RESUMO
The folate biosynthetic pathway offers many druggable targets that have yet to be exploited in tuberculosis therapy. Herein, we have identified a series of small molecules that interrupt Mycobacterium tuberculosis (Mtb) folate metabolism by dual targeting of dihydrofolate reductase (DHFR), a key enzyme in the folate pathway, and its functional analog, Rv2671. We have also compared the antifolate activity of these compounds with that of para-aminosalicylic acid (PAS). We found that the bioactive metabolite of PAS, in addition to previously reported activity against DHFR, inhibits flavin-dependent thymidylate synthase in Mtb, suggesting a multi-targeted mechanism of action for this drug. Finally, we have shown that antifolate treatment in Mtb decreases the production of mycolic acids, most likely due to perturbation of the activated methyl cycle. We conclude that multi-targeting of the folate pathway in Mtb is associated with highly potent anti-mycobacterial activity.
Assuntos
Ácido 4-Aminobenzoico/farmacologia , Antituberculosos/farmacologia , Ácido Fólico/metabolismo , Mycobacterium tuberculosis/química , Bibliotecas de Moléculas Pequenas/farmacologia , Ácido 4-Aminobenzoico/química , Ácido 4-Aminobenzoico/metabolismo , Antituberculosos/química , Antituberculosos/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
AK3 compounds are mitotic arrest agents that induce high levels of γH2AX during mitosis and apoptosis following release from arrest. We synthesized a potent AK3 derivative, AK306, that induced arrest and apoptosis of the HCT116 colon cancer cell line with an EC50 of approximately 50 nmol/L. AK306 was active on a broad spectrum of cancer cell lines with total growth inhibition values ranging from approximately 25 nmol/L to 25 µmol/L. Using biotin and BODIPY-linked derivatives of AK306, binding to clathrin heavy chain (CLTC/CHC) was observed, a protein with roles in endocytosis and mitosis. AK306 inhibited mitosis and endocytosis, while disrupting CHC cellular localization. Cells arrested in mitosis by AK306 showed the formation of multiple microtubule-organizing centers consisting of pericentrin, γ-tubulin, and Aurora A foci, without apparent centrosome amplification. Cells released from AK306 arrest were unable to form bipolar spindles, unlike nocodazole-released cells that reformed spindles and completed division. Like AK306, CHC siRNA knockdown disrupted spindle formation and activated p53. A short-term (3-day) treatment of tumor-bearing APC-mutant mice with AK306 increased apoptosis in tumors, but not normal mucosa. These findings indicate that targeting the mitotic CHC complex can selectively induce apoptosis and may have therapeutic value.Implication: Disruption of clathrin with a small-molecule inhibitor, AK306, selectively induces apoptosis in cancer cells by disrupting bipolar spindle formation. Mol Cancer Res; 16(9); 1361-72. ©2018 AACR.
Assuntos
Cadeias Pesadas de Clatrina/metabolismo , Piperazinas/farmacologia , Fuso Acromático/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Cadeias Pesadas de Clatrina/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Masculino , Camundongos , Mitose/efeitos dos fármacos , Terapia de Alvo Molecular , Piperazinas/química , Fuso Acromático/genética , Fuso Acromático/metabolismo , Relação Estrutura-Atividade , TransfecçãoRESUMO
To develop next generation antifolates for the treatment of trimethoprim-resistant bacteria, synthetic methods were needed to prepare a diverse array of 3-aryl-propynes with various substitutions at the propargyl position. A direct route was sought whereby nucleophilic addition of acetylene to aryl carboxaldehydes would be followed by reduction or substitution of the resulting propargyl alcohol. The direct reduction, methylation, and dimethylation of these readily available alcohols provide efficient access to this uncommon functional array. In addition, an unusual silane exchange reaction was observed in the reduction of the propargylic alcohols.
Assuntos
Álcoois/síntese química , Alcinos/química , Antibacterianos/química , Antagonistas do Ácido Fólico/química , Aldeídos/química , Alcinos/síntese química , Antibacterianos/síntese química , Desenho de Fármacos , Farmacorresistência Bacteriana , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/farmacologia , Humanos , Metilação , Estrutura Molecular , Oxirredução , Estereoisomerismo , Relação Estrutura-Atividade , Trimetoprima/farmacologiaRESUMO
Antibiotic resistance is a rapidly evolving health concern that requires a sustained effort to understand mechanisms of resistance and to develop new agents that overcome those mechanisms. The dihydrofolate reductase (DHFR) inhibitor, trimethoprim (TMP), remains one of the most important orally administered antibiotics. However, resistance through chromosomal mutations and mobile, plasmid-encoded insensitive DHFRs threatens the continued use of this agent. We are pursuing the development of new propargyl-linked antifolate (PLA) DHFR inhibitors designed to evade these mechanisms. While analyzing contemporary TMP-resistant clinical isolates of methicillin-resistant and sensitive Staphylococcus aureus, we discovered two mobile resistance elements, dfrG and dfrK. This is the first identification of these resistance mechanisms in the United States. These resistant organisms were isolated from a variety of infection sites, show clonal diversity, and each contain distinct resistance genotypes for common antibiotics. Several PLAs showed significant activity against these resistant strains by direct inhibition of the TMP resistance elements.
RESUMO
Mycobacterium tuberculosis continues to cause widespread, life-threatening disease. In the last decade, this threat has grown dramatically as multi- and extensively-drug resistant (MDR and XDR) bacteria have spread globally and the number of agents that effectively treat these infections is significantly reduced. We have been developing the propargyl-linked antifolates (PLAs) as potent inhibitors of the essential enzyme dihydrofolate reductase (DHFR) from bacteria and recently found that charged PLAs with partial zwitterionic character showed improved mycobacterial cell permeability. Building on a hypothesis that these PLAs may penetrate the outer membrane of M. tuberculosis and inhibit the essential cytoplasmic DHFR, we screened a group of PLAs for antitubercular activity. In this work, we identified several PLAs as potent inhibitors of the growth of M. tuberculosis with several of the compounds exhibiting minimum inhibition concentrations equal to or less than 1 µg/mL. Furthermore, two of the compounds were very potent inhibitors of MDR and XDR strains. A high resolution crystal structure of one PLA bound to DHFR from M. tuberculosis reveals the interactions of the ligands with the target enzyme.
Assuntos
Antituberculosos/química , Proteínas de Bactérias , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Antagonistas do Ácido Fólico/química , Mycobacterium tuberculosis/enzimologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , HumanosRESUMO
Although classical, negatively charged antifolates such as methotrexate possess high affinity for the dihydrofolate reductase (DHFR) enzyme, they are unable to penetrate the bacterial cell wall, rendering them poor antibacterial agents. Herein, we report a new class of charged propargyl-linked antifolates that capture some of the key contacts common to the classical antifolates while maintaining the ability to passively diffuse across the bacterial cell wall. Eight synthesized compounds exhibit extraordinary potency against Gram-positive S. aureus with limited toxicity against mammalian cells and good metabolic profile. High resolution crystal structures of two of the compounds reveal extensive interactions between the carboxylate and active site residues through a highly organized water network.
RESUMO
Drug-resistant enzymes must balance catalytic function with inhibitor destabilization to provide a fitness advantage. This sensitive balance, often involving very subtle structural changes, must be achieved through a selection process involving a minimal number of eligible point mutations. As part of a program to design propargyl-linked antifolates (PLAs) against trimethoprim-resistant dihydrofolate reductase (DHFR) from Staphylococcus aureus, we have conducted a thorough study of several clinically observed chromosomal mutations in the enzyme at the cellular, biochemical, and structural levels. Through this work, we have identified a promising lead series that displays significantly greater activity against these mutant enzymes and strains than TMP. The best inhibitors have enzyme inhibition and MIC values near or below that of trimethoprim against wild-type S. aureus. Moreover, these studies employ a series of crystal structures of several mutant enzymes bound to the same inhibitor; analysis of the structures reveals a more detailed molecular understanding of drug resistance in this important enzyme.
Assuntos
Antibacterianos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Trimetoprima/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/química , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Immune and inflammatory death ligands expressed within neoplastic tissue could potentially target apoptosis to transformed cells. To develop approaches that accentuate the anti-cancer potential of the inflammatory response, the Chembridge DIVERSet (TM) library was screened for compounds that accentuated apoptosis in a strictly TNF-dependent manner. We identified a number of novel compounds with this activity, the most active of these, AK3 and AK10, sensitized colon cancer cells to TNF at 0.5 µM and 2 µM, respectively, without inducing apoptosis on their own. The activity of these compounds was structure-dependent and general, as they accentuated cell death by TNF or Fas ligation in multiple colon cancer cell lines. Both AK3 and AK10 arrested cells in mitosis, with live cell imaging indicating that mitotically arrested cells were the source of apoptotic bodies. AK3 accentuated caspase-8 and caspase-9 activation with little effect on NFκB target gene activation. Enhanced caspase activation corresponded to an increased expression of TNFR1 on the cell surface. To determine the general interplay between mitotic arrest and TNF sensitivity, Aurora kinase (MLN8054 and MLN8237) and PLK1 (BI2536) inhibitors were tested for their ability to sensitize cells to TNF. PLK1 inhibition was particularly effective and influenced TNFR1 surface presentation and caspase cleavage like AK3, even though it arrested mitosis at an earlier stage. We propose that AK3 and AK10 represent a new class of mitotic inhibitor and that selected mitotic inhibitors may be useful for treating colon cancers or earlier lesions that have a high level of inflammatory cell infiltrate.
Assuntos
Antineoplásicos/farmacologia , Extratos Celulares/farmacologia , Neoplasias do Colo/tratamento farmacológico , Inflamação/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Sinergismo Farmacológico , Células HCT116 , Células HT29 , Humanos , Inflamação/patologia , Ligantes , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Hospital- and community-acquired, complicated skin and soft tissue infections, often attributed to Staphylococcus aureus and Streptococcus pyogenes, present a significant health burden that is associated with increased health care costs and mortality. As these two species are difficult to discern on diagnosis and are associated with differential profiles of drug resistance, the development of an efficacious antibacterial agent that targets both organisms is a high priority. Herein we describe a structure-based drug development effort that has produced highly potent inhibitors of dihydrofolate reductase from both species. Optimized propargyl-linked antifolates containing a key pyridyl substituent display antibacterial activity against both methicillin-resistant S. aureus and S. pyogenes at MIC values below 0.1 µg/mL and minimal cytotoxicity against mammalian cells. Further evaluation against a panel of clinical isolates shows good efficacy against a range of important phenotypes such as hospital- and community-acquired strains as well as strains resistant to vancomycin.