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1.
ChemMedChem ; 18(14): e202300128, 2023 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-37126222

RESUMO

Phenylalanine arginine ß-naphthylamine, or PAßN, is a C-terminus capped dipeptide discovered in 1999 as an RND-type efflux pump inhibitor (EPI). Since then, PAßN has become a standard tool compound in EPI research and development. Despite this, PAßN lacks a detailed or efficient synthesis, and standard parameters for its use in wild-type bacterial strains are inconsistent or non-existent. Therefore, a scalable and chromatography-free synthesis of PAßN was developed using streamlined traditional solution-phase peptide coupling chemistry. With this procedure, gram scale quantities of PAßN were synthesized alongside analogues and stereoisomers to build a focused library to evaluate simple structure activity relationships. While most analogues were less active than the broadly utilized L,L-PAßN itself, we identified that its enantiomer, D,D-PAßN, also provided 8- to 16-fold potentiation of the antibiotic levofloxacin at 40 to 50 µg/mL concentrations of EPI in various wild-type Pseudomonas aeruginosa strains. Additionally, D,D-PAßN was shown to be significantly more hydrolytically stable than L,L-PAßN, indicating that it may be a useful, and now readily synthesized, tool compound facilitating future EPI research.


Assuntos
Antibacterianos , Dipeptídeos , Antibacterianos/farmacologia , Dipeptídeos/farmacologia , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana Múltipla , Proteínas de Bactérias
2.
Org Biomol Chem ; 21(5): 1056-1069, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36628602

RESUMO

Therapeutic agents with unique molecular structures and new mechanisms of action are needed to confront the phenomenon of multidrug resistance among bacteria. Pseudoxylallemycins, cyclic tetrapeptide (CTP) natural products, have exhibited modest antibiotic activity, but their synthesis has proven challenging. Inherent ring strain in CTPs decreases the rate of cyclization in lieu of polymerization and racemization pathways, which has resulted in previous syntheses describing mixtures of diastereomers containing predominantly an undesired epimer. We have optimized the cyclization step of pseudoxylallemycin A to favor production of the natural diastereomer; notably, variation of the base, temperature, and solvent with peptide coupling reagent propylphosphonic anhydride (T3P) afforded exquisite selectivity for the natural product in as high as 97 : 3 DR, and our conditions can provide the natural product in up to 32% overall yield through 8 steps. Employing weaker bases than those typically used in peptide coupling reactions led to the greatest improvement in diastereoselectivity, and these studies demonstrated that the identity of the amine base has enormous impact on the rate of C-terminal epimerization when T3P is used, a variable usually considered of lesser consequence when combined with typical amide coupling reagents. Toward fully characterizing pseudoxylallemycin stereoisomers, variable temperature NMR was described as a tool to more clearly analyze CTPs that exhibit multiple conformational states. These synthetic and spectroscopic insights were applied toward synthesizing several natural product analogues, and their antibacterial activity was examined using microdilution assays.


Assuntos
Produtos Biológicos , Peptídeos Cíclicos , Peptídeos Cíclicos/química , Estrutura Molecular , Conformação Molecular , Estereoisomerismo
3.
ACS Omega ; 7(42): 37907-37916, 2022 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-36312355

RESUMO

Antibiotic resistance among bacteria puts immense strain on public health. The discovery of new antibiotics that work through unique mechanisms is one important pillar toward combating this threat of resistance. A functionalized amino dihydropyrimidine was reported to exhibit antibacterial activity via the inhibition of dihydrofolate reductase, an underexploited antibacterial target. Despite this promise, little is known about its structure-activity relationships (SAR) and mechanism of activity. Toward this goal, the aza-Biginelli reaction was optimized to allow for the preparation of focused libraries of functionalized amino dihydropyridines, which in some cases required the use of variable temperature NMR analysis for the conclusive assignment of compound identity and purity. Antibacterial activity was examined using microdilution assays, and compound interactions with dihydrofolate reductase were assessed using antimicrobial synergy studies alongside in vitro enzyme kinetics, differential scanning fluorimetry, and protein crystallography. Clear antibacterial SAR trends were unveiled (MIC values from >64 to 4 µg/mL), indicating that this compound class has promise for future development as an antibacterial agent. Despite this, the in vitro biochemical and biophysical studies performed alongside the synergy assays call the antibacterial mechanism into question, indicating that further studies will be required to fully evaluate the antibacterial potential of this compound class.

4.
ACS Infect Dis ; 6(4): 649-661, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32037806

RESUMO

Quorum sensing (QS), a bacterial cell-to-cell communication system mediated by small molecules and peptides, has received significant interest as a potential target to block infection. The common pathogen Pseudomonas aeruginosa uses QS to regulate many of its virulence phenotypes at high cell densities, and the LasR QS receptor plays a critical role in this process. Small molecule tools that inhibit LasR activity would serve to illuminate its role in P. aeruginosa virulence, but we currently lack highly potent and selective LasR antagonists, despite considerable research in this area. V-06-018, an abiotic small molecule discovered in a high-throughput screen, represents one of the most potent known LasR antagonists but has seen little study since its initial report. Herein, we report a systematic study of the structure-activity relationships (SARs) that govern LasR antagonism by V-06-018. We synthesized a focused library of V-06-018 derivatives and evaluated the library for bioactivity using a variety of cell-based LasR reporter systems. The SAR trends revealed by these experiments allowed us to design probes with 10-fold greater potency than that of V-06-018 and 100-fold greater potency than other commonly used N-acyl-l-homoserine lactone (AHL)-based LasR antagonists, along with high selectivities for LasR. Biochemical experiments to probe the mechanism of antagonism by V-06-018 and its analogues support these compounds interacting with the native ligand-binding site in LasR and, at least in part, stabilizing an inactive form of the protein. The compounds described herein are the most potent and efficacious antagonists of LasR known and represent robust probes both for characterizing the mechanisms of LuxR-type QS and for chemical biology research in general in the growing QS field.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Transativadores/antagonistas & inibidores , Acil-Butirolactonas/química , Desenho de Fármacos , Concentração Inibidora 50 , Pseudomonas aeruginosa/patogenicidade , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Virulência/efeitos dos fármacos
5.
Cell Chem Biol ; 25(9): 1128-1139.e3, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30033130

RESUMO

Chemical strategies to block quorum sensing (QS) could provide a route to attenuate virulence in bacterial pathogens. Considerable research has focused on this approach in Pseudomonas aeruginosa, which uses the LuxR-type receptor LasR to regulate much of its QS network. Non-native ligands that antagonize LasR have been developed, yet we have little understanding of the mode by which these compounds interact with LasR and alter its function, as the receptor is unstable in their presence. Herein, we report an approach to circumvent this challenge through the study of a series of synthetic LasR agonists with varying levels of potency. Structural investigations of these ligands with the LasR ligand-binding domain reveal that certain agonists can enforce a conformation that deviates from that observed for other, often more potent agonists. These results, when combined with cell-based and biophysical analyses, suggest a functional model for LasR that could guide future ligand design.


Assuntos
Proteínas de Bactérias/agonistas , Proteínas de Bactérias/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Transativadores/agonistas , Transativadores/metabolismo , Proteínas de Bactérias/química , Humanos , Ligantes , Simulação de Acoplamento Molecular , Conformação Proteica/efeitos dos fármacos , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/fisiologia , Transativadores/química
6.
ACS Infect Dis ; 2(1): 32-38, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26807436

RESUMO

Many common bacterial pathogens utilize quorum sensing to coordinate group behaviors and initiate virulence at high cell densities. The use of small molecules to block quorum sensing provides a means of abrogating pathogenic phenotypes, but many known quorum sensing modulators have limitations, including hydrolytic instability and displaying non-monotonic dose curves (indicative of additional targets and/or modes of action). To address these issues, we undertook a structure-based scaffold-hopping approach to develop new chemical modulators of the LasR quorum sensing receptor in Pseudomonas aeruginosa. We combined components from a triphenyl derivative known to strongly agonize LasR with chemical moieties known for LasR antagonism and generated potent LasR antagonists that are hydrolytically stable across a range of pH values. Additionally, many of these antagonists do not exhibit non-monotonic dose effects, delivering probes that inhibit LasR across a wider range of assay conditions relative to known lactone-based ligands.

7.
ACS Chem Biol ; 10(2): 421-32, 2015 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-25384256

RESUMO

Phospholipase D (PLD) hydrolyses cellular lipids to produce the important lipid second messenger phosphatidic acid. A PLD enzyme expressed by Pseudomonas aeruginosa (PldA) has been shown to be important in bacterial infection, and NAPE-PLD has emerged as being key in the synthesis of endocannabinoids. In order to better understand the biology and therapeutic potential of these less explored PLD enzymes, small molecule tools are required. Selective estrogen receptor modulators (SERMs) have been previously shown to inhibit mammalian PLD (PLD1 and PLD2). By targeted screening of a library of SERM analogues, additional parallel synthesis, and evaluation in multiple PLD assays, we discovered a novel desketoraloxifene-based scaffold that inhibited not only the two mammalian PLDs but also structurally divergent PldA and NAPE-PLD. This finding represents an important first step toward the development of small molecules possessing universal inhibition of divergent PLD enzymes to advance the field.


Assuntos
Inibidores Enzimáticos/farmacologia , Fosfolipase D/antagonistas & inibidores , Pseudomonas aeruginosa/enzimologia , Cloridrato de Raloxifeno/análogos & derivados , Cloridrato de Raloxifeno/farmacologia , Animais , Linhagem Celular , Inibidores Enzimáticos/química , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Estrutura Molecular , Fosfolipase D/genética , Fosfolipase D/metabolismo , Cloridrato de Raloxifeno/química
8.
Tetrahedron Lett ; 56(10): 1276-1279, 2015 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-26834294

RESUMO

A short, high-yielding protocol involving the enantioselective α-chlorination of aldehydes has been developed for the enantioselective synthesis of C2-functionalized aziridines and N-alkyl terminal azetidines from a common intermediate. This methodology allows for the rapid preparation of functionalized aziridines in 50-73% overall yields and 88-94% ee, and azetidines in 22-32% overall yields and 84-92% ee. Moreover, we developed a scalable and cost-effective route to the key organocatalyst (54% overall yield, >95% dr).

9.
Bioorg Med Chem Lett ; 24(24): 5553-5557, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25466173

RESUMO

This Letter describes the on-going SAR efforts based on two scaffolds, a PLD1-biased piperidinyl benzimidazolone and a PLD2-biased piperidinyl triazaspirone, with the goal of enhancing PLD inhibitory potency and isoform selectivity. Here, we found that addition of an α-methyl moiety within the PLD2-biased piperidinyl triazaspirone scaffold abolished PLD2 preference, while the incorporation of substituents onto the piperidine moiety of the PLD1-biased piperidinyl benzimidazolone, or replacement with a bioisosteric [3.3.0] core, generally retained PLD1 preference, but at diminished significance. The SAR uncovered within these two allosteric PLD inhibitor series further highlights the inherent challenges of developing isoform selective PLD inhibitors.


Assuntos
Inibidores Enzimáticos/química , Fosfolipase D/antagonistas & inibidores , Animais , Benzimidazóis/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Cinética , Microssomos/metabolismo , Fosfolipase D/metabolismo , Piperidinas/química , Ligação Proteica , Ratos , Relação Estrutura-Atividade
10.
ChemMedChem ; 9(12): 2633-7, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25210004

RESUMO

Further chemical optimization of the halopemide-derived family of dual phospholipase D1/2 (PLD1/2) inhibitors afforded ML395 (VU0468809), a potent, >80-fold PLD2 selective allosteric inhibitor (cellular PLD1, IC50 >30,000 nM; cellular PLD2, IC50 =360 nM). Moreover, ML395 possesses an attractive in vitro DMPK profile, improved physiochemical properties, ancillary pharmacology (Eurofins Panel) cleaner than any other reported PLD inhibitor, and has been found to possess interesting activity as an antiviral agent in cellular assays against a range of influenza strains (H1, H3, H5 and H7).


Assuntos
Antivirais/química , Imidazolidinas/química , Fosfolipase D/antagonistas & inibidores , Compostos de Espiro/química , Animais , Antivirais/farmacocinética , Antivirais/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cães , Meia-Vida , Humanos , Imidazolidinas/farmacocinética , Imidazolidinas/toxicidade , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Subtipo H7N9 do Vírus da Influenza A/efeitos dos fármacos , Células Madin Darby de Rim Canino , Fosfolipase D/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Compostos de Espiro/farmacocinética , Compostos de Espiro/toxicidade , Relação Estrutura-Atividade
11.
Tetrahedron Lett ; 54(28): 3627-3629, 2013 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-24039308

RESUMO

In this Letter, we describe a short, high yielding protocol for the enantioselective (87-96% ee) and general synthesis of ß-fluoroamines and previously difficult to access γ-fluoroamines from commercial aldehydes via organocatalysis.

12.
J Biol Chem ; 288(28): 20477-87, 2013 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-23723068

RESUMO

Phosphatidic acid (PA) is a lipid second messenger located at the intersection of several lipid metabolism and cell signaling events including membrane trafficking, survival, and proliferation. Generation of signaling PA has long been primarily attributed to the activation of phospholipase D (PLD). PLD catalyzes the hydrolysis of phosphatidylcholine into PA. A variety of both receptor-tyrosine kinase and G-protein-coupled receptor stimulations have been shown to lead to PLD activation and PA generation. This study focuses on profiling the PA pool upon P2Y6 receptor signaling manipulation to determine the major PA producing enzymes. Here we show that PLD, although highly active, is not responsible for the majority of stable PA being produced upon UDP stimulation of the P2Y6 receptor and that PA levels are tightly regulated. By following PA flux in the cell we show that PLD is involved in an initial increase in PA upon receptor stimulation; however, when PLD is blocked, the cell compensates by increasing PA production from other sources. We further delineate the P2Y6 signaling pathway showing that phospholipase Cß3 (PLCß3), PLCδ1, DGKζ and PLD are all downstream of receptor activation. We also show that DGKζ is a novel negative regulator of PLD activity in this system that occurs through an inhibitory mechanism with PKCα. These results further define the downstream events resulting in PA production in the P2Y6 receptor signaling pathway.


Assuntos
Ácidos Fosfatídicos/biossíntese , Fosfatidilcolinas/metabolismo , Fosfolipase D/metabolismo , Receptores Purinérgicos P2/metabolismo , 1-Butanol/farmacologia , Western Blotting , Linhagem Celular Tumoral , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Diglicerídeos/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Humanos , Hidrólise , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Espectrometria de Massas , Modelos Biológicos , Fosfolipase C delta/genética , Fosfolipase C delta/metabolismo , Fosfolipase D/antagonistas & inibidores , Fosfolipase D/genética , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Interferência de RNA , Receptores Purinérgicos P2/genética , Transdução de Sinais/efeitos dos fármacos , Difosfato de Uridina/farmacologia
13.
J Med Chem ; 56(6): 2695-9, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23445448

RESUMO

An iterative parallel synthesis effort identified a PLD2 selective inhibitor, ML298 (PLD1 IC50 > 20000 nM, PLD2 IC50 = 355 nM) and a dual PLD1/2 inhibitor, ML299 (PLD1 IC50 = 6 nM, PLD2 IC50 = 20 nM). SAR studies revealed that a small structural change (incorporation of a methyl group) increased PLD1 activity within this classically PLD2-preferring core and that the effect was enantiospecific. Both probes decreased invasive migration in U87-MG glioblastoma cells.


Assuntos
Benzamidas/química , Benzamidas/farmacologia , Movimento Celular/efeitos dos fármacos , Descoberta de Drogas , Glioblastoma/patologia , Fosfolipase D/antagonistas & inibidores , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Benzamidas/metabolismo , Benzamidas/farmacocinética , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Humanos , Invasividade Neoplásica , Compostos de Espiro/metabolismo , Compostos de Espiro/farmacocinética
14.
Tetrahedron Lett ; 53(13): 1539-1542, 2012 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-22919116

RESUMO

In this Letter, we describe a novel three-step, one-pot procedure for the enantioselective synthesis of N-benzyl protected morpholines and orthogonally N,N'-protected piperazines with chiral alkyl groups installed at the C2 position of each heterocyclic core via organocatalysis. This methodology allows for the rapid preparation of functionalized morpholines and piperazines that are not readily accessible through any other chemistry in good to excellent % ee (55-98% ee).

15.
Org Lett ; 14(11): 2910-3, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22616979

RESUMO

A short, high yielding protocol has been developed for the enantioselective and general synthesis of C2-functionalized, benzyl protected morpholines and orthogonally N,N'-protected piperazines from a common intermediate.


Assuntos
Morfolinas/síntese química , Piperazinas/síntese química , Ciclização , Estrutura Molecular , Morfolinas/química , Piperazinas/química , Estereoisomerismo
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