RESUMEN
Mevalonate kinase is a key regulator of the mevalonate pathway, subject to feedback inhibition by the downstream metabolite farnesyl pyrophosphate. In this study, we validated the hypothesis that monophosphonate compounds mimicking farnesyl pyrophosphate can inhibit mevalonate kinase. Exploring compounds originally synthesized as allosteric inhibitors of farnesyl pyrophosphate synthase, we discovered mevalonate kinase inhibitors with nanomolar activity. Kinetic characterization of the two most potent inhibitors demonstrated Ki values of 3.1 and 22 nm. Structural comparison suggested features of these inhibitors likely responsible for their potency. Our findings introduce the first class of nanomolar inhibitors of human mevalonate kinase, opening avenues for future research. These compounds might prove useful as molecular tools to study mevalonate pathway regulation and evaluate mevalonate kinase as a potential therapeutic target.
Asunto(s)
Inhibidores Enzimáticos , Fosfotransferasas (Aceptor de Grupo Alcohol) , Humanos , Regulación Alostérica/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Cinética , Geraniltranstransferasa/antagonistas & inhibidores , Geraniltranstransferasa/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Sesquiterpenos/farmacología , Sesquiterpenos/metabolismo , Sesquiterpenos/químicaRESUMEN
This study analysed the occupational dose in Korean pressurized heavy-water reactors (PHWRs) and identified tasks involving high radiation exposure. The average individual dose was sufficiently low to be below the annual effective dose limit for radiation workers and is even lower than the dose limit for the general public. However, some workers received relatively higher doses than others. Furthermore, most PHWR workers are exposed to radiation during planned maintenance periods. In this study, the radiation dose was normalized (radiation dose per unit time) to determine the high-radiation-exposure tasks in Korean PHWRs. Consequently, end-fitting lapping, delayed neutron tube work and fuel channel fixed-end change tasks were identified as high-radiation-exposure tasks in Korean PHWRs. If appropriate radiation protection measures are prioritized for the identified high-dose exposure tasks, optimization of radiological protection will be effectively achieved by reducing the dose that is relatively higher than the average.
Asunto(s)
Exposición Profesional , Dosis de Radiación , Monitoreo de Radiación , Exposición Profesional/análisis , Exposición Profesional/prevención & control , Humanos , República de Corea , Monitoreo de Radiación/métodos , Monitoreo de Radiación/instrumentación , Protección Radiológica/instrumentación , Exposición a la Radiación/análisis , Reactores Nucleares , AguaRESUMEN
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.
Asunto(s)
Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Geranilgeranil-Difosfato Geranilgeraniltransferasa/antagonistas & inhibidores , Mieloma Múltiple/tratamiento farmacológico , Pirimidinas/uso terapéutico , Tiofenos/uso terapéutico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Antineoplásicos/toxicidad , Células de la Médula Ósea/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/toxicidad , Femenino , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/metabolismo , Geranilgeranil-Difosfato Geranilgeraniltransferasa/metabolismo , Humanos , Hígado/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Estructura Molecular , Unión Proteica , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Pirimidinas/toxicidad , Ratas , Saccharomyces cerevisiae/enzimología , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/metabolismo , Tiofenos/toxicidadRESUMEN
Plazomicin is currently the only next-generation aminoglycoside approved for clinical use that has the potential of evading the effects of widespread enzymatic resistance factors. However, plazomicin is still susceptible to the action of the resistance enzyme AAC(2')-Ia from Providencia stuartii. As the clinical use of plazomicin begins to increase, the spread of resistance factors will undoubtedly accelerate, rendering this aminoglycoside increasingly obsolete. Understanding resistance to plazomicin is an important step to ensure this aminoglycoside remains a viable treatment option for the foreseeable future. Here, we present three crystal structures of AAC(2')-Ia from P. stuartii, two in complex with acetylated aminoglycosides tobramycin and netilmicin, and one in complex with a non-substrate aminoglycoside, amikacin. Together, with our previously reported AAC(2')-Ia-acetylated plazomicin complex, these structures outline AAC(2')-Ia's specificity for a wide range of aminoglycosides. Additionally, our survey of AAC(2')-I homologues highlights the conservation of residues predicted to be involved in aminoglycoside binding, and identifies the presence of plasmid-encoded enzymes in environmental strains that confer resistance to the latest next-generation aminoglycoside. These results forecast the likely spread of plazomicin resistance and highlight the urgency for advancements in next-generation aminoglycoside design.
Asunto(s)
Acetiltransferasas/química , Antibacterianos/química , Proteínas Bacterianas/química , Farmacorresistencia Bacteriana/genética , Providencia/enzimología , Sisomicina/análogos & derivados , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Amicacina/química , Amicacina/metabolismo , Amicacina/farmacología , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Cinética , Modelos Moleculares , Netilmicina/química , Netilmicina/metabolismo , Netilmicina/farmacología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Providencia/química , Providencia/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sisomicina/química , Sisomicina/metabolismo , Sisomicina/farmacología , Especificidad por Sustrato , Tobramicina/química , Tobramicina/metabolismo , Tobramicina/farmacologíaRESUMEN
Macrolides are a class of antibiotics widely used in both medicine and agriculture. Unsurprisingly, as a consequence of their exensive usage a plethora of resistance mechanisms have been encountered in pathogenic bacteria. One of these resistance mechanisms entails the enzymatic cleavage of the macrolides' macrolactone ring by erythromycin esterases (Eres). The most frequently identified Ere enzyme is EreA, which confers resistance to the majority of clinically used macrolides. Despite the role Eres play in macrolide resistance, research into this family enzymes has been sparse. Here, we report the first three-dimensional structures of an erythromycin esterase, EreC. EreC is an extremely close homologue of EreA, displaying more than 90% sequence identity. Two structures of this enzyme, in conjunction with in silico flexible docking studies and previously reported mutagenesis data allowed for the proposal of a detailed catalytic mechanism for the Ere family of enzymes, labeling them as metal-independent hydrolases. Also presented are substrate spectrum assays for different members of the Ere family. The results from these assays together with an examination of residue conservation for the macrolide binding site in Eres, suggests two distinct active site archetypes within the Ere enzyme family.
Asunto(s)
Antibacterianos/química , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Esterasas/química , Esterasas/genética , Macrólidos/química , Antibacterianos/farmacología , Bacterias/enzimología , Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Eritromicina/química , Genes Bacterianos , Macrólidos/farmacología , Simulación del Acoplamiento Molecular , Conformación Proteica , Difracción de Rayos XRESUMEN
Genetic screening has identified numerous variants of the endosomal solute carrier family 9 member A6 (SLC9A6)/(Na+,K+)/H+ exchanger 6 (NHE6) gene that cause Christianson syndrome, a debilitating X-linked developmental disorder associated with a range of neurological, somatic, and behavioral symptoms. Many of these variants cause complete loss of NHE6 expression, but how subtler missense substitutions or nonsense mutations that partially truncate its C-terminal cytoplasmic regulatory domain impair NHE6 activity and endosomal function are poorly understood. Here, we describe the molecular and cellular consequences of six unique mutations located in the N-terminal cytoplasmic segment (A9S), the membrane ion translocation domain (L188P and G383D), and the C-terminal regulatory domain (E547*, R568Q, and W570*) of human NHE6 that purportedly cause disease. Using a heterologous NHE6-deficient cell expression system, we show that the biochemical, catalytic, and cellular properties of the A9S and R568Q variants were largely indistinguishable from those of the WT transporter, which obscured their disease significance. By contrast, the L188P, G383D, E547*, and W570* mutants exhibited variable deficiencies in biosynthetic post-translational maturation, membrane sorting, pH homeostasis in recycling endosomes, and cargo trafficking, and they also triggered apoptosis. These findings broaden our understanding of the molecular dysfunctions of distinct NHE6 variants associated with Christianson syndrome.
Asunto(s)
Ataxia , Endosomas , Epilepsia , Enfermedades Genéticas Ligadas al Cromosoma X , Discapacidad Intelectual , Microcefalia , Mutación Missense , Trastornos de la Motilidad Ocular , Intercambiadores de Sodio-Hidrógeno , Sustitución de Aminoácidos , Animales , Ataxia/genética , Ataxia/metabolismo , Cricetinae , Endosomas/química , Endosomas/genética , Endosomas/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Microcefalia/genética , Microcefalia/metabolismo , Trastornos de la Motilidad Ocular/genética , Trastornos de la Motilidad Ocular/metabolismo , Dominios Proteicos , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
Aminoglycoside antibiotics have lost much of their effectiveness due to widespread resistance, primarily via covalent modification. One of the most ubiquitous enzymes responsible for aminoglycoside resistance is aminoglycoside O-nucleotidyltransferase(2â³), which catalyzes a nucleotidylation reaction. Due to its clinical importance, much research has focused on dissecting the mechanism of action, some of it dating back more than 30 years. Here, we present structural data for catalytically informative states of the enzyme, i.e., ANT(2â³) in complex with adenosine monophosphate (AMP) and tobramycin (inactive-intermediate state) and in complex with adenylyl-2â³-tobramycin, pyrophosphate, and Mn2+(product-bound state). These two structures in conjunction with our previously reported structure of ANT(2â³)'s substrate-bound complex capture clinical states along ANT(2â³)'s reaction coordinate. Additionally, isothermal titration calorimetry (ITC)-based studies are presented that assess the order of substrate binding and product release. Combined, these results outline a kinetic mechanism for ANT(2â³) that contradicts what has been previously reported. Specifically, we show that the release of adenylated aminoglycoside precedes pyrophosphate. Furthermore, the ternary complex structures provide additional details on the catalytic mechanism, which reveals extensive similarities to the evolutionarily related DNA polymerase-ß superfamily.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Farmacorresistencia Microbiana/efectos de los fármacos , Nucleotidiltransferasas/metabolismo , Adenosina Monofosfato/química , Secuencia de Aminoácidos , Catálisis , Difosfatos/química , Cinética , Manganeso/química , Modelos Moleculares , Conformación Molecular , Unión Proteica , Transducción de Señal , Relación Estructura-Actividad , Especificidad por Sustrato , Tobramicina/químicaRESUMEN
Phosphonates and bisphosphonates have proven their pharmacological utility as inhibitors of enzymes that metabolize phosphate and pyrophosphate substrates. The blockbuster class of drugs nitrogen-containing bisphosphonates represent one of the best-known examples. Widely used to treat bone-resorption disorders, these drugs work by inhibiting the enzyme farnesyl pyrophosphate synthase. Playing a key role in the isoprenoid biosynthetic pathway, this enzyme is also a potential anticancer target. Here, we provide a comprehensive overview of the research efforts to identify new inhibitors of farnesyl pyrophosphate synthase for various therapeutic applications. While the majority of these efforts have been directed against the human enzyme, some have been targeted on its homologs from other organisms, such as protozoan parasites and insects. Our particular focus is on the structures of the target enzymes and how the structural information has guided the drug discovery efforts.
RESUMEN
Thienopyrimidine-based allosteric inhibitors of the human farnesyl pyrophosphate synthase (hFPPS), characterized by a chiral α-aminophosphonic acid moiety, were synthesized as enantiomerically enriched pairs, and their binding mode was investigated by X-ray crystallography. A general consensus in the binding orientation of all (R)- and (S)-enantiomers was revealed. This finding is a prerequisite for establishing a reliable structure-activity relationship (SAR) model.
Asunto(s)
Ácido Aminoetilfosfónico/química , Ácido Aminoetilfosfónico/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ligasas/antagonistas & inhibidores , Ligasas/química , Fosfatos de Poliisoprenilo/metabolismo , Sesquiterpenos/metabolismo , Regulación Alostérica/efectos de los fármacos , Humanos , Ligasas/metabolismo , Modelos Moleculares , Conformación Proteica , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: High tibial osteotomy (HTO) is often performed to postpone or avoid the need for subsequent total knee arthroplasty (TKA). We designed the present study to investigate the incidence rate and risk factors for subsequent revision in patients treated with HTO compared with those managed with TKA. METHODS: In this retrospective nationwide cohort study, we reviewed the South Korean National Health Insurance claims database from January 1, 2009, to August 31, 2017. We evaluated patients ≥41 years old who had undergone TKA or HTO as the primary surgical procedure without a history of having undergone either procedure during the preceding 2 years. By including only new interventions without such prior surgery, we could eliminate the influence of previous TKA and HTO treatments. Multivariable logistic regression models were used to compare the risk of revision between the groups after propensity score matching with inverse probability of treatment weighting (IPTW). Revision was defined as conversion to revision TKA after primary TKA and conversion to TKA after HTO. RESULTS: After applying the IPTW, a total of 436,538 patients with TKA and 452,724 patients with HTO were identified. The risk of revision during the entire study period was higher for patients with HTO than for patients with TKA (adjusted hazard ratio [HR], 2.47). The Kaplan-Meier 8-year survival was 97.8% in the TKA group and 91.5% in the HTO group. Compared with patients with TKA, patients with HTO had an increased risk of revision in cases of advanced age (HR of 1.85 for patients who were ≥61 to 69 years old and HR of 4.17 for those who were ≥70 years old), female sex (HR, 2.90), recipients of Medical Aid program benefits (HR, 4.77), the presence of hyperlipidemia (HR, 3.70), the presence of diabetes (HR, 4.86), and the presence of osteoporosis (HR, 3.53). However, younger patients with HTO (≤60 years old) had a lower risk of subsequent revision (HR, 0.64). CONCLUSIONS: The risk of revision was higher for patients with HTO than for patients with TKA. The risk factors for subsequent revision in patients with HTO in our cohort of patients were advanced age (>60 years), female sex, receipt of Medical Aid, and the presence of comorbidities, such as diabetes, osteoporosis, and hyperlipidemia. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.
Asunto(s)
Artroplastia de Reemplazo de Rodilla/efectos adversos , Osteoartritis de la Rodilla/cirugía , Osteotomía/efectos adversos , Complicaciones Posoperatorias/epidemiología , Reoperación , Tibia/cirugía , Adulto , Anciano , Femenino , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Osteoartritis de la Rodilla/complicaciones , Complicaciones Posoperatorias/cirugía , Puntaje de Propensión , República de Corea , Estudios Retrospectivos , Factores de Riesgo , Resultado del TratamientoRESUMEN
Understanding the principles of protein dynamics will help guide engineering of protein function: altering protein motions may be a barrier to success or may be an enabling tool for protein engineering. The impact of dynamics on protein function is typically reported over a fraction of the full scope of motional timescales. If motional patterns vary significantly at different timescales, then only by monitoring motions broadly will we understand the impact of protein dynamics on engineering functional proteins. Using an integrative approach combining experimental and in silico methodologies, we elucidate protein dynamics over the entire span of fast to slow timescales (ps to ms) for a laboratory-engineered system composed of five interrelated ß-lactamases: two natural homologs and three laboratory-recombined variants. Fast (ps-ns) and intermediate (ns-µs) dynamics were mostly conserved. However, slow motions (µs-ms) were few and conserved in the natural homologs yet were numerous and widely dispersed in their recombinants. Nonetheless, modified slow dynamics were functionally tolerated. Crystallographic B-factors from high-resolution X-ray structures were partly predictive of the conserved motions but not of the new slow motions captured in our solution studies. Our inspection of protein dynamics over a continuous range of timescales vividly illustrates the complexity of dynamic impacts of protein engineering as well as the functional tolerance of an engineered enzyme system to new slow motions.
Asunto(s)
Modelos Moleculares , Conformación Proteica , Ingeniería de Proteínas , beta-Lactamasas/química , Sitios de Unión , Cristalografía por Rayos X , Cinética , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , beta-Lactamasas/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0214279.].
RESUMEN
There is debate in the literature whether rotating hinge knee (RHK) or constrained condylar knee (CCK) prostheses lead to better clinical outcomes and survival rates in patients undergoing revision total knee arthroplasty (RTKA). The purpose of this meta-analysis is to compare the survivorship and clinical outcomes of RHK and CCK prostheses. In this meta-analysis, we reviewed studies that evaluated pain and function scores, range of motion (ROM), complications, and survival rates in patients treated with RHK or CCK with short-term (<5 years) or midterm (5-10 years) follow-up. The survivorship was considered as the time to additional surgical intervention such as removal or revision of the components. A total of 12 studies (one randomized study and 11 non-randomized studies) met the inclusion criteria and were analyzed in detail. The proportion of the knees in which short-term (<5 years) survival rates (RHK, 83/95; CCK, 111/148; odds ratio [OR] 0.52; 95% CI, 0.24-1.11; P = 0.09) and midterm (5-10 years) survival rates (RHK, 104/128; CCK, 196/234; OR 1.05; 95% CI, 0.56-1.97; P = 0.88) were evaluated did not differ significantly between RHK and CCK prostheses. In addition, there were no significant differences in ROM (95% CI: -0.40 to 9.93; P = 0.07) and complication rates (95% CI: 0.66 to 2.49; P = 0.46). In contrast, CCK groups reported significantly better pain score (95% CI: 0.50 to 2.73; P = 0.005) and function score (95% CI: 0.01 to 2.00; P = 0.05) than RHK groups. This meta-analysis revealed that 87.4% of RHK and 75.0% of CCK prostheses survive at short-term (<5 years), while 81.3% of RHK and 83.8% of CCK prostheses survive at midterm (5-10 years). The differences in standardized mean pain and function scores we detected were likely to be imperceptible to patients and almost certainly below the minimum clinically important level, despite a significant difference in both groups. Based on the findings of the current meta-analysis, RHK prostheses continue to be an option in complex RTKA with reasonable midterm survivorship.
Asunto(s)
Artroplastia de Reemplazo de Rodilla/instrumentación , Articulación de la Rodilla/cirugía , Prótesis de la Rodilla , Falla de Prótesis , Fenómenos Biomecánicos , Femenino , Humanos , Masculino , Dimensión del Dolor , Diseño de Prótesis , Rango del Movimiento Articular , Reoperación , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Vías Biosintéticas/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Geraniltranstransferasa/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Descubrimiento de Drogas , Inhibidores Enzimáticos/uso terapéutico , Farnesiltransferasa/metabolismo , Geraniltranstransferasa/metabolismo , Humanos , Ácido Mevalónico/metabolismo , Modelos Moleculares , Neoplasias/metabolismo , Fosfatos de Poliisoprenilo/antagonistas & inhibidores , Fosfatos de Poliisoprenilo/metabolismo , Prenilación de Proteína/efectos de los fármacos , Sesquiterpenos/antagonistas & inhibidores , Sesquiterpenos/metabolismoRESUMEN
Loss-of-function mutations in the recycling endosomal (Na+,K+)/H+ exchanger gene SLC9A6/NHE6 result in overacidification and dysfunction of endosomal-lysosomal compartments, and cause a neurodevelopmental and degenerative form of X-linked intellectual disability called Christianson Syndrome (CS). However, knowledge of the disease heterogeneity of CS is limited. Here, we describe the clinical features and underlying molecular and cellular mechanisms associated with a CS patient carrying a de novo missense variant (p.Gly218Arg; G218R) of a conserved residue in its ion translocation domain that results in a potential gain-of-function. The patient manifested several core symptoms typical of CS, including pronounced cognitive impairment, mutism, epilepsy, ataxia and microcephaly; however, deterioration of motor function often observed after the first decade of life in CS children with total loss of SLC9A6/NHE6 function was not evident. In transfected non-neuronal cells, complex glycosylation and half-life of the G218R were significantly decreased compared to the wild-type transporter. This correlated with elevated ubiquitination and partial proteasomal-mediated proteolysis of G218R. However, a major fraction was delivered to the plasma membrane and endocytic pathways. Compared to wild-type, G218R-containing endosomes were atypically alkaline and showed impaired uptake of recycling endosomal cargo. Moreover, instead of accumulating in recycling endosomes, G218R was redirected to multivesicular bodies/late endosomes and ejected extracellularly in exosomes rather than progressing to lysosomes for degradation. Attenuated acidification and trafficking of G218R-containing endosomes were also observed in transfected hippocampal neurons, and correlated with diminished dendritic branching and density of mature mushroom-shaped spines and increased appearance of filopodia-like protrusions. Collectively, these findings expand our understanding of the genetic diversity of CS and further elucidate a critical role for SLC9A6/NHE6 in fine-tuning recycling endosomal pH and cargo trafficking, processes crucial for the maintenance of neuronal polarity and mature synaptic structures.
Asunto(s)
Ataxia/genética , Ataxia/patología , Endosomas/metabolismo , Epilepsia/genética , Epilepsia/patología , Mutación con Ganancia de Función , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Microcefalia/genética , Microcefalia/patología , Neuronas/patología , Trastornos de la Motilidad Ocular/genética , Trastornos de la Motilidad Ocular/patología , Intercambiadores de Sodio-Hidrógeno/genética , Adulto , Animales , Atrofia , Cricetulus , Dendritas/patología , Vesículas Extracelulares/metabolismo , Células HeLa , Hipocampo/patología , Humanos , Masculino , Mutación Missense , Intercambiadores de Sodio-Hidrógeno/química , Adulto JovenRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Geranilgeranil-Difosfato Geranilgeraniltransferasa/antagonistas & inhibidores , Mieloma Múltiple/patología , Prenilación de Proteína/efectos de los fármacos , Apoptosis/efectos de los fármacos , Dominio Catalítico , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/química , Geranilgeranil-Difosfato Geranilgeraniltransferasa/química , Geranilgeranil-Difosfato Geranilgeraniltransferasa/metabolismo , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Pirimidinas/química , Pirimidinas/farmacología , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
Human farnesyl pyrophosphate synthase (hFPPS) catalyzes the production of the 15-carbon isoprenoid farnesyl pyrophosphate. The enzyme is a key regulator of the mevalonate pathway and a well-established drug target. Notably, it was elucidated as the molecular target of nitrogen-containing bisphosphonates, a class of drugs that have been widely successful against bone resorption disorders. More recently, research has focused on the anticancer effects of these inhibitors. In order to achieve increased non-skeletal tissue exposure, we created phenylaminopyridine bisphosphonates (PNP-BPs) that have bulky hydrophobic side chains through a structure-based approach. Some of these compounds have proven to be more potent than the current clinical drugs in a number of antiproliferation assays using multiple myeloma cell lines. In the present work, we characterized the binding of our most potent PNP-BPs to the target enzyme, hFPPS. Co-crystal structures demonstrate that the molecular interactions designed to elicit tighter binding are indeed established. We carried out thermodynamic studies as well; the newly introduced protein-ligand interactions are clearly reflected in the enthalpy of binding measured, which is more favorable for the new PNP-BPs than for the lead compound. These studies also indicate that the affinity of the PNP-BPs to hFPPS is comparable to that of the current drug risedronate. Risedronate forms additional polar interactions via its hydroxyl functional group and thus exhibits more favorable binding enthalpy; however, the entropy of binding is more favorable for the PNP-BPs, owing to the greater desolvation effects resulting from their large hydrophobic side chains. These results therefore confirm the overall validity of our drug design strategy. With a distinctly different molecular scaffold, the PNP-BPs described in this report represent an interesting new group of future drug candidates. Further investigation should follow to characterize the tissue distribution profile and assess the potential clinical benefits of these compounds.
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Difosfonatos/metabolismo , Geraniltranstransferasa/química , Geraniltranstransferasa/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Cristalografía por Rayos X , Difosfonatos/química , Humanos , Unión Proteica , TermodinámicaRESUMEN
The human farnesyl pyrophosphate synthase (hFPPS), a key regulatory enzyme in the mevalonate pathway, catalyzes the biosynthesis of the C-15 isoprenoid farnesyl pyrophosphate (FPP). FPP plays a crucial role in the post-translational prenylation of small GTPases that perform a plethora of cellular functions. Although hFPPS is a well-established therapeutic target for lytic bone diseases, the currently available bisphosphonate drugs exhibit poor cellular uptake and distribution into nonskeletal tissues. Recent drug discovery efforts have focused primarily on allosteric inhibition of hFPPS and the discovery of non-bisphosphonate drugs for potentially treating nonskeletal diseases. Hit-to-lead optimization of a new series of thienopyrimidine-based monosphosphonates (ThP-MPs) led to the identification of analogs with nanomolar potency in inhibiting hFPPS. Their interactions with the allosteric pocket of the enzyme were characterized by crystallography, and the results provide further insight into the pharmacophore requirements for allosteric inhibition.
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Inhibidores Enzimáticos/farmacología , Geraniltranstransferasa/antagonistas & inhibidores , Pirimidinas/farmacología , Descubrimiento de Drogas , HumanosRESUMEN
Farnesyl pyrophosphate synthase (FPPS) is an enzyme of the mevalonate pathway and a well-established therapeutic target. Recent research has focused around a newly identified druggable pocket near the enzyme's active site. Pharmacological exploitation of this pocket is deemed promising; however, its natural biological function, if any, is yet unknown. Here we report that the product of FPPS, farnesyl pyrophosphate (FPP), can bind to this pocket and lock the enzyme in an inactive state. The Kd for this binding is 5-6 µM, within a catalytically relevant range. These results indicate that FPPS activity is sensitive to the product concentration. Kinetic analysis shows that the enzyme is inhibited through FPP accumulation. Having a specific physiological effector, FPPS is a bona fide allosteric enzyme. This allostery offers an exquisite mechanism for controlling prenyl pyrophosphate levels in vivo and thus contributes an additional layer of regulation to the mevalonate pathway.
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Regulación Alostérica , Geraniltranstransferasa/química , Geraniltranstransferasa/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Sesquiterpenos/metabolismo , Dominio Catalítico , Humanos , Cinética , Ácido Mevalónico/química , Ácido Mevalónico/metabolismo , Fosfatos de Poliisoprenilo/química , Sesquiterpenos/químicaRESUMEN
In order to explore the interactions of bisphosphonate ligands with the active site and an allosteric pocket of the human farnesyl pyrophosphate synthase (hFPPS), substituted indole and azabenzimidazole bisphosphonates were designed as chameleon ligands. NMR and crystallographic studies revealed that these compounds can occupy both sub-pockets of the active site cavity, as well as the allosteric pocket of hFPPS in the presence of the enzyme's Mg(2+) ion cofactor. These results are consistent with the previously proposed hypothesis that the allosteric pocket of hFPPS, located near the active site, plays a feed-back regulatory role for this enzyme.