RESUMO
Allosteric regulation of inosine 5'-monophosphate dehydrogenase (IMPDH), an essential enzyme of purine metabolism, contributes to the homeostasis of adenine and guanine nucleotides. However, the precise molecular mechanism of IMPDH regulation in bacteria remains unclear. Using biochemical and cryo-EM approaches, we reveal the intricate molecular mechanism of the IMPDH allosteric regulation in mycobacteria. The enzyme is inhibited by both GTP and (p)ppGpp, which bind to the regulatory CBS domains and, via interactions with basic residues in hinge regions, lock the catalytic core domains in a compressed conformation. This results in occlusion of inosine monophosphate (IMP) substrate binding to the active site and, ultimately, inhibition of the enzyme. The GTP and (p)ppGpp allosteric effectors bind to their dedicated sites but stabilize the compressed octamer by a common mechanism. Inhibition is relieved by the competitive displacement of GTP or (p)ppGpp by ATP allowing IMP-induced enzyme expansion. The structural knowledge and mechanistic understanding presented here open up new possibilities for the development of allosteric inhibitors with antibacterial potential.
Assuntos
Guanosina Trifosfato , IMP Desidrogenase , IMP Desidrogenase/metabolismo , IMP Desidrogenase/química , IMP Desidrogenase/antagonistas & inibidores , Regulação Alostérica , Guanosina Trifosfato/metabolismo , Microscopia Crioeletrônica , Domínio Catalítico , Modelos Moleculares , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Guanosina Pentafosfato/metabolismo , Inosina Monofosfato/metabolismo , Inosina Monofosfato/química , Ligação Proteica , Trifosfato de Adenosina/metabolismo , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/metabolismoRESUMO
The human inosine monophosphate dehydrogenase (hIMPDH) is a metabolic enzyme that possesses a unique ability to self-assemble into higher-order structures, forming cytoophidia. The hIMPDH II isoform is more active in chronic myeloid leukemia (CML) cancer cells, making it a promising target for anti-leukemic therapy. However, the structural details and molecular mechanisms of the dynamics of hIMPDHcytoophidia assembly in vitro need to be better understood, and it is crucial to reconstitute the computational nucleoplasm model with cytophilic-like polymers in vitro to characterize their structure and function. Finally, a computational model and its dynamics of the nucleoplasm for CML cells have been proposed in this short review. This research on nucleoplasm aims to aid the scientific community's understanding of how metabolic enzymes like hIMPDH function in cancer and normal cells. However, validating and justifying the computational results from modeling and simulation with experimental data is essential. The new insights gained from this research could explain the structure/topology, geometrical, and electronic consequences of hIMPDH inhibitors on leukemic and normal cells. They could lead to further advancements in the knowledge of nucleoplasmic chemical reaction dynamics.
Assuntos
Antineoplásicos , IMP Desidrogenase , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , IMP Desidrogenase/antagonistas & inibidores , IMP Desidrogenase/química , IMP Desidrogenase/metabolismo , Núcleo Celular/metabolismo , Leucemia/tratamento farmacológico , Simulação de Dinâmica Molecular , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Desenho de Fármacos , Modelos MolecularesRESUMO
The main structural difference between the mutation-susceptible retinal isoforms of inosine 5´-monophosphate dehydrogenase-1 (IMPDH-1) with the canonical form resides in the C- and N-terminal peptide extensions with unknown structural/functional impacts. In this report, we aimed to experimentally evaluate the functional impact of these extensions on the specific/non-specific single-stranded DNA (ssDNA)-binding activities relative to those of the canonical form. Our in silico findings indicated the possible contribution of the C-terminal segment to the reduced flexibility of the Bateman domain of the enzyme. In addition, the in silico data indicated that the N-terminal tail acts by altering the distance between the tetramers in the concave octamer complex (the native form) of the enzyme. The overall impact of these predicted structural variations became evident, first, through higher Km values with respect to either of the substrates relative to the canonical isoform, as reported previously (Andashti et al. in Mol Cell Biochem 465(1):155-164, 2020). Secondary, the binding of the recombinant mouse retinal isoform IMPDH1 (603) to its specific Rhodopsin target gene was significantly augmented while its binding to non-specific ssDNA was lower than that of the canonical isoform. The DNA-binding activity of the other mouse retinal isoform, IMPDH1(546), to specific and non-specific ssDNA was lower than that of the canonical form most probably due to the in silico predicted rigidity created in the Bateman domain by the C-terminal peptide extension. Furthermore, the DNA binding to the Rhodopsin target gene by each of the IMPDH isoforms influenced in the presence of GTP (Guanosine triphosphate) and ATP (Adenosine triphosphate).
Assuntos
IMP Desidrogenase , IMP Desidrogenase/metabolismo , IMP Desidrogenase/química , IMP Desidrogenase/genética , Animais , Camundongos , Isoenzimas/metabolismo , Isoenzimas/química , Isoenzimas/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Retina/metabolismo , Retina/enzimologia , Ligação Proteica , HumanosRESUMO
IMP dehydrogenase and GMP reductase are enzymes from the same protein family with analogous structures and catalytic mechanisms that have gained attention because of their essential roles in nucleotide metabolism and as potential drug targets. This study focusses on GuaB3, a less-explored enzyme within this family. Phylogenetic analysis uncovers GuaB3's independent evolution from other members of the family and it predominantly occurs in Cyanobacteria. Within this group, GuaB3 functions as a unique IMP dehydrogenase, while its counterpart in Actinobacteria has a yet unknown function. Synechocystis sp. PCC6803 GuaB3 structures demonstrate differences in the active site compared to canonical IMP dehydrogenases, despite shared catalytic mechanisms. These findings highlight the essential role of GuaB3 in Cyanobacteria, provide insights into the diversity and evolution of the IMP dehydrogenase protein family, and reveal a distinctive characteristic in nucleotide metabolism, potentially aiding in combating harmful cyanobacterial blooms-a growing concern for humans and wildlife.
Assuntos
Cianobactérias , IMP Desidrogenase , Humanos , IMP Desidrogenase/química , IMP Desidrogenase/metabolismo , Filogenia , Catálise , Nucleotídeos/metabolismo , Cianobactérias/genéticaRESUMO
Onchocerciasis is a vector-borne disease caused by the filarial nematode Onchocerca volvulus, which is responsible for most of the visual impairments recorded in Africa, Asia and the Americas. It is known that O. volvulus has similar molecular and biological characteristics as Onchocerca ochengi in cattle. This study was designed to screen for immunogenic epitopes and binding pockets of O. ochengi IMPDH and GMPR ligands using immunoinformatic approaches. In this study, a total of 23 B cell epitopes for IMPDH and 7 B cell epitopes for GMPR were predicted using ABCpred tool, Bepipred 2.0 and Kolaskar and Tongaonkar methods. The CD4+ Th computational results showed 16 antigenic epitopes from IMPDH with strong binding affinity for DRB1_0301, DRB3_0101, DRB1_0103 and DRB1_1501 MHC II alleles while 8 antigenic epitopes from GMPR were predicted to bind DRB1_0101 and DRB1_0401 MHC II alleles, respectively. For the CD8+ CTLs analysis, 8 antigenic epitopes from IMPDH showed strong binding affinity to human leukocyte antigen HLA-A*26:01, HLA-A*03:01, HLA-A*24:02 and HLA-A*01:01 MHC I alleles while 2 antigenic epitopes from GMPR showed strong binding affinity to HLA-A*01:01 allele, respectively. The immunogenic B cell and T cell epitopes were further evaluated for antigenicity, non-alllergernicity, toxicity, IFN-gamma, IL4 and IL10. The docking score revealed favorable binding free energy with IMP and MYD scoring the highest binding affinity at -6.6 kcal/mol with IMPDH and -8.3 kcal/mol with GMPR. This study provides valuable insight on IMPDH and GMPR as potential drug targets and for the development of multiple epitope vaccine candidates.Communicated by Ramaswamy H. Sarma.
Assuntos
Onchocerca , Vacinas , Humanos , Animais , Bovinos , Onchocerca/metabolismo , Imunoinformática , GMP Redutase/química , GMP Redutase/metabolismo , IMP Desidrogenase/química , IMP Desidrogenase/metabolismo , Epitopos de Linfócito B , Epitopos de Linfócito T , Guanosina , Inosina , Antígenos HLA-ARESUMO
Inosine-5'-monophosphate dehydrogenase (IMPDH), a key regulatory enzyme in purine nucleotide biosynthesis, dynamically assembles filaments in response to changes in metabolic demand. Humans have two isoforms: IMPDH2 filaments reduce sensitivity to feedback inhibition, while IMPDH1 assembly remains uncharacterized. IMPDH1 plays a unique role in retinal metabolism, and point mutants cause blindness. Here, in a series of cryogenic-electron microscopy structures we show that human IMPDH1 assembles polymorphic filaments with different assembly interfaces in extended and compressed states. Retina-specific splice variants introduce structural elements that reduce sensitivity to GTP inhibition, including stabilization of the extended filament form. Finally, we show that IMPDH1 disease mutations fall into two classes: one disrupts GTP regulation and the other has no effect on GTP regulation or filament assembly. These findings provide a foundation for understanding the role of IMPDH1 in retinal function and disease and demonstrate the diverse mechanisms by which metabolic enzyme filaments are allosterically regulated.
Assuntos
IMP Desidrogenase/genética , Retina/enzimologia , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sítios de Ligação , Domínio Catalítico , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , IMP Desidrogenase/química , IMP Desidrogenase/ultraestrutura , Modelos Moleculares , NAD/metabolismo , Doenças Retinianas/genéticaAssuntos
Grânulos Citoplasmáticos/metabolismo , IMP Desidrogenase/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animais , Biocatálise , Linhagem Celular Tumoral , Células Cultivadas , Grânulos Citoplasmáticos/química , Imunofluorescência/métodos , Humanos , IMP Desidrogenase/química , Inosina Monofosfato/metabolismo , Secreção de Insulina , Multimerização Proteica , Ribonucleotídeos/metabolismo , Xantina/metabolismoRESUMO
Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo guanine nucleotide biosynthesis. Its activity is negatively regulated by the binding of GTP. IMPDH can form a membraneless subcellular structure termed the cytoophidium in response to certain changes in the metabolic status of the cell. The polymeric form of IMPDH, which is the subunit of the cytoophidium, has been shown to be more resistant to the inhibition by GTP at physiological concentrations, implying a functional correlation between cytoophidium formation and the upregulation of GTP biosynthesis. Herein we demonstrate that zebrafish IMPDH1b and IMPDH2 isoforms can assemble abundant cytoophidium in most of cultured cells under stimuli, while zebrafish IMPDH1a shows distinctive properties of forming the cytoophidium in different cell types. Point mutations that disrupt cytoophidium structure in mammalian models also prevent the aggregation of zebrafish IMPDHs. In addition, we discover the presence of the IMPDH cytoophidium in various tissues of larval and adult fish under normal growth conditions. Our results reveal that polymerization and cytoophidium assembly of IMPDH can be a regulatory machinery conserved among vertebrates, and with specific physiological purposes.
Assuntos
Estruturas Citoplasmáticas/ultraestrutura , IMP Desidrogenase/química , Proteínas de Peixe-Zebra/química , Peixe-Zebra/metabolismo , Animais , Linhagem Celular , Estruturas Citoplasmáticas/química , Expressão Gênica , Guanosina Trifosfato/biossíntese , Guanosina Trifosfato/metabolismo , Humanos , IMP Desidrogenase/genética , IMP Desidrogenase/metabolismo , Isoenzimas/química , Isoenzimas/genética , Mutação Puntual , Regulação para Cima , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Defects in inosine monophosphate dehydrogenase-1 (IMPDH1) lead to insufficient biosyntheses of purine nucleotides. In eyes, these defects are believed to cause retinitis pigmentosa (RP). Major retinal isoforms of IMPDH1 are structurally distinct from those in other tissues, by bearing terminal extensions. Using recombinant mouse IMPDH1 (mH1), we evaluated the kinetics and oligomerization states of the retinal isoforms. Moreover, we adopted molecular simulation tools to study the possible effect of terminal tails on the function of major enzyme isoforms with the aim to find structural evidence in favor of contradictory observations on retinal IMPDH1 function. Our findings indicated higher catalytic activity for the major mouse retinal isoform (mH1603) along with lower fibrillation capacity under the influence of ATP. However, higher mass oligomerization products were formed by the mH1 (603) isoform in the presence of the enzyme inhibitors such as GTP and/or MPA. Collectively, our findings demonstrate that the structural differences between the retinal isoforms have led to functional variations possibly to justify the retinal cells' requirements.
Assuntos
IMP Desidrogenase/metabolismo , Retina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Humanos , Ligação de Hidrogênio , IMP Desidrogenase/antagonistas & inibidores , IMP Desidrogenase/química , IMP Desidrogenase/genética , Cinética , Camundongos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/químicaRESUMO
Cryptosporidiosis accounts for a surge in infant (<5 years) mortality and morbidity. To date, several drug discovery efforts have been put in place to develop effective therapeutic options against the causative parasite. Based on a recent report, P131 spares inosine monophosphate dehydrogenase (IMPDH) in a eukaryotic model (mouse IMPDH (mIMPDH)) while binding selectively to the NAD+ site in Cryptosporidium parvum (CpIMPDH). However, no structural detail exists on the underlining mechanisms of P131-CpIMPDH selective targeting till date. To this effect, we investigate the selective inhibitory dynamics of P131 in CpIMPDH relative to mIMPDH via molecular biocomputation methods. Pairwise sequence alignment revealed prominent variations at the NAD+ binding regions of both proteins that accounted for disparate P131 binding activities. The influence of these variations was further revealed by the MM/PBSA energy estimations coupled with per-residue energy decomposition which monitored the systematic binding of the compound. Furthermore, relative high-affinity interactions occurred at the CpIMPDH NAD+ site which were majorly mediated by SER22, VAL24, PRO26, SER354, GLY357, and TYR358 located on chain D. These residues are unique to the parasite IMPDH form and not in the eukaryotic protein, highlighting variations that account for preferential P131 binding. Molecular insights provided herein corroborate previous experimental reports and further underpin the basis of CpIMPDH inhibitor selectivity. Findings from this study could present attractive prospects toward the design of novel anticryptosporidials with improved selectivity and binding affinity against parasitic targets.
Assuntos
Criptosporidiose/tratamento farmacológico , Cryptosporidium parvum/efeitos dos fármacos , Desenho de Fármacos , Inibidores Enzimáticos/química , IMP Desidrogenase/química , Simulação de Dinâmica Molecular , Animais , Sítios de Ligação , Biologia Computacional , Cryptosporidium parvum/metabolismo , Modelos Animais de Doenças , IMP Desidrogenase/metabolismo , Camundongos , Modelos Teóricos , NAD/química , Ligação Proteica , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Citrus huanglongbing (HLB) is a destructive disease that causes significant damage to many citrus producing areas worldwide. To date, no strategy against this disease has been established. Inosine 5'-monophosphate dehydrogenase (IMPDH) plays crucial roles in the de novo synthesis of guanine nucleotides. This enzyme is used as a potential target to treat bacterial infection. In this study, the crystal structure of a deletion mutant of CLas IMPDHΔ98-201 in the apo form was determined. Eight known bioactive compounds were used as ligands for molecular docking. The results showed that bronopol and disulfiram bound to CLas IMPDHΔ98-201 with high affinity. These compounds were tested for their inhibition against CLas IMPDHΔ98-201 activity. Bronopol and disulfiram showed high inhibition at nanomolar concentrations, and bronopol was found to be the most potent molecule (Ki = 234 nM). The Ki value of disulfiram was 616 nM. These results suggest that bronopol and disulfiram can be considered potential candidate agents for the development of CLas inhibitors.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/química , Dissulfiram/química , Inibidores Enzimáticos/química , IMP Desidrogenase/química , Propilenoglicóis/química , Antibacterianos/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Citrus/efeitos dos fármacos , Citrus/microbiologia , Clonagem Molecular , Cristalografia por Raios X , Dissulfiram/metabolismo , Inibidores Enzimáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , IMP Desidrogenase/antagonistas & inibidores , IMP Desidrogenase/metabolismo , Cinética , Liberibacter/enzimologia , Liberibacter/genética , Liberibacter/patogenicidade , Ligantes , Simulação de Acoplamento Molecular , Doenças das Plantas/microbiologia , Doenças das Plantas/terapia , Propilenoglicóis/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5'-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.
Assuntos
Coenzimas/química , Cristalização , IMP Desidrogenase/química , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Guanosina Monofosfato , Modelos Moleculares , Conformação Proteica , Células Sf9 , Trypanosoma brucei brucei/genéticaRESUMO
Inosine monophosphate dehydrogenase (IMPDH) mediates the first committed step in guanine nucleotide biosynthesis and plays important roles in cellular proliferation and the immune response. IMPDH reversibly polymerizes in cells and tissues in response to changes in metabolic demand. Self-assembly of metabolic enzymes is increasingly recognized as a general mechanism for regulating activity, typically by stabilizing specific conformations of an enzyme, but the regulatory role of IMPDH filaments has remained unclear. Here, we report a series of human IMPDH2 cryo-EM structures in both active and inactive conformations. The structures define the mechanism of filament assembly, and reveal how filament-dependent allosteric regulation of IMPDH2 makes the enzyme less sensitive to feedback inhibition, explaining why assembly occurs under physiological conditions that require expansion of guanine nucleotide pools. Tuning sensitivity to an allosteric inhibitor distinguishes IMPDH from other metabolic filaments, and highlights the diversity of regulatory outcomes that can emerge from self-assembly.
Assuntos
Microscopia Crioeletrônica/métodos , IMP Desidrogenase/metabolismo , Regulação Alostérica , Humanos , IMP Desidrogenase/química , Conformação ProteicaRESUMO
Mutations in the retinal inosine monophosphate dehydrogenase1 (IMPDH1) gene is believed to be one cause of retinitis pigmentosa (RP). The main structural difference between the mutation-susceptible retinal isoforms with canonical one resides in the C- and N-terminal extensions. There are limited studies on the structure and function of terminal peptide extensions of the IMPDH1 retinal isoforms. Using recombinant murine IMPDH1 (mH1), we evaluated the kinetics of the retinal isoforms along with inhibition by some of the purine nucleotides. Molecular modeling tools were also applied to study the probable effect(s) of the terminal peptide tails on the function of the retinal isoforms. Molecular dynamic simulations indicated the possible impact of the end-terminal segments on the enzyme function through interactions with the enzyme's finger domain, affecting its critical pseudo barrel structure. The higher experimentally-determined Km and Ki values of the retinal mIMPDH1 (546) and mIMPDH1 (603) relative to that of the canonical isoform, mIMPDH1 (514), might clearly be due to these interactions. Furthermore and despite of the canonical isoform, the retinal isoforms of mH1 exhibited no NAD+ substrate inhibition. The resent data would certainly provide the ground for future evaluation of the physiological significance of these variations.
Assuntos
IMP Desidrogenase/química , Modelos Moleculares , Retina/enzimologia , Animais , IMP Desidrogenase/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , CamundongosRESUMO
IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the de novo guanine nucleotide biosynthetic pathway. Because of its involvement in the control of cell division and proliferation, IMPDH represents a therapeutic for managing several diseases, including microbial infections and cancer. IMPDH must be tightly regulated, but the molecular mechanisms responsible for its physiological regulation remain unknown. To this end, we recently reported an important role of adenine and guanine mononucleotides that bind to the regulatory Bateman domain to allosterically modulate the catalytic activity of eukaryotic IMPDHs. Here, we have used enzyme kinetics, X-ray crystallography, and small-angle X-ray scattering (SAXS) methodologies to demonstrate that adenine/guanine dinucleoside polyphosphates bind to the Bateman domain of IMPDH from the fungus Ashbya gossypii with submicromolar affinities. We found that these dinucleoside polyphosphates modulate the catalytic activity of IMPDHs in vitro by efficiently competing with the adenine/guanine mononucleotides for the allosteric sites. These results suggest that dinucleoside polyphosphates play important physiological roles in the allosteric regulation of IMPDHs by adding an additional mechanism for fine-tuning the activities of these enzymes. We propose that these findings may have important implications for the design of therapeutic strategies to inhibit IMPDHs.
Assuntos
Fosfatos de Dinucleosídeos/química , IMP Desidrogenase/química , Conformação Proteica , Domínios Proteicos/genética , Regulação Alostérica/genética , Infecções Bacterianas/genética , Infecções Bacterianas/microbiologia , Sítios de Ligação/genética , Catálise , Cristalografia por Raios X , Fosfatos de Dinucleosídeos/genética , Eremothecium/genética , Nucleotídeos de Guanina , Humanos , IMP Desidrogenase/genética , IMP Desidrogenase/ultraestrutura , Modelos Moleculares , Neoplasias/genética , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Members of a large family of Ankyrin Repeat Domain (ANKRD) proteins regulate numerous cellular processes by binding to specific protein targets and modulating their activity, stability, and other properties. The same ANKRD protein may interact with different targets and regulate distinct cellular pathways. The mechanisms responsible for switches in the ANKRDs' behavior are often unknown. We show that cells' metabolic state can markedly alter interactions of an ANKRD protein with its target and the functional outcomes of this interaction. ANKRD9 facilitates degradation of inosine monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme in GTP biosynthesis. Under basal conditions ANKRD9 is largely segregated from the cytosolic IMPDH2 in vesicle-like structures. Upon nutrient limitation, ANKRD9 loses its vesicular pattern and assembles with IMPDH2 into rodlike filaments, in which IMPDH2 is stable. Inhibition of IMPDH2 activity with ribavirin favors ANKRD9 binding to IMPDH2 rods. The formation of ANKRD9/IMPDH2 rods is reversed by guanosine, which restores ANKRD9 associations with the vesicle-like structures. The conserved Cys109Cys110 motif in ANKRD9 is required for the vesicle-to-rods transition as well as binding and regulation of IMPDH2. Oppositely to overexpression, ANKRD9 knockdown increases IMPDH2 levels and prevents formation of IMPDH2 rods upon nutrient limitation. Taken together, the results suggest that a guanosine-dependent metabolic switch determines the mode of ANKRD9 action toward IMPDH2.
Assuntos
IMP Desidrogenase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sítios de Ligação , Vesículas Citoplasmáticas/metabolismo , Guanosina/metabolismo , Células HEK293 , Células HeLa , Humanos , IMP Desidrogenase/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Nutrientes/metabolismo , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
Tuberculosis (TB) is a major infectious disease associated increasingly with drug resistance. Thus, new anti-tubercular agents with novel mechanisms of action are urgently required for the treatment of drug-resistant TB. In prior work, we identified compound 1 (cyclohexyl(4-(isoquinolin-5-ylsulfonyl)piperazin-1-yl)methanone) and showed that its anti-tubercular activity is attributable to inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) in Mycobacterium tuberculosis. In the present study, we explored the structure-activity relationship around compound 1 by synthesizing and evaluating the inhibitory activity of analogues against M. tuberculosis IMPDH in biochemical and whole-cell assays. X-ray crystallography was performed to elucidate the mode of binding of selected analogues to IMPDH. We establish the importance of the cyclohexyl, piperazine and isoquinoline rings for activity, and report the identification of an analogue with IMPDH-selective activity against a mutant of M. tuberculosis that is highly resistant to compound 1. We also show that the nitrogen in urea analogues is required for anti-tubercular activity and identify benzylurea derivatives as promising inhibitors that warrant further investigation.
Assuntos
Antituberculosos/farmacologia , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Isoquinolinas/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Piperazinas/farmacologia , Antituberculosos/síntese química , Antituberculosos/química , Domínio Catalítico , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , IMP Desidrogenase/química , Isoquinolinas/síntese química , Isoquinolinas/química , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Piperazinas/síntese química , Piperazinas/química , Relação Estrutura-AtividadeRESUMO
Ribavirin (RBV) is a guanosine analogue triazole most commonly used in the treatment of chronic hepatitis C (HCV) infection. Although its mechanism of action is a matter of debate, several possibilities have been proposed, including depletion of guanine nucleotides through inhibition of inosine monophosphate dehydrogenase (IMPDH). IMPDH has been shown to assemble into micron-scale rod- and ring-shaped structures (rods/rings or RR), also called "IMPDH filaments," both in vitro and in vivo. Formation of RR structures can occur naturally, potentially to influence IMPDH activity, or when de novo guanosine monophosphate biosynthesis or IMPDH itself are inhibited by nutrient deprivation or drugs like RBV. Numerous studies have also reported the occurrence of autoantibodies targeting RR structures (anti-RR) in HCV patients previously treated or under treatment with interferon-α and ribavirin (IFN/RBV) combination therapy. For this brief study, we considered the strong association between RR autoantibodies and IFN/RBV treatment, and the lack of data assessing how RBV affects RR formation in a variety of tissues in vivo. First, RR structures formed in the spleen and pancreas of normal mice without any treatment. Then, in RBV-treated mice, we detected RR structures in a number of tissues, including stomach, liver, spleen, kidney, brain, skin, and cardiac and skeletal muscle. We made several intriguing observations: predominance of RR structures in the mucosa and submucosa layers of the stomach wall; a high proportion of RR-positive cells in the cerebral cortex, suggesting that RBV actually crosses the blood-brain barrier; and a higher ratio of rings to rods in the epidermis compared to the dermis layer of the skin. Screening for RR structures appears to be a useful method to track tissue penetration of RBV and the many RR-inducing drugs previously identified.
Assuntos
Antivirais/farmacocinética , IMP Desidrogenase/química , Ribavirina/farmacocinética , Animais , Barreira Hematoencefálica , Córtex Cerebral/química , Hepacivirus/efeitos dos fármacos , Rim/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pâncreas/química , Baço/químicaRESUMO
Rods and rings (RRs) are large linear- or circular-shaped structures typically described as polymers of IMPDH (inosine monophosphate dehydrogenase). They have been observed across a wide variety of cell types and species and can be induced to form by inhibitors of IMPDH. RRs are thought to play a role in the regulation of de novo guanine nucleotide synthesis; however, the function and regulation of RRs is poorly understood. Here we show that the regulatory GTPase, ARL2, a subset of its binding partners, and several resident proteins at the endoplasmic reticulum (ER) also localize to RRs. We also have identified two new inducers of RR formation: AICAR and glucose deprivation. We demonstrate that RRs can be disassembled if guanine nucleotides can be generated by salvage synthesis regardless of the inducer. Finally, we show that there is an ordered addition of components as RRs mature, with IMPDH first forming aggregates, followed by ARL2, and only later calnexin, a marker of the ER. These findings suggest that RRs are considerably more complex than previously thought and that the function(s) of RRs may include involvement of a regulatory GTPase, its effectors, and potentially contacts with intracellular membranes.
Assuntos
IMP Desidrogenase/química , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Carbono-Nitrogênio Ligases/metabolismo , Linhagem Celular , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/ultraestrutura , Proteínas de Ligação ao GTP/metabolismo , Glucose/deficiência , Guanosina/farmacologia , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Cinética , Síndrome de Lesch-Nyhan/patologia , Camundongos , Ácido Micofenólico/farmacologia , Transporte Proteico , Ribonucleotídeos/farmacologiaRESUMO
New drugs and molecular targets are urgently needed to address the emergence and spread of drug-resistant tuberculosis. Mycobacterium tuberculosis ( Mtb) inosine 5'-monophosphate dehydrogenase 2 ( MtbIMPDH2) is a promising yet controversial potential target. The inhibition of MtbIMPDH2 blocks the biosynthesis of guanine nucleotides, but high concentrations of guanine can potentially rescue the bacteria. Herein we describe an expansion of the structure-activity relationship (SAR) for the benzoxazole series of MtbIMPDH2 inhibitors and demonstrate that minimum inhibitory concentrations (MIC) of ≤1 µM can be achieved. The antibacterial activity of the most promising compound, 17b (Q151), is derived from the inhibition of MtbIMPDH2 as demonstrated by conditional knockdown and resistant strains. Importantly, guanine does not change the MIC of 17b, alleviating the concern that guanine salvage can protect Mtb in vivo. These findings suggest that MtbIMPDH2 is a vulnerable target for tuberculosis.