RESUMO
Lysophospholipids are deacylated derivatives of their bilayer forming phospholipid counterparts that are present at low concentrations in cells. Phosphatidylglycerol (PG) is the principal membrane phospholipid in Staphylococcus aureus and lysophosphatidylglycerol (LPG) is detected in low abundance. Here, we used a mass spectrometry screen to identify locus SAUSA300_1020 as the gene responsible for maintaining low concentrations of 1-acyl-LPG in S. aureus. The SAUSA300_1020 gene encodes a protein with a predicted amino terminal transmembrane α-helix attached to a globular glycerophosphodiester phosphodiesterase (GDPD) domain. We determined that the purified protein lacking the hydrophobic helix (LpgDΔN) possesses cation-dependent lysophosphatidylglycerol phospholipase D activity that generates both lysophosphatidic acid (LPA) and cyclic-LPA products and hydrolyzes cyclic-LPA to LPA. Mn2+ was the highest affinity cation and stabilized LpgDΔN to thermal denaturation. LpgDΔN was not specific for the phospholipid headgroup and degraded 1-acyl-LPG, but not 2-acyl-LPG. Furthermore, a 2.1 Å crystal structure shows that LpgDΔN adopts the GDPD variation of the TIM barrel architecture except for the length and positioning of helix α6 and sheet ß7. These alterations create a hydrophobic diffusion path for LPG to access the active site. The LpgD active site has the canonical GDPD metal binding and catalytic residues, and our biochemical characterization of site-directed mutants support a two-step mechanism involving a cyclic-LPA intermediate. Thus, the physiological function of LpgD in S. aureus is to convert LPG to LPA, which is re-cycled into the PG biosynthetic pathway at the LPA acyltransferase step to maintain membrane PG molecular species homeostasis.
Assuntos
Fosfolipase D , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Lisofosfolipídeos/metabolismo , FosfatidilgliceróisRESUMO
Pantothenate kinase-associated neurodegeneration (PKAN) is characterized by a motor disorder with combinations of dystonia, parkinsonism, and spasticity, leading to premature death. PKAN is caused by mutations in the PANK2 gene that result in loss or reduction of PANK2 protein function. PANK2 is one of three kinases that initiate and regulate coenzyme A biosynthesis from vitamin B5, and the ability of BBP-671, an allosteric activator of pantothenate kinases, to enter the brain and elevate coenzyme A was investigated. The metabolic stability, protein binding, and membrane permeability of BBP-671 all suggest that it has the physical properties required to cross the blood-brain barrier. BBP-671 was detected in plasma, liver, cerebrospinal fluid, and brain following oral administration in rodents, demonstrating the ability of BBP-671 to penetrate the brain. The pharmacokinetic and pharmacodynamic properties of orally administered BBP-671 evaluated in cannulated rats showed that coenzyme A (CoA) concentrations were elevated in blood, liver, and brain. BBP-671 elevation of whole-blood acetyl-CoA served as a peripheral pharmacodynamic marker and provided a suitable method to assess target engagement. BBP-671 treatment elevated brain coenzyme A concentrations and improved movement and body weight in a PKAN mouse model. Thus, BBP-671 crosses the blood-brain barrier to correct the brain CoA deficiency in a PKAN mouse model, resulting in improved locomotion and survival and providing a preclinical foundation for the development of BBP-671 as a potential treatment of PKAN. SIGNIFICANCE STATEMENT: The blood-brain barrier represents a major hurdle for drugs targeting brain metabolism. This work describes the pharmacokinetic/pharmacodynamic properties of BBP-671, a pantothenate kinase activator. BBP-671 crosses the blood-brain barrier to correct the neuron-specific coenzyme A (CoA) deficiency and improve motor function in a mouse model of pantothenate kinase-associated neurodegeneration. The central role of CoA and acetyl-CoA in intermediary metabolism suggests that pantothenate kinase activators may be useful in modifying neurological metabolic disorders.
Assuntos
Neurodegeneração Associada a Pantotenato-Quinase , Camundongos , Animais , Ratos , Neurodegeneração Associada a Pantotenato-Quinase/tratamento farmacológico , Neurodegeneração Associada a Pantotenato-Quinase/genética , Acetilcoenzima A/metabolismo , Acetilcoenzima A/uso terapêutico , Coenzima A/metabolismo , Modelos Animais de Doenças , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Encéfalo/metabolismoRESUMO
Propionic acidemia (PA, OMIM 606054) is a devastating inborn error of metabolism arising from mutations that reduce the activity of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). The defects in PCC reduce the concentrations of nonesterified coenzyme A (CoASH), thus compromising mitochondrial function and disrupting intermediary metabolism. Here, we use a hypomorphic PA mouse model to test the effectiveness of BBP-671 in correcting the metabolic imbalances in PA. BBP-671 is a high-affinity allosteric pantothenate kinase activator that counteracts feedback inhibition of the enzyme to increase the intracellular concentration of CoA. Liver CoASH and acetyl-CoA are depressed in PA mice and BBP-671 treatment normalizes the cellular concentrations of these two key cofactors. Hepatic propionyl-CoA is also reduced by BBP-671 leading to an improved intracellular C3:C2-CoA ratio. Elevated plasma C3:C2-carnitine ratio and methylcitrate, hallmark biomarkers of PA, are significantly reduced by BBP-671. The large elevations of malate and α-ketoglutarate in the urine of PA mice are biomarkers for compromised tricarboxylic acid cycle activity and BBP-671 therapy reduces the amounts of both metabolites. Furthermore, the low survival of PA mice is restored to normal by BBP-671. These data show that BBP-671 relieves CoA sequestration, improves mitochondrial function, reduces plasma PA biomarkers, and extends the lifespan of PA mice, providing the preclinical foundation for the therapeutic potential of BBP-671.
Assuntos
Acidemia Propiônica , Camundongos , Animais , Acidemia Propiônica/genética , Metilmalonil-CoA Descarboxilase/genética , Metilmalonil-CoA Descarboxilase/metabolismo , Modelos Animais de Doenças , Mitocôndrias/metabolismo , CarnitinaRESUMO
Pantothenate kinase (PANK) is the critical regulator of intracellular levels of coenzyme A and has emerged as an attractive target for treating neurological and metabolic disorders. This report describes the optimization, synthesis, and full structure-activity relationships of a new chemical series of pantothenate competitive PANK inhibitors. Potent drug-like molecules were obtained by optimizing a high throughput screening hit, using lipophilic ligand efficiency (LipE) derived from human PANK3 IC50 values to guide ligand development. X-ray crystal structures of PANK3 with index inhibitors from the optimization were determined to rationalize the emerging structure activity relationships. The analysis revealed a key bidentate hydrogen bonding interaction between pyridazine and R306' as a major contributor to the LipE gain observed in the optimization. A tractable series of PANK3 modulators with nanomolar potency, excellent LipE values, desirable physicochemical properties, and a well-defined structural binding mode was produced from this study.
Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Piridazinas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Ligação de Hidrogênio , Ligantes , Estrutura Molecular , Piridazinas/síntese química , Piridazinas/química , Relação Estrutura-AtividadeRESUMO
Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis-targeting chimera (PROTAC) approaches, owing to favorable drug-like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs are known to be inherently unstable, readily undergoing hydrolysis in body fluids. Here we show that IMiDs and IMiD-based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy. We designed novel CRBN binders, phenyl glutarimide (PG) analogues, and showed that they retained affinity for CRBN with high ligand efficiency (LE >0.48) and displayed improved chemical stability. Our efforts led to the discovery of PG PROTAC 4 c (SJ995973), a uniquely potent degrader of bromodomain and extra-terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4-11 cells at low picomolar concentrations (IC50 =3â pM; BRD4 DC50 =0.87â nM). These findings strongly support the utility of PG derivatives in the design of CRBN-directed PROTACs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Piperidonas/química , Ubiquitina-Proteína Ligases/química , Humanos , Hidrólise , ProteóliseRESUMO
Pantothenate kinase is the master regulator of CoA biosynthesis and is feedback-inhibited by acetyl-CoA. Comparison of the human PANK3·acetyl-CoA complex to the structures of PANK3 in four catalytically relevant complexes, 5'-adenylyl-ß,γ-imidodiphosphate (AMPPNP)·Mg2+, AMPPNP·Mg2+·pantothenate, ADP·Mg2+·phosphopantothenate, and AMP phosphoramidate (AMPPN)·Mg2+, revealed a large conformational change in the dimeric enzyme. The amino-terminal nucleotide binding domain rotates to close the active site, and this allows the P-loop to engage ATP and facilitates required substrate/product interactions at the active site. Biochemical analyses showed that the transition between the inactive and active conformations, as assessed by the binding of either ATP·Mg2+ or acyl-CoA to PANK3, is highly cooperative indicating that both protomers move in concert. PANK3(G19V) cannot bind ATP, and biochemical analyses of an engineered PANK3/PANK3(G19V) heterodimer confirmed that the two active sites are functionally coupled. The communication between the two protomers is mediated by an α-helix that interacts with the ATP-binding site at its amino terminus and with the substrate/inhibitor-binding site of the opposite protomer at its carboxyl terminus. The two α-helices within the dimer together with the bound ligands create a ring that stabilizes the assembly in either the active closed conformation or the inactive open conformation. Thus, both active sites of the dimeric mammalian pantothenate kinases coordinately switch between the on and off states in response to intracellular concentrations of ATP and its key negative regulators, acetyl(acyl)-CoA.
Assuntos
Acil Coenzima A/química , Mutação de Sentido Incorreto , Fosfotransferases (Aceptor do Grupo Álcool)/química , Acil Coenzima A/metabolismo , Regulação Alostérica , Substituição de Aminoácidos , Humanos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Domínios Proteicos , Estrutura Secundária de ProteínaRESUMO
Nucleophosmin (NPM1) is a multifunctional phospho-protein with critical roles in ribosome biogenesis, tumor suppression, and nucleolar stress response. Here we show that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating conformational states ranging from a highly ordered, folded pentamer to a highly disordered monomer. The monomer-pentamer equilibrium is modulated by posttranslational modification and protein binding. Phosphorylation drives the equilibrium in favor of monomeric forms, and this effect can be reversed by Npm-N binding to its interaction partners. We have identified a short, arginine-rich linear motif in NPM1 binding partners that mediates Npm-N oligomerization. We propose that the diverse functional repertoire associated with NPM1 is controlled through a regulated unfolding mechanism signaled through posttranslational modifications and intermolecular interactions.
Assuntos
Biopolímeros/química , Proteínas Nucleares/química , Sequência de Aminoácidos , Biopolímeros/metabolismo , Cromatografia em Gel , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Eletroforese em Gel de Poliacrilamida Nativa , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/metabolismo , Nucleofosmina , Fosforilação , Ligação Proteica , Conformação ProteicaRESUMO
The sulfonamide class of antibiotics has been in continuous use for over 70years. They are thought to act by directly inhibiting dihydropteroate synthase (DHPS), and also acting as prodrugs that sequester pterin pools by forming dead end pterin-sulfonamide conjugates. In this study, eight pterin-sulfonamide conjugates were synthesized using a novel synthetic strategy and their biochemical and microbiological properties were investigated. The conjugates were shown to competitively inhibit DHPS, and inhibition was enhanced by the presence of pyrophosphate that is crucial to catalysis and is known to promote an ordering of the DHPS active site. The co-crystal structure of Yersinia pestis DHPS bound to one of the more potent conjugates revealed a mode of binding that is similar to that of the enzymatic product analog pteroic acid. The antimicrobial activities of the pterin-sulfonamide conjugates were measured against Escherichia coli in the presence and absence of folate precursors and dependent metabolites. These results show that the conjugates have appreciable antibacterial activity and act by an on target, anti-folate pathway mechanism rather than as simple dead end products.
Assuntos
Antibacterianos/química , Di-Hidropteroato Sintase/antagonistas & inibidores , Pterinas/química , Sulfonamidas/química , Antibacterianos/síntese química , Antibacterianos/farmacologia , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Di-Hidropteroato Sintase/metabolismo , Escherichia coli/efeitos dos fármacos , Ácido Fólico/química , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Yersinia pestis/enzimologiaRESUMO
Following DNA damage, nuclear p53 induces the expression of PUMA, a BH3-only protein that binds and inhibits the antiapoptotic BCL-2 repertoire, including BCL-xL. PUMA, unique among BH3-only proteins, disrupts the interaction between cytosolic p53 and BCL-xL, allowing p53 to promote apoptosis via direct activation of the BCL-2 effector molecules BAX and BAK. Structural investigations using NMR spectroscopy and X-ray crystallography revealed that PUMA binding induced partial unfolding of two α-helices within BCL-xL. Wild-type PUMA or a PUMA mutant incapable of causing binding-induced unfolding of BCL-xL equivalently inhibited the antiapoptotic BCL-2 repertoire to sensitize for death receptor-activated apoptosis, but only wild-type PUMA promoted p53-dependent, DNA damage-induced apoptosis. Our data suggest that PUMA-induced partial unfolding of BCL-xL disrupts interactions between cytosolic p53 and BCL-xL, releasing the bound p53 to initiate apoptosis. We propose that regulated unfolding of BCL-xL provides a mechanism to promote PUMA-dependent signaling within the apoptotic pathways.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Desdobramento de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína bcl-X/metabolismo , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Humanos , Modelos Moleculares , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/química , Proteína bcl-X/químicaRESUMO
Emerging influenza viruses are a serious threat to human health because of their pandemic potential. A promising target for the development of novel anti-influenza therapeutics is the PA protein, whose endonuclease activity is essential for viral replication. Translation of viral mRNAs by the host ribosome requires mRNA capping for recognition and binding, and the necessary mRNA caps are cleaved or "snatched" from host pre-mRNAs by the PA endonuclease. The structure-based development of inhibitors that target PA endonuclease is now possible with the recent crystal structure of the PA catalytic domain. In this study, we sought to understand the molecular mechanism of inhibition by several compounds that are known or predicted to block endonuclease-dependent polymerase activity. Using an in vitro endonuclease activity assay, we show that these compounds block the enzymatic activity of the isolated PA endonuclease domain. Using X-ray crystallography, we show how these inhibitors coordinate the two-metal endonuclease active site and engage the active site residues. Two structures also reveal an induced-fit mode of inhibitor binding. The structures allow a molecular understanding of the structure-activity relationship of several known influenza inhibitors and the mechanism of drug resistance by a PA mutation. Taken together, our data reveal new strategies for structure-based design and optimization of PA endonuclease inhibitors.
Assuntos
Desenho de Fármacos , Endorribonucleases , Inibidores Enzimáticos/química , Virus da Influenza A Subtipo H5N1/enzimologia , Simulação de Acoplamento Molecular , RNA Polimerase Dependente de RNA , Proteínas Virais , Animais , Linhagem Celular , Embrião de Galinha , Galinhas , Cristalografia por Raios X , Cães , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/química , Inibidores Enzimáticos/farmacologia , Humanos , Influenza Aviária/tratamento farmacológico , Influenza Aviária/enzimologia , Estrutura Terciária de Proteína , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/química , Relação Estrutura-Atividade , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/químicaRESUMO
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is an essential enzyme in the microbial folate biosynthetic pathway. This pathway has proven to be an excellent target for antimicrobial development, but widespread resistance to common therapeutics including the sulfa drugs has stimulated interest in HPPK as an alternative target in the pathway. A screen of a pterin-biased compound set identified several HPPK inhibitors that contain an aryl substituted 8-thioguanine scaffold, and structural analyses showed that these compounds engage the HPPK pterin-binding pocket and an induced cryptic pocket. A preliminary structure activity relationship profile was developed from biophysical and biochemical characterizations of derivative molecules. Also, a similarity search identified additional scaffolds that bind more tightly within the HPPK pterin pocket. These inhibitory scaffolds have the potential for rapid elaboration into novel lead antimicrobial agents.
Assuntos
Difosfotransferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Tioguanina/farmacologia , Cristalografia por Raios X , Difosfotransferases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Tioguanina/análogos & derivados , Tioguanina/químicaRESUMO
Conversion of pantothenate to phosphopantothenate in humans is the first dedicated step in the coenzyme A (CoA) biosynthesis pathway and is mediated by four isoforms of pantothenate kinase. These enzymes are allosterically regulated by acyl-CoA levels, which control the rate of CoA biosynthesis. Small molecule activators of the PANK enzymes that overcome feedback suppression increase CoA levels in cultured cells and animals and have shown great potential for the treatment of pantothenate kinase-associated neurodegeneration and propionic acidemias. In this study, we detail the further optimization of PANK pyridazine activators using structure-guided design and focus on the cellular CoA activation potential, metabolic stability, and solubility as the primary drivers of the structure-activity relationship. These studies led to the prioritization of three late-stage preclinical lead PANK modulators with improved pharmacokinetic profiles and the ability to substantially increase brain CoA levels. Compound 22 (BBP-671) eventually advanced into clinical testing for the treatment of PKAN and propionic acidemia.
Assuntos
Encéfalo , Fosfotransferases (Aceptor do Grupo Álcool) , Piridazinas , Humanos , Animais , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Piridazinas/farmacocinética , Piridazinas/farmacologia , Piridazinas/química , Piridazinas/síntese química , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Relação Estrutura-Atividade , Ratos , Ativadores de Enzimas/farmacologia , Ativadores de Enzimas/química , Ativadores de Enzimas/farmacocinética , Ativadores de Enzimas/síntese química , Coenzima A/metabolismo , CamundongosRESUMO
Extensive preclinical studies have shown that colchicine-binding site inhibitors (CBSIs) are promising drug candidates for cancer therapy. Although numerous CBSIs were generated and evaluated, but so far the FDA has not approved any of them due to undesired adverse events or insufficient efficacies. We previously reported two very potent CBSIs, the dihydroquinoxalinone compounds 5 m and 5t. In this study, we further optimized the structures of compounds 5 m and 5t and integrated them to generate a new analog, SB226. X-ray crystal structure studies and a tubulin polymerization assay confirmed that SB226 is a CBSI that could disrupt the microtubule dynamics and interfere with microtubule assembly. Biophysical measurements using surface plasmon resonance (SPR) spectroscopy verified the high binding affinity of SB226 to tubulin dimers. The in vitro studies showed that SB226 possessed sub-nanomolar anti-proliferative activities with an average IC50 of 0.76 nM against a panel of cancer cell lines, some of which are paclitaxel-resistant, including melanoma, breast cancer and prostate cancer cells. SB226 inhibited the colony formation and migration of Taxol-resistant A375/TxR cells, and induced their G2/M phase arrest and apoptosis. Our subsequent in vivo studies confirmed that 4 mg/kg SB226 strongly inhibited the tumor growth of A375/TxR melanoma xenografts in mice and induced necrosis, anti-angiogenesis, and apoptosis in tumors. Moreover, SB226 treatment significantly inhibited spontaneous axillary lymph node, lung, and liver metastases originating from subcutaneous tumors in mice without any obvious toxicity to the animals' major organs, demonstrating the therapeutic potential of SB226 as a novel anticancer agent for cancer therapy.
Assuntos
Antineoplásicos , Melanoma , Moduladores de Tubulina , Animais , Humanos , Masculino , Camundongos , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Colchicina/farmacologia , Melanoma/tratamento farmacológico , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Polimerização/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Moduladores de Tubulina/uso terapêuticoRESUMO
Polymerization of tubulin dimers to form microtubules is one of the key events in cell proliferation. The inhibition of this event has long been recognized as a potential treatment option for various types of cancer. Compound 1e was previously developed by our team as a potent inhibitor of tubulin polymerization that binds to the colchicine site. To further improve the potency and therapeutic properties of compound 1e, we hypothesized based on the X-ray crystal structure that modification of the pyrimidine dihydroquinoxalinone scaffold with additional hetero-atom (N, O, and S) substituents could allow the resulting new compounds to bind more tightly to the colchicine site and display greater efficacy in cancer therapy. We therefore synthesized a series of new pyrimidine dihydroquinoxalinone derivatives, compounds 10, 12b-c, 12e, 12h, and 12j-l, and evaluated their cytotoxicity and relative ability to inhibit proliferation, resulting in the discovery of new tubulin-polymerization inhibitors. Among these, the most potent new inhibitor was compound 12k, which exhibited high cytotoxic activity in vitro, a longer half-life than the parental compound in liver microsomes (IC50 = 0.2 nM, t 1/2 = >300 min), and significant potency against a wide range of cancer cell lines including those from melanoma and breast, pancreatic, and prostate cancers. High-resolution X-ray crystal structures of the best compounds in this scaffold series, 12e, 12j, and 12k, confirmed their direct binding to the colchicine site of tubulin and revealed their detailed molecular interactions. Further evaluation of 12k in vivo using a highly taxane-resistant prostate cancer xenograft model, PC-3/TxR, demonstrated the strong tumor growth inhibition at the low dose of 2.5 mg/kg (i.v., twice per week). Collectively, these results strongly support further preclinical evaluations of 12k as a potential candidate for development.
RESUMO
Chimeric transcription factors drive lineage-specific oncogenesis but are notoriously difficult to target. Alveolar rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue sarcoma transformed by the pathognomonic Paired Box 3-Forkhead Box O1 (PAX3-FOXO1) fusion protein, which governs a core regulatory circuitry transcription factor network. Here, we show that the histone lysine demethylase 4B (KDM4B) is a therapeutic vulnerability for PAX3-FOXO1+ RMS. Genetic and pharmacologic inhibition of KDM4B substantially delayed tumor growth. Suppression of KDM4 proteins inhibited the expression of core oncogenic transcription factors and caused epigenetic alterations of PAX3-FOXO1-governed superenhancers. Combining KDM4 inhibition with cytotoxic chemotherapy led to tumor regression in preclinical PAX3-FOXO1+ RMS subcutaneous xenograft models. In summary, we identified a targetable mechanism required for maintenance of the PAX3-FOXO1-related transcription factor network, which may translate to a therapeutic approach for fusion-positive RMS.
Assuntos
Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Carcinogênese/genética , Linhagem Celular Tumoral , Criança , Proteína Forkhead Box O1/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição Box Pareados/uso terapêutico , Rabdomiossarcoma/genética , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologiaRESUMO
Dihydropteroate synthase (DHPS) is the classical target of the sulfonamide class of antimicrobial agents, whose use has been limited by widespread resistance and pharmacological side effects. We have initiated a structure-based drug design approach for the development of novel DHPS inhibitors that bind to the highly conserved and structured pterin subsite rather than to the adjacent p-aminobenzoic acid binding pocket that is targeted by the sulfonamide class of antibiotics. To facilitate these studies, a robust pterin site-specific fluorescence polarization (FP) assay has been developed and is discussed herein. These studies include the design, synthesis, and characterization of two fluorescent probes, and the development and validation of a rapid DHPS FP assay. This assay has excellent DMSO tolerance and is highly reproducible as evidenced by a high Z' factor. This assay offers significant advantages over traditional radiometric or phosphate release assays against this target, and is suitable for site-specific high-throughput and fragment-based screening studies.
Assuntos
Bacillus anthracis/enzimologia , Di-Hidropteroato Sintase/metabolismo , Polarização de Fluorescência/métodos , Corantes Fluorescentes/química , Pterinas/química , Sítios de Ligação , Ligação Competitiva , Di-Hidropteroato Sintase/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Pterinas/síntese química , Pterinas/metabolismo , Sensibilidade e EspecificidadeRESUMO
The increasing emergence of resistant bacteria drives us to design and develop new antimicrobial agents. Pursuant to that goal, a new targeting approach of the dihydropteroate synthase enzyme, which serves as the site of action for the sulfonamide class of antimicrobial agents, is being explored. Using structural information, a new class of transition state mimics has been designed and synthesized that have the capacity to bind to the pterin, phosphate and para-amino binding sites. The design, synthesis and evaluation of these compounds as inhibitors of Bacillusanthracis dihydropteroate synthase is described herein. Outcomes from this work have identified the first trivalent inhibitors of dihydropteroate synthase whose activity displayed slow binding inhibition. The most active compounds in this series contained an oxidized pterin ring. The binding of these inhibitors was modeled into the dihydropteroate synthase active site and demonstrated a good correlation with the observed bioassay data, as well as provided important insight for the future design of higher affinity transition state mimics.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Di-Hidropteroato Sintase/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Sondas Moleculares , Organofosfonatos/síntese química , Pirimidinonas/síntese química , Antibacterianos/química , Bacillus anthracis/efeitos dos fármacos , Bacillus anthracis/enzimologia , Di-Hidropteroato Sintase/metabolismo , Desenho de Fármacos , Modelos Moleculares , Estrutura Molecular , Organofosfonatos/química , Organofosfonatos/farmacologia , Pterinas/química , Pirimidinonas/química , Pirimidinonas/farmacologia , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Propionic acidemia (PA) is a rare autosomal-recessive metabolic disease that arises from mutations in propionyl-CoA (C3-CoA) carboxylase. Reduced enzyme activity slows C3-CoA metabolism, leading to an elevated plasma C3:C2-carnitine ratio, the hallmark biomarker of PA. The metabolic imbalances experienced in PA are however poorly defined. Here, we used a hypomorphic PA mouse model to demonstrate that C3-CoA accumulation in liver reduced non-esterified CoA (CoASH) and acetyl-CoA (C2-CoA). Tricarboxylic acid (TCA) cycle intermediates that are normally metabolized instead accumulated in urine, providing direct evidence for compromised mitochondrial function in PA. Pantothenate kinase (PanK) is known to catalyze the rate-controlling step in CoA biosynthesis, and its inhibition by C3-CoA prevents an increase in CoA biosynthesis to alleviate CoASH sequestration. PZ-3022 is an allosteric PanK activator that counteracts C3-CoA inhibition. PZ-3022 therapy increased hepatic CoASH and C2-CoA and decreased C3-CoA in the PA mouse model, leading to improved intracellular C3:C2-CoA and plasma C3:C2-carnitine ratios. Elevated urinary malate is a major component of the metabolic signature for TCA cycle dysfunction in the PA mouse, and the 80% reduction in urine malate by PZ-3022 therapy indicates the restoration of mitochondrial function. Thus, CoASH sequestration in PA leads to reduced TCA cycle activity that is relieved by PZ-3022, providing preclinical proof of concept for PanK activators as a therapy to attenuate the underlying mitochondrial defect in PA.
Assuntos
Acidemia Propiônica , Animais , Coenzima A , Camundongos , Mitocôndrias , Fosfotransferases (Aceptor do Grupo Álcool) , Acidemia Propiônica/tratamento farmacológicoRESUMO
We previously reported a potent tubulin inhibitor CH-2-77. In this study, we optimized the structure of CH-2-77 by blocking metabolically labile sites and synthesized a series of CH-2-77 analogues. Two compounds, 40a and 60c, preserved the potency while improving the metabolic stability over CH-2-77 by 3- to 4-fold (46.8 and 29.4 vs 10.8 min in human microsomes). We determined the high-resolution X-ray crystal structures of 40a (resolution 2.3 Å) and 60c (resolution 2.6 Å) in complex with tubulin and confirmed their direct binding at the colchicine-binding site. In vitro, 60c maintained its mode of action by inhibiting tubulin polymerization and was effective against P-glycoprotein-mediated multiple drug resistance and taxol resistance. In vivo, 60c exhibited a strong inhibitory effect on tumor growth and metastasis in a taxol-resistant A375/TxR xenograft model without obvious toxicity. Collectively, this work showed that 60c is a promising lead compound for further development as a potential anticancer agent.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Piridinas/uso terapêutico , Moduladores de Tubulina/uso terapêutico , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Estabilidade de Medicamentos , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Metástase Neoplásica/prevenção & controle , Piridinas/síntese química , Piridinas/metabolismo , Piridinas/farmacocinética , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Small molecules that interact with the colchicine binding site in tubulin have demonstrated therapeutic efficacy in treating cancers. We report the design, syntheses, and antitumor efficacies of new analogues of pyridopyrimidine and hydroquinoxalinone compounds with improved drug-like characteristics. Eight analogues, 5j, 5k, 5l, 5m, 5n, 5r, 5t, and 5u, showed significant improvement in metabolic stability and demonstrated strong antiproliferative potency in a panel of human cancer cell lines, including melanoma, lung cancer, and breast cancer. We report crystal structures of tubulin in complex with five representative compounds, 5j, 5k, 5l, 5m, and 5t, providing direct confirmation for their binding to the colchicine site in tubulin. A quantitative structure-activity relationship analysis of the synthesized analogues showed strong ability to predict potency. In vivo, 5m (4 mg/kg) and 5t (5 mg/kg) significantly inhibited tumor growth as well as melanoma spontaneous metastasis into the lung and liver against a highly paclitaxel-resistant A375/TxR xenograft model.