RESUMO
We previously discovered first-in-class multitargeted 5-substituted pyrrolo[3,2-d]pyrimidine antifolates that inhibit serine hydroxymethyltransferase 2 (SHMT2), resulting in potent in vitro and in vivo antitumor efficacies. In this report, we present crystallographic structures for SHMT2 in complex with an expanded series of pyrrolo[3,2-d]pyrimidine compounds with variations in bridge length (3-5 carbons) and the side chain aromatic ring (phenyl, thiophene, fluorine-substituted phenyl, and thiophene). We evaluated structural features of the inhibitor-SHMT2 complexes and correlations to inhibitor potencies (i.e., Kis), highlighting conserved polar contacts and identifying 5-carbon bridge lengths as key determinants of inhibitor potency. Based on the analysis of SHMT2 structural data, we investigated the impact of mutation of Tyr105 in SHMT2 kinetic analysis and studies with HCT116 cells with inducible expression of wild-type and Y105F SHMT2. Increased enzyme inhibition potency by the pyrrolo[3,2-d]pyrimidine inhibitors with Phe105 SHMT2 accompanied an increased growth inhibition of Phe105-expressing HCT116 cells compared to wild-type SHMT2. Pyrrolo[3,2-d]pyrimidine inhibitors with polyglutamate modifications were evaluated for potencies against SHMT2. We determined the crystal structures of SHMT2 in complex with our lead antifolate AGF347 lacking L-glutamate, or as a diglutamate and triglutamate, for comparison with parent AGF347. These data provide the first insights into the influence of antifolate polyglutamylation on SHMT2:inhibitor interactions. Collectively, our results provide new insights into the critical structural determinants of SHMT2 binding by pyrrolo[3,2-d]pyrimidine inhibitors as novel antitumor agents, as well as the first structural characterization of human SHMT2 in complex with polyglutamates of an SHMT2-targeted antifolate.
RESUMO
Folate-dependent one-carbon (C1) metabolism encompasses distinct cytosolic and mitochondrial pathways connected by an interchange between serine, glycine and formate. In both the cytosol and mitochondria, folates exist as polyglutamates with polyglutamylation catalyzed by folylpolyglutamate synthetase (FPGS), including cytosolic and mitochondrial isoforms. Serine is metabolized by serine hydroxymethyltransferase (SHMT) 2 in the mitochondria and generates glycine and C1 units for cellular biosynthesis in the cytosol. AGF347 is a novel pyrrolo[3,2-d]pyrimidine antifolate that targets SHMT2 in the mitochondria, and SHMT1 and de novo purine biosynthesis in the cytosol. FPGS is expressed in primary pancreatic cancer specimens and FPGS levels correlate with in vitro efficacies of AGF347 toward human pancreatic cancer cells. MIA PaCa-2 pancreatic cancer cells with CRISPR knockout of FPGS were engineered to express doxycycline-inducible FPGS exclusively in the cytosol (cFPGS) or in both the cytosol and mitochondria (mFPGS). Folate and AGF347 accumulations increased in both the cytosol and mitochondria with increased mFPGS but were restricted to the cytosol with cFPGS. AGF347-Glu5 inhibited SHMT2 ~19-fold greater than AGF347 By metabolomics analysis, mFPGS stimulated the C1 flux from serine in the mitochondria and de novo purine and dTTP synthesis far greater than cFPGS. mFPGS enhanced in vitro inhibition of MIA PaCa-2 cell proliferation by AGF347 (~30-fold) more than cFPGS (~4.9-fold). Similar results were seen with other pyrrolo[3,2-d]pyrimidine antifolates (AGF291, AGF320); however, elevated mFPGS adversely impacted inhibition by the non-classical SHMT2/SHMT1 inhibitor, SHIN1. These results suggest a critical role of mFPGS levels in determining anti-tumor efficacies of mitochondrial-targeted pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer. Significance Statement AGF347 is a novel pyrrolo[3,2-d]pyrimidine antifolate that targets serine hydroxymethyltransferase (SHMT) 2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. AGF347 accumulation increases with folylpolyglutamate synthetase (FPGS) levels in both the cytosol and mitochondria. Increased mitochondrial FPGS stimulated one-carbon metabolic fluxes in the cytosol and mitochondria and substantially enhanced in vitro inhibition of pancreatic cancer cells by AGF347 Mitochondrial FPGS levels play important roles in determining the anti-tumor efficacies of pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer.
RESUMO
The human proton-coupled folate transporter (PCFT; SLC46A1) or hPCFT was identified in 2006 as the principal folate transporter involved in the intestinal absorption of dietary folates. A rare autosomal recessive hereditary folate malabsorption syndrome is attributable to human SLC46A1 variants. The recognition that hPCFT was highly expressed in many tumors stimulated substantial interest in its potential for cytotoxic drug targeting, taking advantage of its high-level transport activity under acidic pH conditions that characterize many tumors and its modest expression in most normal tissues. To better understand the basis for variations in hPCFT levels between tissues including human tumors, studies have examined the transcriptional regulation of hPCFT including the roles of CpG hypermethylation and critical transcription factors and cis elements. Additional focus involved identifying key structural and functional determinants of hPCFT transport that, combined with homology models based on structural homologies to the bacterial transporters GlpT and LacY, have enabled new structural and mechanistic insights. Recently, cryo-electron microscopy structures of chicken PCFT in a substrate-free state and in complex with the antifolate pemetrexed were reported, providing further structural insights into determinants of (anti)folate recognition and the mechanism of pH-regulated (anti)folate transport by PCFT. Like many major facilitator proteins, hPCFT exists as a homo-oligomer, and evidence suggests that homo-oligomerization of hPCFT monomeric proteins may be important for its intracellular trafficking and/or transport function. Better understanding of the structure, function and regulation of hPCFT should facilitate the rational development of new therapeutic strategies for conditions associated with folate deficiency, as well as cancer.
Assuntos
Ácido Fólico/metabolismo , Transportador de Folato Acoplado a Próton/metabolismo , Animais , Antagonistas do Ácido Fólico/metabolismo , Humanos , Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologiaRESUMO
Complex natural products that bind to tubulin/microtubules come under the broad category of microtubule binding agents. The design of simplified analogs of previously reported bicyclic, microtubule depolymerizer, pyrrolo[2,3-d]pyrimidine, provided valuable structure-activity relationship data and led to the identification of novel monocyclic pyrimidine analogs of which 12 was 47-fold more potent (EC50 123 nM) for cellular microtubule depolymerization activity and 7.5-fold more potent (IC50 24.4 nM) at inhibiting the growth of MDA-MB-435 cancer cells, suggesting significantly better binding of the target within the colchicine site of tubulin compared to lead compound 1. This compound and others of this series of monocyclic pyrimidine analogs were able to overcome multidrug resistance due to the expression of the ßIII-isotype of tubulin and P-glycoprotein. In vivo evaluation of the most potent analog 12 in an MDA-MB-435 xenograft mouse model indicated, along with paclitaxel, that both compounds showed a trend towards lower tumor volume however neither compound showed significant antitumor activity in the trial. To our knowledge these are the first examples of simple substituted monocyclic pyrimidines as colchicine site binding antitubulin compounds with potent antitumor activity.
Assuntos
Antineoplásicos , Colchicina , Humanos , Camundongos , Animais , Colchicina/farmacologia , Colchicina/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Microtúbulos/metabolismo , Relação Estrutura-Atividade , Pirimidinas/química , Antineoplásicos/química , Linhagem Celular Tumoral , Sítios de Ligação , Proliferação de CélulasRESUMO
A series of eleven 4-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines were designed and synthesized and their biological activities were evaluated. Synthesis involved the Gewald reaction to synthesize ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate ring, and SNAr reactions. Compound 4 was 1.6- and ~7-fold more potent than the lead compound 1 in cell proliferation and microtubule depolymerization assays, respectively. Compounds 4, 5 and 7 showed the most potent antiproliferative effects (IC50 values < 40 nM), while compounds 6, 8, 10, 12 and 13 had lower antiproliferative potencies (IC50 values of 53-125 nM). Additionally, compounds 4-8, 10 and 12-13 circumvented Pgp and ßIII-tubulin mediated drug resistance, mechanisms that diminish the clinical efficacy of paclitaxel (PTX). In the NCI-60 cell line panel, compound 4 exhibited an average GI50 of ~10 nM in the 40 most sensitive cell lines. Compound 4 demonstrated statistically significant antitumor effects in a murine MDA-MB-435 xenograft model.
Assuntos
Técnicas de Química Sintética , Desenho de Fármacos , Pirimidinas/química , Pirimidinas/farmacologia , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Multimerização Proteica/efeitos dos fármacos , Pirimidinas/síntese química , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntese químicaRESUMO
The design, synthesis, and biological evaluation of a series novel N1methyl pyrazolo[4,3-d]pyrimidines as inhibitors of tubulin polymerization and colchicine binding were described here. Synthesis of target compounds involved alkylation of the pyrazolo scaffold, which afforded two regioisomers. These were separated, characterized and identified with 1H NMR and NOESY spectroscopy. All compounds, except 10, inhibited [3H]colchicine binding to tubulin, and the potent inhibition was similar to that obtained with CA-4. Compounds 9 and 11-13 strongly inhibited the polymerization of tubulin, with IC50 values of 0.45, 0.42, 0.49 and 0.42⯵M, respectively. Compounds 14-16 inhibited the polymerization of tubulin with IC50s near â¼1⯵M. Compounds 9, 12, 13 and 16 inhibited MCF-7 breast cancer cell lines and circumvented ßIII-tubulin mediated cancer cell resistance to taxanes and other MTAs, and compounds 9-17 circumvented Pgp-mediated drug resistance. In the standard NCI testing protocol, compound 9 exhibited excellent potency with low to sub nanomolar GI50 values (≤10â¯nM) against most tumor cell lines, including several multidrug resistant phenotypes. Compound 9 was significantly (Pâ¯<â¯0.0001) better than paclitaxel at reducing MCF-7 TUBB3 (ßIII-tubulin overexpressing) tumors in a mouse xenograft model. Collectively, these studies support the further preclinical development of the pyrazolo[4,3-d]pyrimidine scaffold as a new generation of tubulin inhibitors and 9 as an anticancer agent with advantages over paclitaxel.
Assuntos
Antineoplásicos/farmacologia , Microtúbulos , Pirimidinas/farmacologia , Moduladores de Tubulina/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Pirimidinas/química , Relação Estrutura-Atividade , Moduladores de Tubulina/químicaRESUMO
The efficacy of quinazoline-based antiglioma agents has been attributed to their effects on microtubule dynamics.1,2 The design, synthesis and biological evaluation of quinazolines as potent inhibitors of multiple intracellular targets, including microtubules and multiple RTKs, is described. In addition to the known ability of quinazolines 1 and 2 to cause microtubule depolymerization, they were found to be low nanomolar inhibitors of EGFR, VEGFR-2 and PDGFR-ß. Low nanomolar inhibition of EGFR was observed for 1-3 and 9-10. Compounds 1 and 4 inhibited VEGFR-2 kinase with activity better than or equal to that of sunitinib. In addition, compounds 1 and 2 had similar potency to sunitinib in the CAM angiogenesis assay. Multitarget activities of compounds in the present study demonstrates that the quinazolines can affect multiple pathways and could lead to these agents having antitumor potential caused by their activity against multiple targets.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Quinazolinas/síntese química , Quinazolinas/química , Relação Estrutura-Atividade , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
A series of methoxy naphthyl substituted cyclopenta[d]pyrimidine compounds, 4-10, were designed and synthesized to study the influence of the 3-D conformation on microtubule depolymerizing and antiproliferative activities. NOESY studies with the N,2-dimethyl-N-(6'-methoxynaphthyl-1'-amino)-cyclopenta[d]pyrimidin-4-amine (4) showed hindered rotation of the naphthyl ring around the cyclopenta[d]pyrimidine scaffold. In contrast, NOESY studies with N,2-dimethyl-N-(5'-methoxynaphthyl-2'-amino)-cyclopenta[d]pyrimidin-4-amine (5) showed free rotation of the naphthyl ring around the cyclopenta[d]pyrimidine scaffold. The rotational flexibility and conformational dissimilarity between 4 and 5 led to a significant difference in biological activities. Compound 4 is inactive while 5 is the most potent in this series with potent microtubule depolymerizing effects and low nanomolar IC50 values in vitro against a variety of cancer cell lines. The ability of 5 to inhibit tumor growth in vivo was investigated in a U251 glioma xenograft model. The results show that 5 had better antitumor effects than the positive control temozolomide and have identified 5 as a potential preclinical candidate for further studies. The influence of conformation on the microtubule depolymerizing and antitumor activity forms the basis for the development of conformation-activity relationships for the cyclopenta[d]pyrimidine class of microtubule targeting agents.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Ciclopentanos/farmacologia , Glioma/tratamento farmacológico , Microtúbulos/efeitos dos fármacos , Pirimidinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Neoplasias Encefálicas/patologia , Proliferação de Células/efeitos dos fármacos , Ciclopentanos/síntese química , Ciclopentanos/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Glioma/patologia , Humanos , Masculino , Camundongos , Camundongos Nus , Modelos Moleculares , Conformação Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
We discovered 6-substituted thieno[2,3-d]pyrimidine compounds (3-9) with 3-4 bridge carbons and side-chain thiophene or furan rings for dual targeting one-carbon (C1) metabolism in folate receptor- (FR) expressing cancers. Synthesis involved nine steps starting from the bromo-aryl carboxylate. From patterns of growth inhibition toward Chinese hamster ovary cells expressing FRα or FRß, the proton-coupled folate transporter or reduced folate carrier, specificity for uptake by FRs was confirmed. Anti-proliferative activities were demonstrated toward FRα-expressing KB tumor cells and NCI-IGROV1 ovarian cancer cells. Inhibition of de novo purine biosynthesis at both 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and glycinamide ribonucleotide formyltransferase (GARFTase) was confirmed by metabolite rescue, metabolomics and enzyme assays. X-ray crystallographic structures were obtained with compounds 3-5 and human GARFTase. Our studies identify first-in-class C1 inhibitors with selective uptake by FRs and dual inhibition of enzyme targets in de novo purine biosynthesis, resulting in anti-tumor activity. This series affords an exciting new platform for selective multi-targeted anti-tumor agents.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Fosforribosilaminoimidazolcarboxamida Formiltransferase/antagonistas & inibidores , Fosforribosilglicinamido Formiltransferase/antagonistas & inibidores , Pirimidinas/farmacologia , Tiofenos/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Células CHO , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cricetulus , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Receptores de Folato com Âncoras de GPI/metabolismo , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Fosforribosilaminoimidazolcarboxamida Formiltransferase/metabolismo , Fosforribosilglicinamido Formiltransferase/metabolismo , Ligação Proteica , Pirimidinas/síntese química , Pirimidinas/metabolismo , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/metabolismoRESUMO
Folate-dependent one-carbon (C1) metabolism is compartmentalized in the mitochondria and cytosol and is a source of critical metabolites for proliferating tumors. Mitochondrial C1 metabolism including serine hydroxymethyltransferase 2 (SHMT2) generates glycine for de novo purine nucleotide and glutathione biosynthesis and is an important source of NADPH, ATP, and formate, which affords C1 units as 10-formyl-tetrahydrofolate and 5,10-methylene-tetrahydrofolate for nucleotide biosynthesis in the cytosol. We previously discovered novel first-in-class multitargeted pyrrolo[3,2-d]pyrimidine inhibitors of SHMT2 and de novo purine biosynthesis at glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase with potent in vitro and in vivo antitumor efficacy toward pancreatic adenocarcinoma cells. In this report, we extend our findings to an expanded panel of pancreatic cancer models. We used our lead analog AGF347 [(4-(4-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)butyl)-2-fluorobenzoyl)-l-glutamic acid] to characterize pharmacodynamic determinants of antitumor efficacy for this series and demonstrated plasma membrane transport into the cytosol, uptake from cytosol into mitochondria, and metabolism to AGF347 polyglutamates in both cytosol and mitochondria. Antitumor effects of AGF347 downstream of SHMT2 and purine biosynthesis included suppression of mammalian target of rapamycin signaling, and glutathione depletion with increased levels of reactive oxygen species. Our results provide important insights into the cellular pharmacology of novel pyrrolo[3,2-d]pyrimidine inhibitors as antitumor compounds and establish AGF347 as a unique agent for potential clinical application for pancreatic cancer, as well as other malignancies. SIGNIFICANCE STATEMENT: This study establishes the antitumor efficacies of novel inhibitors of serine hydroxymethyltransferase 2 and of cytosolic targets toward a panel of clinically relevant pancreatic cancer cells and demonstrates the important roles of plasma membrane transport, mitochondrial accumulation, and metabolism to polyglutamates of the lead compound AGF347 to drug activity. We also establish that loss of serine catabolism and purine biosynthesis resulting from AGF347 treatment impacts mammalian target of rapamycin signaling, glutathione pools, and reactive oxygen species, contributing to antitumor efficacy.
Assuntos
Antineoplásicos/farmacologia , Citosol/efeitos dos fármacos , Glicina Hidroximetiltransferase/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Pirimidinas/farmacologia , Pirróis/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citosol/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Técnicas de Inativação de Genes , Glutationa/biossíntese , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Humanos , Leucovorina/análogos & derivados , Leucovorina/metabolismo , Mitocôndrias/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Nucleotídeos de Purina/biossíntese , Pirimidinas/química , Pirimidinas/uso terapêutico , Pirróis/química , Pirróis/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Serina/metabolismo , Tetra-Hidrofolatos/metabolismoRESUMO
Tumor-targeted 6-substituted pyrrolo[2,3-d]pyrimidine benzoyl compounds based on 2 were isosterically modified at the 4-carbon bridge by replacing the vicinal (C11) carbon by heteroatoms N (4), O (5) or S (6), or with an N-substituted formyl (7), trifluoroacetyl (8) or acetyl (9). Replacement with sulfur (6) afforded the most potent KB tumor cell inhibitor, ~6-fold better than the parent 2. In addition, 6 retained tumor transport selectivity via folate receptor (FR) α and -ß over the ubiquitous reduced folate carrier (RFC). FRα-mediated cell inhibition for 6 was generally equivalent to 2, while the FRß-mediated activity was improved by 16-fold over 2. N (4) and O (5) substitutions afforded similar tumor cell inhibitions as 2, with selectivity for FRα and -ß over RFC. The N-substituted analogs 7-9 also preserved transport selectivity for FRα and -ß over RFC. For FRα-expressing CHO cells, potencies were in the order of 8 > 7 > 9. Whereas 8 and 9 showed similar results with FRß-expressing CHO cells, 7 was ~16-fold more active than 2. By nucleoside rescue experiments, all the compounds inhibited de novo purine biosynthesis, likely at the step catalyzed by glycinamide ribonucleotide formyltransferase. Thus, heteroatom replacements of the CH2 in the bridge of 2 afford analogs with increased tumor cell inhibition that could provide advantages over 2, as well as tumor transport selectivity over clinically used antifolates including methotrexate and pemetrexed.
Assuntos
Antineoplásicos/síntese química , Desenho de Fármacos , Receptor 1 de Folato/metabolismo , Receptor 2 de Folato/metabolismo , Ácido Fólico/metabolismo , Pirimidinas/química , Pirróis/química , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Sítios de Ligação , Células CHO , Domínio Catalítico , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Receptor 1 de Folato/química , Receptor 1 de Folato/genética , Receptor 2 de Folato/química , Receptor 2 de Folato/genética , Ácido Fólico/química , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Humanos , Simulação de Acoplamento Molecular , Fosforribosilglicinamido Formiltransferase/química , Fosforribosilglicinamido Formiltransferase/metabolismo , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Pirróis/metabolismo , Pirróis/farmacologia , Relação Estrutura-AtividadeRESUMO
Tumors can be therapeutically targeted with novel antifolates (e.g. AGF94) that are selectively transported by the human proton-coupled folate transporter (hPCFT). Studies were performed to determine the transcription regulation of hPCFT in tumors and identify possible mechanisms that contribute to the highly disparate levels of hPCFT in HepG2 versus HT1080 tumor cells. Transfection of hPCFT-null HT1080 cells with hPCFT restored transport and sensitivity to AGF94 Progressive deletions of the hPCFT promoter construct (-2005 to +96) and reporter gene assays in HepG2 and HT1080 cells confirmed differences in hPCFT transactivation and localized a minimal promoter to between positions -50 and +96. The minimal promoter included KLF15, GC-Box and NRF-1 cis-binding elements whose functional importance was confirmed by promoter deletions and mutations of core consensus sequences and reporter gene assays. In HepG2 cells, NRF-1, KLF15 and Sp1 transcripts were increased over HT1080 cells by â¼5.1-, â¼44-, and â¼2.4-fold, respectively. In Drosophila SL2 cells, transfection with KLF15 and NRF-1 synergistically activated the hPCFT promoter; Sp1 was modestly activating or inhibitory. Chromatin immunoprecipitation and electrophoretic mobility shift assay (EMSA) and supershifts confirmed differential binding of KLF15, Sp1, and NRF-1 to the hPCFT promoter in HepG2 and HT1080 cells that paralleled hPCFT levels. Treatment of HT1080 nuclear extracts (NE) with protein kinase A increased Sp1 binding to its consensus sequence by EMSA, suggesting a role for Sp1 phosphorylation in regulating hPCFT transcription. A better understanding of determinants of hPCFT transcriptional control may identify new therapeutic strategies for cancer by modulating hPCFT levels in combination with hPCFT-targeted antifolates.
Assuntos
Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Transportador de Folato Acoplado a Próton/biossíntese , Elementos de Resposta , Fator de Transcrição Sp1/metabolismo , Animais , Drosophila melanogaster , Células HeLa , Células Hep G2 , Humanos , Fatores de Transcrição Kruppel-Like/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Fator 1 Nuclear Respiratório/genética , Transportador de Folato Acoplado a Próton/genética , Fator de Transcrição Sp1/genéticaRESUMO
Pneumocystis pneumonia (PCP) caused by Pneumocystis jirovecii (pj) can lead to serious health consequences in patients with an immunocompromised system. Trimethoprim (TMP), used as first-line therapy in combination with sulfamethoxazole, is a selective but only moderately potent pj dihydrofolate reductase (pjDHFR) inhibitor, whereas non-clinical pjDHFR inhibitors, such as, piritrexim and trimetrexate are potent but non-selective pjDHFR inhibitors. To meet the clinical needs for a potent and selective pjDHFR inhibitor for PCP treatment, fourteen 6-substituted pyrido[3,2-d]pyrimidines were developed. Comparison of the amino acid residues in the active site of pjDHFR and human DHFR (hDHFR) revealed prominent amino acid differences which could be exploited to structurally design potent and selective pjDHFR inhibitors. Molecular modeling followed by enzyme assays of the compounds revealed 15 as the best compound of the series with an IC50 of 80â¯nM and 28-fold selectivity for inhibiting pjDHFR over hDHFR. Compound 15 serves as the lead analog for further structural variations to afford more potent and selective pjDHFR inhibitors.
Assuntos
Antagonistas do Ácido Fólico/uso terapêutico , Pneumocystis carinii/patogenicidade , Pneumocystis/efeitos dos fármacos , Pirimidinas/uso terapêutico , Trimetoprima/uso terapêutico , Antagonistas do Ácido Fólico/farmacologia , Humanos , Modelos Moleculares , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Trimetoprima/farmacologiaRESUMO
We previously showed that classical 6-substituted pyrrolo[2,3-d]pyrimidine antifolates bind to folate receptor (FR) α and the target purine biosynthetic enzyme glycinamide ribonucleotide formyltransferase (GARFTase) with different cis and trans conformations. In this study, we designed novel analogs of this series with an amide moiety in the bridge region that can adopt both the cis and trans lowest energy conformations. This provides entropic benefit, by restricting the number of side-chain conformations of the unbound ligand to those most likely to promote binding to FRα and the target enzyme required for antitumor activity. NMR of the most active compound 7 showed both cis and trans amide bridge conformations in ~1:1 ratio. The bridge amide group in the best docked poses of 7 in the crystal structures of FRα and GARFTase adopted both cis and trans conformations, with the lowest energy conformations predicted by Maestro and evidenced by NMR within 1â¯kcal/mol. Compound 7 showed ~3-fold increased inhibition of FRα-expressing cells over its non-restricted parent analog 1 and was selectively internalized by FRα over the reduced folate carrier (RFC), resulting in significant in vitro antitumor activity toward FRα-expressing KB human tumor cells. Antitumor activity of 7 was abolished by treating cells with adenosine but was incompletely protected by 5-aminoimidazole-4-carboxamide (AICA) at higher drug concentrations, suggesting GARFTase and AICA ribonucleotide formyltransferase (AICARFTase) in de novo purine biosynthesis as the likely intracellular targets. GARFTase inhibition by compound 7 was confirmed by an in situ cell-based activity assay. Our results identify a "first-in-class" classical antifolate with a novel amide linkage between the scaffold and the side chain aryl L-glutamate that affords exclusive selectivity for transport via FRα over RFC and antitumor activity resulting from inhibition of GARFTase and likely AICARFTase. Compound 7 offers significant advantages over clinically used inhibitors of this class that are transported by the ubiquitous RFC, resulting in dose-limiting toxicities.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Piridinas/química , Piridinas/farmacologia , Pirróis/química , Pirróis/farmacologia , Amidas/química , Amidas/farmacocinética , Amidas/farmacologia , Animais , Antineoplásicos/farmacocinética , Vias Biossintéticas/efeitos dos fármacos , Células CHO , Linhagem Celular Tumoral , Cricetulus , Receptor 1 de Folato/metabolismo , Antagonistas do Ácido Fólico/farmacocinética , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Nucleotídeos de Purina/metabolismo , Piridinas/farmacocinética , Pirróis/farmacocinéticaRESUMO
The design, synthesis and biological evaluation of 4-substituted 5-methyl-furo[2,3-d]pyrimidines is described. The Ullmann coupling of 5-methyl-furo[2,3-d]pyrimidine with aryl iodides was successfully optimized to synthesize these analogs. Compounds 6-10 showed single-digit nanomolar inhibition of EGFR kinase. Compounds 1 and 6-10 inhibited VEGFR-2 kinase better than or equal to sunitinib. Compounds 1 and 3-10 were more potent inhibitors of PDGFR-ß kinase than sunitinib. In addition, compounds 4-11 had higher potency in the CAM angiogenesis assay than sunitinib. Compound 1 showed in vivo efficacy in an A498 renal xenograft model in mice. Multiple RTK and tubulin inhibitory attributes of 1, 4, 6 and 8 indicates that these compounds may be valuable preclinical single agents targeting multiple intracellular targets.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Humanos , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirimidinas/síntese química , Pirimidinas/química , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Relação Estrutura-Atividade , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
We report a series of tubulin targeting agents, some of which demonstrate potent antiproliferative activities. These analogs were designed to optimize the antiproliferative activity of 1 by varying the heteroatom substituent at the 4'-position, the basicity of the 4-position amino moiety, and conformational restriction. The potential metabolites of the active compounds were also synthesized. Some compounds demonstrated single digit nanomolar IC50 values for antiproliferative effects in MDA-MB-435 melanoma cells. Particularly, the S-methyl analog 3 was more potent than 1 in MDA-MB-435 cells (IC50â¯=â¯4.6â¯nM). Incubation of 3 with human liver microsomes showed that the primary metabolite of the S-methyl moiety of 3 was the methyl sulfinyl group, as in analog 5. This metabolite was equipotent with the lead compound 1 in MDA-MB-435 cells (IC50â¯=â¯7.9â¯nM). Molecular modeling and electrostatic surface area were determined to explain the activities of the analogs. Most of the potent compounds overcome multiple mechanisms of drug resistance and compound 3 emerged as the lead compound for further SAR and preclinical development.
Assuntos
Antineoplásicos/farmacologia , Pirimidinas/farmacologia , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Conformação Molecular , Simulação de Acoplamento Molecular , Ligação Proteica , Pirimidinas/síntese química , Pirimidinas/química , Pirimidinas/metabolismo , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismoRESUMO
To combine the potency of trimetrexate (TMQ) or piritrexim (PTX) with the species selectivity of trimethoprim (TMP), target based design was carried out with the X-ray crystal structure of human dihydrofolate reductase (hDHFR) and the homology model of Pneumocystis jirovecii DHFR (pjDHFR). Using variation of amino acids such as Met33/Phe31 (in pjDHFR/hDHFR) that affect the binding of inhibitors due to their distinct positive or negative steric effect at the active binding site of the inhibitor, we designed a series of substituted-pyrrolo[2,3-d]pyrimidines. The best analogs displayed better potency (IC50) than PTX and high selectivity for pjDHFR versus hDHFR, with 4 exhibiting a selectivity for pjDHFR of 24-fold.
Assuntos
Aminoácidos/química , Antibacterianos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Antibacterianos/síntese química , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Ensaios Enzimáticos , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/metabolismo , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Estrutura Molecular , Pneumocystis carinii/enzimologia , Ligação Proteica , Pirimidinas/síntese química , Pirimidinas/química , Pirimidinas/metabolismo , Pirróis/síntese química , Pirróis/química , Pirróis/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
The discovery, synthesis and biological evaluations of a series of nine N5-substituted-pyrrolo[3,2-d]pyrimidin-4-amines are reported. Novel compounds with microtubule depolymerizing activity were identified. Some of these compounds also circumvent clinically relevant drug resistance mechanisms (expression of P-glycoprotein and ßIII tubulin). Compounds 4, 5, and 8-13 were one to two-digit nanomolar (IC50) inhibitors of cancer cells in culture. Contrary to recent reports (Banerjee et al. J. Med. Chem.2018, 61, 1704-1718), the conformation of the most active compounds determined by 1H NMR and molecular modeling are similar to that reported previously and in keeping with recently reported X-ray crystal structures. Compound 11, freely water soluble as the HCl salt, afforded statistically significant inhibition of tumor growth in three xenograft models [MDA-MB-435, MDA-MB-231 and NCI/ADR-RES] compared with controls. Compound 11 did not display indications of animal toxicity and is currently slated for further preclinical development.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Pirróis/química , Pirróis/farmacologia , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos Nus , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , Estereoisomerismo , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/uso terapêuticoRESUMO
In an effort to optimize the structural requirements for combined cytostatic and cytotoxic effects in single agents, a series of 5-(arylthio)-9H-pyrimido[4,5-b]indole-2,4-diamines 3-7 were synthesized and evaluated as inhibitors of receptor tyrosine kinases (RTKs) as well as thymidylate synthase (TS). The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-bromo/5-chloro-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate aryl thiols. A novel four step synthetic scheme to the common intermediate was developed which is more efficient relative to the previously reported six-step sequence. Biological evaluation of these compounds indicated dual activity in RTKs and human TS (hTS). In the VEGFR-2 assay, compound 5 was equipotent to the standard compound semaxanib and was better than standard TS inhibitor pemetrexed, in the hTS assay. Compounds 3, 6 and 7 were nanomolar inhibitors of hTS and were several fold better than pemetrexed.
Assuntos
Antineoplásicos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Timidilato Sintase/antagonistas & inibidores , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Cisplatino/farmacologia , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/farmacologia , Compostos Heterocíclicos com 3 Anéis/síntese química , Humanos , Indóis/síntese química , Camundongos , Pemetrexede/farmacologia , Inibidores de Proteínas Quinases/síntese química , Pirimidinas/síntese química , Pirróis/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To identify the structural features of 9H-pyrimido[4,5-b]indoles as microtubule depolymerizers, pyrimido[4,5-b]indoles 2-8 with varied substituents at the 2-, 4- and 5-positions were designed and synthesized. Nucleophilic displacement of 2,5-substituted-4-chloro-pyrimido[4,5-b]indoles with appropriate arylamines was the final step employed in the synthesis of target compounds 2-8. Compounds 2 and 6 had two-digit nanomolar potency (IC50) against MDA-MB-435, SK-OV-3 and HeLa cancer cells in vitro. Compounds 2 and 6 also depolymerized microtubules comparable to the lead compound 1. Compounds 2, 3, 6 and 8 were effective in cells expressing P-glycoprotein or the ßIII isotype of tubulin, mechanisms that are associated with clinical drug resistance to microtubule targeting drugs. Proton NMR and molecular modeling studies were employed to identify the structural basis for the microtubule depolymerizing activity of pyrimido[4,5-b]indoles.