation on the chemoattractant response to C5a. Simvastatin treatment inhibits the cytoskeletal remodeling associated with a chemotactic response. We showed that the chemoattractant response to C5a was dependent on geranylgeranylation, and proteomic analysis identified several geranylgeranylated proteins that are involved in C5a receptor signaling and cytoskeletal remodeling. Furthermore, they establish the role of geranylgeranylation in mediating the response to chemoattractant C5a.
Assuntos
Fosfatos de Poli-Isoprenil , Fosfatos de Poli-Isoprenil/farmacologia , Fosfatos de Poli-Isoprenil/metabolismo , Humanos , Sinvastatina/farmacologia , Fatores Quimiotáticos/farmacologia , Fatores Quimiotáticos/metabolismo , Fagócitos/efeitos dos fármacos , Fagócitos/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Complemento C5a/metabolismo , Prenilação de Proteína/efeitos dos fármacos , Animais , Camundongos , SesquiterpenosRESUMO
Prenylation is an irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Dysregulation of prenylation contributes to multiple disorders, including cancers and vascular and neurodegenerative diseases. Prenyltransferases tether isoprenoid lipids to proteins via a thioether linkage during prenylation. Pharmacological inhibition of the lipid synthesis pathway by statins is a therapeutic approach to control hyperlipidemia. Building on our previous finding that statins inhibit membrane association of G protein γ (Gγ) in a subtype-dependent manner, we investigated the molecular reasoning for this differential inhibition. We examined the prenylation of carboxy-terminus (Ct) mutated Gγ in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged Gγ subunits and their mutants using live-cell confocal imaging. Reversible optogenetic unmasking-masking of Ct residues was used to probe their contribution to prenylation and membrane interactions of the prenylated proteins. Our findings suggest that specific Ct residues regulate membrane interactions of the Gγ polypeptide, statin sensitivity, and extent of prenylation. Our results also show a few hydrophobic and charged residues at the Ct are crucial determinants of a protein's prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different Gγ subtypes, our findings indicate a plausible mechanism allowing for statins to differentially perturb heterotrimeric G protein signaling in cells depending on their Gγ-subtype composition. Our results may also provide molecular reasoning for repurposing statins as Ras oncogene inhibitors and the failure of using prenyltransferase inhibitors in cancer treatment.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP , Prenilação de Proteína , Humanos , Motivos de Aminoácidos , Resistência a Medicamentos/genética , Células HeLa , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Modelos Moleculares , Mutação , Prenilação de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Bisphosphonates drugs target the skeleton and are used globally for the treatment of common bone disorders. Nitrogen-containing bisphosphonates act by inhibiting the mevalonate pathway in bone-resorbing osteoclasts but, surprisingly, also appear to reduce the risk of death from pneumonia. We overturn the long-held belief that these drugs act only in the skeleton and show that a fluorescently labelled bisphosphonate is internalised by alveolar macrophages and large peritoneal macrophages in vivo. Furthermore, a single dose of a nitrogen-containing bisphosphonate (zoledronic acid) in mice was sufficient to inhibit the mevalonate pathway in tissue-resident macrophages, causing the build-up of a mevalonate metabolite and preventing protein prenylation. Importantly, one dose of bisphosphonate enhanced the immune response to bacterial endotoxin in the lung and increased the level of cytokines and chemokines in bronchoalveolar fluid. These studies suggest that bisphosphonates, as well as preventing bone loss, may boost immune responses to infection in the lung and provide a mechanistic basis to fully examine the potential of bisphosphonates to help combat respiratory infections that cause pneumonia.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Pulmão/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Ácido Zoledrônico/farmacologia , Animais , Conservadores da Densidade Óssea/administração & dosagem , Quimiocinas/metabolismo , Citocinas/metabolismo , Feminino , Lipopolissacarídeos/toxicidade , Pulmão/metabolismo , Ácido Mevalônico/metabolismo , Camundongos Endogâmicos C57BL , Prenilação de Proteína/efeitos dos fármacos , Ácido Zoledrônico/administração & dosagemRESUMO
Macrothrombocytopenia is a common pathology of missense mutations in genes regulating actin dynamics. Takenouchi-Kosaki syndrome (TKS) harboring the c.191A > G, Tyr64Cys (Y64C) variant in Cdc42 exhibits a variety of clinical manifestations, including immunological and hematological anomalies. In the present study, we investigated the functional abnormalities of the Y64C mutant in HEK293 cells and elucidated the mechanism of macrothrombocytopenia, one of the symptoms of TKS patients, by monitoring the production of platelet-like particles (PLP) using MEG-01 cells. We found that the Y64C mutant was concentrated at the membrane compartment due to impaired binding to Rho-GDI and more active than the wild-type. The Y64C mutant also had lower association with its effectors Pak1/2 and N-WASP. Y64C mutant-expressing MEG-01 cells demonstrated short cytoplasmic protrusions with aberrant F-actin and microtubules, and reduced PLP production. This suggested that the Y64C mutant facilitates its activity and membrane localization, resulting in impaired F-actin dynamics for proplatelet extension, which is necessary for platelet production. Furthermore, such dysfunction was ameliorated by either suppression of Cdc42 activity or prenylation using chemical inhibitors. Our study may lead to pharmacological treatments for TKS patients.
Assuntos
Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trombocitopenia/metabolismo , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Alquil e Aril Transferases/antagonistas & inibidores , Benzamidas/farmacologia , Plaquetas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células HEK293 , Humanos , Mutação , Prenilação de Proteína/efeitos dos fármacos , Pirazóis/farmacologia , Transdução de Sinais/genética , Sulfonamidas/farmacologia , Síndrome , Trombocitopenia/genética , Trombopoese/efeitos dos fármacos , Trombopoese/genética , Transfecção , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Quinases Ativadas por p21/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismoRESUMO
Vascular development is essential for the establishment of the circulatory system during embryonic development and requires the proliferation of endothelial cells. However, the underpinning regulatory mechanisms are not well understood. Here, we report that geranylgeranyl pyrophosphate (GGPP), a metabolite involved in protein geranylgeranylation, plays an indispensable role in embryonic vascular development. GGPP is synthesized by geranylgeranyl pyrophosphate synthase (GGPPS) in the mevalonate pathway. The selective knockout of Ggpps in endothelial cells led to aberrant vascular development and embryonic lethality, resulting from the decreased proliferation and enhanced apoptosis of endothelial cells during vasculogenesis. The defect in protein geranylgeranylation induced by GGPP depletion inhibited the membrane localization of RhoA and enhanced yes-associated protein (YAP) phosphorylation, thereby prohibiting the entry of YAP into the nucleus and the expression of YAP target genes related to cell proliferation and the antiapoptosis process. Moreover, inhibition of the mevalonate pathway by simvastatin induced endothelial cell proliferation defects and apoptosis, which were ameliorated by GGPP. Geranylgeraniol (GGOH), a precursor of GGPP, ameliorated the harmful effects of simvastatin on vascular development of developing fetuses in pregnant mice. These results indicate that GGPP-mediated protein geranylgeranylation is essential for endothelial cell proliferation and the antiapoptosis process during embryonic vascular development.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Farnesiltranstransferase/genética , Complexos Multienzimáticos/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Embrião de Mamíferos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Camundongos , Morfogênese/genética , Fosfatos de Poli-Isoprenil/metabolismo , Fosfatos de Poli-Isoprenil/farmacologia , Gravidez , Prenilação de Proteína/efeitos dos fármacos , Prenilação de Proteína/genética , Proteínas de Sinalização YAP/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
The review discusses a new approach to the prevention and treatment of viral infections based on the use of pine needles polyprenyl phosphate (PPP) and associated with the infringement of prenylation process-the attachment of farnesol or geranyl geraniol to the viral protein. Currently, prenylation has been detected in type 1 adenovirus, hepatitis C virus, several herpes viruses, influenza virus, HIV. However, this list is far from complete, given that prenylated proteins play an extremely important role in the activity of the virus. We assume that the interferon produced in response to PPP may suppress expression of the SREBP2 transcription factor. As a result, the mevalonic acid pathway is violated and, as a result, the formation of early polyprenols precursors (geraniol, geranyl geraniol, farnesol), which are necessary for the prenylation of viral proteins, is blocked and the formation of mature, virulent virus particles is broken. As a consequence, the maturation of viral particles is inhibited, and defective particles are formed. Polyprenol was extracted from greenery (pine, fir and spruce needles, mulberry leaves, etc.), purified by chromatography, phosphorylated and identified by HPLC and NMR. Obtained PPP was used as antiviral in some experimental models in vitro and in vivo. During numerous studies, it was found that PPP manifested versatile antiviral effects, both in vitro and in vivo. The maximum effect was observed with viruses in which the presence of prenylated proteins was established, namely influenza A virus, HIV-1, tick-borne encephalitis virus, hepatitis A and C viruses, herpes simplex viruses type 1 and 2, some coronavirus. The available data obtained both in the experimental conditions and during clinical trials allow us to regard PPPs as safe and effective medicine for prevention and treatment of viral diseases.
Assuntos
Antivirais/farmacologia , Pinus/química , Fosfatos de Poli-Isoprenil/farmacologia , Prenilação de Proteína/efeitos dos fármacos , Viroses/tratamento farmacológico , Animais , Antivirais/uso terapêutico , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Interferons/metabolismo , Microscopia Eletrônica , Fosfatos de Poli-Isoprenil/uso terapêutico , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Resultado do Tratamento , Proteínas Virais/metabolismo , Vírion/efeitos dos fármacos , Vírion/ultraestrutura , Viroses/imunologia , Viroses/prevenção & controle , Replicação Viral/efeitos dos fármacos , Replicação Viral/imunologiaRESUMO
Geranylgeranyl diphosphate synthase (GGDPS), an enzyme in the isoprenoid biosynthetic pathway (IBP), produces the isoprenoid (geranylgeranyl pyrophosphate, GGPP) used in protein geranylgeranylation reactions. Our prior studies utilizing triazole bisphosphonate-based GGDPS inhibitors (GGSIs) have revealed that these agents represent a novel strategy by which to induce cancer cell death, including multiple myeloma and pancreatic cancer. Statins inhibit the rate-limiting enzyme in the IBP and potentiate the effects of GGSIs in vitro. The in vivo effects of combination therapy with statins and GGSIs have not been determined. Here we evaluated the effects of combining VSW1198, a novel GGSI, with a statin (lovastatin or pravastatin) in CD-1 mice. Twice-weekly dosing with VSW1198 at the previously established maximally tolerated dose in combination with a statin led to hepatotoxicity, while once-weekly VSW1198-based combinations were feasible. No abnormalities in kidney, spleen, brain or skeletal muscle were observed with combination therapy. Combination therapy disrupted protein geranylgeranylation in vivo. Evaluation of hepatic isoprenoid levels revealed decreased GGPP levels in the single drug groups and undetectable GGPP levels in the combination groups. Additional studies with combinations using 50% dose-reductions of either VSW1198 or lovastatin revealed minimal hepatotoxicity with expected on-target effects of diminished GGPP levels and disruption of protein geranylgeranylation. Combination statin/GGSI therapy significantly slowed tumor growth in a myeloma xenograft model. Collectively, these studies are the first to demonstrate that combination IBP inhibitor therapy alters isoprenoid levels and disrupts protein geranylgeranylation in vivo as well as slows tumor growth in a myeloma xenograft model, thus providing the framework for future clinical exploration.
Assuntos
Vias Biossintéticas/efeitos dos fármacos , Diterpenos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Prenilação de Proteína/efeitos dos fármacos , Terpenos/metabolismo , Triazóis/administração & dosagem , Animais , Vias Biossintéticas/fisiologia , Linhagem Celular Tumoral , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Diterpenos/toxicidade , Avaliação Pré-Clínica de Medicamentos/métodos , Quimioterapia Combinada , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/toxicidade , Farnesiltranstransferase/antagonistas & inibidores , Farnesiltranstransferase/metabolismo , Feminino , Inibidores de Hidroximetilglutaril-CoA Redutases/toxicidade , Lovastatina/administração & dosagem , Lovastatina/toxicidade , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pravastatina/administração & dosagem , Pravastatina/toxicidade , Prenilação de Proteína/fisiologia , Terpenos/antagonistas & inibidores , Triazóis/toxicidade , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Fungal pathogens pose an increasing threat to global food security through devastating effects on staple crops and contamination of food supplies with carcinogenic toxins. Widespread deployment of agricultural fungicides has increased crop yields but is driving increasingly frequent resistance to available agents and creating environmental reservoirs of drug-resistant fungi that can also infect susceptible human populations. To uncover non-cross-resistant modes of antifungal action, we leveraged the unique chemical properties of boron chemistry to synthesize novel 6-thiocarbamate benzoxaboroles with broad spectrum activity against diverse fungal plant pathogens. Through whole genome sequencing of Saccharomyces cerevisiae isolates selected for stable resistance to these compounds, we identified mutations in the protein prenylation-related genes, CDC43 and ERG20. Allele-swapping experiments confirmed that point mutations in CDC43, which encodes an essential catalytic subunit within geranylgeranyl transferase I (GGTase I) complex, were sufficient to confer resistance to the benzoxaboroles. Mutations in ERG20, which encodes an upstream farnesyl pyrophosphate synthase in the geranylgeranylation pathway, also conferred resistance. Consistent with impairment of protein prenylation, the compounds disrupted membrane localization of the classical geranylgeranylation substrate Cdc42. Guided by molecular docking predictions, which favored Cdc43 as the most likely direct target, we overexpressed and purified functional GGTase I complex to demonstrate direct binding of benzoxaboroles to it and concentration-dependent inhibition of its transferase activity. Further development of the boron-containing scaffold described here offers a promising path to the development of GGTase I inhibitors as a mechanistically distinct broad spectrum fungicide class with reduced potential for cross-resistance to antifungals in current use.
Assuntos
Antifúngicos/farmacologia , Compostos de Boro/farmacologia , Prenilação de Proteína/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Tiocarbamatos/farmacologia , Alquil e Aril Transferases/antagonistas & inibidores , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Antifúngicos/síntese química , Antifúngicos/metabolismo , Compostos de Boro/síntese química , Compostos de Boro/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/metabolismo , Farmacorresistência Fúngica/genética , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Fungos/efeitos dos fármacos , Fungos/genética , Simulação de Acoplamento Molecular , Mutação Puntual , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/genética , Tiocarbamatos/síntese química , Tiocarbamatos/metabolismo , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismoRESUMO
The RAS genes, which include H, N, and KRAS, comprise the most frequently mutated family of oncogenes in cancer. Mutations in KRAS - such as the G12C mutation - are found in most pancreatic, half of colorectal and a third of lung cancer cases and is thus responsible for a substantial proportion of cancer deaths. Consequently, KRAS has been the subject of exhaustive drug-targeting efforts over the past 3-4 decades. These efforts have included targeting the KRAS protein itself but also its posttranslational modifications, membrane localization, protein-protein interactions and downstream signalling pathways. Most of these strategies have failed and no KRAS-specific drugs have yet been approved. However, for one specific mutation, KRASG12C , there is light on the horizon. MRTX849 was recently identified as a potent, selective and covalent KRASG12C inhibitor that possesses favourable drug-like properties. MRTX849 selectively modifies the mutant cysteine residue in GDP-bound KRASG12C and inhibits GTP-loading and downstream KRAS-dependent signalling. The drug inhibits the in vivo growth of multiple KRASG12C -mutant cell line xenografts, causes tumour regression in patient-derived xenograft models and shows striking responses in combination with other agents. It has also produced objective responses in patients with mutant-specific lung and colorectal cancer. In this review, we discuss the history of RAS drug-targeting efforts, the discovery of MRTX849, and how this drug provides an exciting and long-awaited opportunity to selectively target mutant KRAS in patients.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Ensaios Clínicos como Assunto , Inibidores Enzimáticos/farmacologia , Humanos , Mutação , Prenilação de Proteína/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
Aberrant activation of GTPases is common in cervical cancer, and their proper biological functions largely depend on a post-translational modification termed prenylation. Simvastatin is a cholesterol-lowering drug via inhibiting HMG-CoA reductase, thereby inhibiting protein prenylation. In this study, we show that simvastatin selectively inhibits proliferation and induces apoptosis in cervical cancer cells while sparing normal cervical epithelial cells. This is achieved by depleting geranylgeranyl pyrophosphate, inhibiting prenylation, decreasing GTPases activities and suppressing the activation of downstream Ras and RhoA signaling. The combination of simvastatin and paclitaxel remarkably augments in vitro as well as in vivo efficacy of either drug alone in cellular system and xenograft mouse model. Importantly, we show that cervical cancer cells have higher level of HMG-CoA reductase and elevated activities of GTPases, suggesting that cervical cancer cells may be more dependent on prenylation than normal cervical epithelial cells. This might explain the selective inhibitory effects of simvastatin in cervical cancer. Since simvastatin is already available for clinic use, these results suggest that simvastatin is a promising drug candidate in combination with chemotherapy for the treatment of cervical cancer. Our findings also emphasize the therapeutic value of prenylation inhibition and provide preclinical evidence to evaluate prenylation-targeted drugs in cervical cancer.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Paclitaxel/farmacologia , Sinvastatina/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Hidroximetilglutaril-CoA Redutases/efeitos dos fármacos , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Camundongos SCID , Paclitaxel/administração & dosagem , Fosfatos de Poli-Isoprenil/metabolismo , Prenilação de Proteína/efeitos dos fármacos , Sinvastatina/administração & dosagem , Neoplasias do Colo do Útero/enzimologia , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The members of Rho family of GTPases, RhoA and Rac1 regulate endothelial cytoskeleton dynamics and hence barrier integrity. The spatial activities of these GTPases are regulated by post-translational prenylation. In the present study, we investigated the effect of prenylation inhibition on the endothelial cytoskeleton and barrier properties. The study was carried out in human umbilical vein endothelial cells (HUVEC) and protein prenylation is manipulated with various pharmacological inhibitors. Inhibition of either complete prenylation using statins or specifically geranylgeranylation but not farnesylation has a biphasic effect on HUVEC cytoskeleton and permeability. Short-term treatment inhibits the spatial activity of RhoA/Rho kinase (Rock) to actin cytoskeleton resulting in adherens junctions (AJ) stabilization and ameliorates thrombin-induced barrier disruption whereas long-term inhibition results in collapse of endothelial cytoskeleton leading to increased basal permeability. These effects are reversed by supplementing the cells with geranylgeranyl but not farnesyl pyrophosphate. Moreover, long-term inhibition of protein prenylation results in basal hyper activation of RhoA/Rock signaling that is antagonized by a specific Rock inhibitor or an activation of cAMP signaling. In conclusion, inhibition of geranylgeranylation in endothelial cells (ECs) exerts biphasic effect on endothelial barrier properties. Short-term inhibition stabilizes AJs and hence barrier function whereas long-term treatment results in disruption of barrier properties.
Assuntos
Endotélio/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Permeabilidade da Membrana Celular , Citoesqueleto/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio/efeitos dos fármacos , Humanos , Junções Intercelulares/metabolismo , Modelos Biológicos , Prenilação de Proteína/efeitos dos fármacos , Quinases Associadas a rho/metabolismoRESUMO
Benzimidazole carboxyphosphonates and bisphosphonates have been prepared and evaluated for their activity as inhibitors of protein prenylation or isoprenoid biosynthesis. The nature of the phosphonate head group was found to dictate enzyme specificity. The lead carboxyphosphonate inhibits geranylgeranyl transferase II while its corresponding bisphosphonate analogue potently inhibits farnesyl diphosphate synthase. The most active inhibitors effectively disrupted protein prenylation in human multiple myeloma cells.
Assuntos
Benzimidazóis/antagonistas & inibidores , Benzimidazóis/uso terapêutico , Organofosfonatos/antagonistas & inibidores , Organofosfonatos/uso terapêutico , Prenilação de Proteína/efeitos dos fármacos , Benzimidazóis/farmacologia , Humanos , Organofosfonatos/farmacologiaRESUMO
The clinical management of pediatric acute myeloid leukemia (AML) is still challenging and identification of drugs that can enhance the efficacy of standard of care is a potential therapeutic strategy. We show that pamidronate, a FDA-approved drug used for bone disorders, is an attractive candidate for AML treatment. Pamidronate inhibits proliferation and induces apoptosis of AML cells regardless of cellular and genetic heterogeneity. Pamidronate displays selective anti-AML activity by preferentially inhibiting survival and colony formation of AML CD34+ cells while normal bone marrow CD34+ cells are largely unaffected. Importantly, pamidronate remarkably enhances the inhibitory effects of all tested AML standard of care at subtoxic concentration. Mechanism studies show that pamidronate inhibits protein prenylation via dual action on geranylgeranylation and farnesylation, and subsequently decreases Ras activity. The rescue studies using overexpression of constitutively active Ras further confirm that pamidronate augments the efficacy of AML standard of care through inhibiting Ras. Since pamidronate is already used in clinic, our preclinical findings suggest that it may be an effective addition to treatment armamentarium for AML.
Assuntos
Antineoplásicos/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Pamidronato/farmacologia , Prenilação de Proteína/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Proteínas ras/antagonistas & inibidores , Anilidas/farmacologia , Anexina A5/genética , Anexina A5/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzotiazóis/farmacologia , Conservadores da Densidade Óssea/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Criança , Citarabina/farmacologia , Daunorrubicina/farmacologia , Reposicionamento de Medicamentos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Compostos de Fenilureia/farmacologia , Cultura Primária de Células , Piridinas/farmacologia , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Apicomplexan parasites possess a plastid organelle called the apicoplast. Inhibitors that selectively target apicoplast housekeeping functions, including DNA replication and protein translation, are lethal for the parasite, and several (doxycycline, clindamycin, and azithromycin) are in clinical use as antimalarials. A major limitation of such drugs is that treated parasites only arrest one intraerythrocytic development cycle (approximately 48 hours) after treatment commences, a phenotype known as the 'delayed death' effect. The molecular basis of delayed death is a long-standing mystery in parasitology, and establishing the mechanism would aid rational clinical implementation of apicoplast-targeted drugs. Parasites undergoing delayed death transmit defective apicoplasts to their daughter cells and cannot produce the sole, blood-stage essential metabolic product of the apicoplast: the isoprenoid precursor isopentenyl-pyrophosphate. How the isoprenoid precursor depletion kills the parasite remains unknown. We investigated the requirements for the range of isoprenoids in the human malaria parasite Plasmodium falciparum and characterised the molecular and morphological phenotype of parasites experiencing delayed death. Metabolomic profiling reveals disruption of digestive vacuole function in the absence of apicoplast derived isoprenoids. Three-dimensional electron microscopy reveals digestive vacuole fragmentation and the accumulation of cytostomal invaginations, characteristics common in digestive vacuole disruption. We show that digestive vacuole disruption results from a defect in the trafficking of vesicles to the digestive vacuole. The loss of prenylation of vesicular trafficking proteins abrogates their membrane attachment and function and prevents the parasite from feeding. Our data show that the proximate cause of delayed death is an interruption of protein prenylation and consequent cellular trafficking defects.
Assuntos
Apicoplastos/metabolismo , Espaço Intracelular/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Antimaláricos/farmacologia , Morte Celular/efeitos dos fármacos , Hemiterpenos/metabolismo , Hemiterpenos/farmacologia , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/parasitologia , Malária Falciparum/parasitologia , Metabolômica/métodos , Compostos Organofosforados/metabolismo , Compostos Organofosforados/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/fisiologia , Prenilação de Proteína/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/parasitologiaRESUMO
Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates the localization and function of over 100 proteins, including the Ras isoforms, and is a therapeutic target in diseases including cancer and infection. Here, we report global and selective profiling of prenylated proteins in living cells enabled by the development of isoprenoid analogues YnF and YnGG in combination with quantitative chemical proteomics. Eighty prenylated proteins were identified in a single human cell line, 64 for the first time at endogenous abundance without metabolic perturbation. We further demonstrate that YnF and YnGG enable direct identification of post-translationally processed prenylated peptides, proteome-wide quantitative analysis of prenylation dynamics and alternative prenylation in response to four different prenyltransferase inhibitors, and quantification of defective Rab prenylation in a model of the retinal degenerative disease choroideremia.
Assuntos
Alcinos/química , Sondas Moleculares/química , Prenilação de Proteína , Proteínas/análise , Proteoma/análise , Proteômica/métodos , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Espectrometria de Massas , Camundongos Knockout , Prenilação de Proteína/efeitos dos fármacos , Proteínas/química , Proteoma/químicaRESUMO
INTRODUCTION: Bisphosphonates (BPs) are widely used to manage a variety of bone disorders, including osteoporosis, metastatic bone disease and myeloma bone disease. The nitrogen-containing BPs (NBPs) target osteoclast activity by disrupting protein prenylation via inhibition of farnesyl diphosphate synthase (FDPS). AREAS COVERED: This review summarizes the recent advances in BPs with a focus on the latest patents (2015-2018). Patents involving novel BPs, new modes of BP delivery, as well as use of BPs to deliver other drugs to bone are discussed. A review of phosphonate-based drugs targeting geranylgeranyl diphosphate synthase (GGDPS) or geranylgeranyl transferase II (GGTase II) as alternative strategies to disrupt protein geranylgeranylation is provided. EXPERT OPINION: While the NBPs remain the mainstay of treatment for most bone disorders, further understanding of their pharmacological properties could lead to further refinement of their chemical structures and optimization of efficacy and safety profiles. In addition, the development of NBP analogs or drug delivery mechanisms that allow for nonbone tissue exposure could allow for the use of these drugs as direct anticancer agents. The development of GGDPS and GGTase II inhibitors represents alternative heterocycle phosphonate-based strategies to disrupt protein geranylgeranylation and may have potential as anticancer agents and/or as bone-targeted therapies.
Assuntos
Doenças Ósseas/tratamento farmacológico , Difosfonatos/uso terapêutico , Prenilação de Proteína/efeitos dos fármacos , Animais , Doenças Ósseas/fisiopatologia , Difosfonatos/farmacologia , Sistemas de Liberação de Medicamentos , Desenvolvimento de Medicamentos , Farnesiltranstransferase/antagonistas & inibidores , Geraniltranstransferase/antagonistas & inibidores , Humanos , Osteoclastos/efeitos dos fármacos , Patentes como Assunto , Transferases/antagonistas & inibidoresRESUMO
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/metabolismoRESUMO
Guanine nucleotide-binding proteins (G proteins) facilitate the transduction of external signals to the cell interior, regulate most eukaryotic signaling, and thus have become crucial disease drivers. G proteins largely function at the inner leaflet of the plasma membrane (PM) using covalently attached lipid anchors. Both small monomeric and heterotrimeric G proteins are primarily prenylated, either with a 15-carbon farnesyl or a 20-carbon geranylgeranyl polyunsaturated lipid. The mevalonate [3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase] pathway synthesizes lipids for G-protein prenylation. It is also the source of the precursor lipids for many biomolecules, including cholesterol. Consequently, the rate-limiting enzymes of the mevalonate pathway are major targets for cholesterol-lowering medications and anticancer drug development. Although prenylated G protein γ (Gγ) is essential for G protein-coupled receptor (GPCR)-mediated signaling, how mevalonate pathway inhibitors, statins, influence subcellular distribution of Gßγ dimer and Gαßγ heterotrimer, as well as their signaling upon GPCR activation, is poorly understood. The present study shows that clinically used statins not only significantly disrupt PM localization of Gßγ but also perturb GPCR-G protein signaling and associated cell behaviors. The results also demonstrate that the efficiency of prenylation inhibition by statins is Gγ subtype-dependent and is more effective toward farnesylated Gγ types. Since Gγ is required for Gßγ signaling and shows a cell- and tissue-specific subtype distribution, the present study can help understand the mechanisms underlying clinical outcomes of statin use in patients. This work also reveals the potential of statins as clinically usable drugs to control selected GPCR-G protein signaling.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colesterol/metabolismo , Células HeLa , Humanos , Ácido Mevalônico/farmacologia , Camundongos , Prenilação de Proteína/efeitos dos fármacos , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacosRESUMO
Recent research focusing on brown adipose tissue (BAT) function emphasizes its importance in systemic metabolic homeostasis. We show here that genetic and pharmacological inhibition of the mevalonate pathway leads to reduced human and mouse brown adipocyte function in vitro and impaired adipose tissue browning in vivo. A retrospective analysis of a large patient cohort suggests an inverse correlation between statin use and active BAT in humans, while we show in a prospective clinical trial that fluvastatin reduces thermogenic gene expression in human BAT. We identify geranylgeranyl pyrophosphate as the key mevalonate pathway intermediate driving adipocyte browning in vitro and in vivo, whose effects are mediated by geranylgeranyltransferases (GGTases), enzymes catalyzing geranylgeranylation of small GTP-binding proteins, thereby regulating YAP1/TAZ signaling through F-actin modulation. Conversely, adipocyte-specific ablation of GGTase I leads to impaired adipocyte browning, reduced energy expenditure, and glucose intolerance under obesogenic conditions, highlighting the importance of this pathway in modulating brown adipocyte functionality and systemic metabolism.