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1.
Nature ; 538(7625): 344-349, 2016 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27602946

RESUMO

Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.


Assuntos
Antimaláricos/síntese química , Antimaláricos/farmacologia , Azetidinas/uso terapêutico , Descoberta de Drogas , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Animais , Antimaláricos/administração & dosagem , Antimaláricos/uso terapêutico , Compostos Azabicíclicos/administração & dosagem , Compostos Azabicíclicos/síntese química , Compostos Azabicíclicos/farmacologia , Compostos Azabicíclicos/uso terapêutico , Azetidinas/administração & dosagem , Azetidinas/efeitos adversos , Azetidinas/farmacologia , Citosol/enzimologia , Modelos Animais de Doenças , Feminino , Fígado/efeitos dos fármacos , Fígado/parasitologia , Macaca mulatta/parasitologia , Malária Falciparum/prevenção & controle , Malária Falciparum/transmissão , Masculino , Camundongos , Fenilalanina-tRNA Ligase/antagonistas & inibidores , Compostos de Fenilureia/administração & dosagem , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Plasmodium falciparum/citologia , Plasmodium falciparum/enzimologia , Segurança
2.
J Am Chem Soc ; 139(32): 11300-11306, 2017 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-28732448

RESUMO

The development of new antimalarial therapeutics is necessary to address the increasing resistance to current drugs. Bicyclic azetidines targeting Plasmodium falciparum phenylalanyl-tRNA synthetase comprise one promising new class of antimalarials, especially due to their activities against three stages of the parasite's life cycle, but a lengthy synthetic route to these compounds may affect the feasibility of delivering new therapeutic agents within the cost constraints of antimalarial drugs. Here, we report an efficient synthesis of antimalarial compound BRD3914 (EC50 = 15 nM) that hinges on a Pd-catalyzed, directed C(sp3)-H arylation of azetidines at the C3 position. This newly developed protocol exhibits a broad substrate scope and provides access to valuable, stereochemically defined building blocks. BRD3914 was evaluated in P. falciparum-infected mice, providing a cure after four oral doses.


Assuntos
Antimaláricos/síntese química , Antimaláricos/uso terapêutico , Azetidinas/síntese química , Azetidinas/uso terapêutico , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Azetidinas/química , Azetidinas/farmacologia , Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/química , Compostos Bicíclicos com Pontes/farmacologia , Compostos Bicíclicos com Pontes/uso terapêutico , Catálise , Modelos Animais de Doenças , Células Hep G2 , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Paládio/química , Estereoisomerismo
3.
J Infect Dis ; 211(7): 1097-103, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25336726

RESUMO

BACKGROUND: The emergence and spread of drug resistance to current antimalarial therapies remains a pressing concern, escalating the need for compounds that demonstrate novel modes of action. Diversity-Oriented Synthesis (DOS) libraries bridge the gap between conventional small molecule and natural product libraries, allowing the interrogation of more diverse chemical space in efforts to identify probes of novel parasite pathways. METHODS: We screened and optimized a probe from a DOS library using whole-cell phenotypic assays. Resistance selection and whole-genome sequencing approaches were employed to identify the cellular target of the compounds. RESULTS: We identified a novel macrocyclic inhibitor of Plasmodium falciparum with nanomolar potency and identified the reduction site of cytochrome b as its cellular target. Combination experiments with reduction and oxidation site inhibitors showed synergistic inhibition of the parasite. CONCLUSIONS: The cytochrome b oxidation center is a validated antimalarial target. We show that the reduction site of cytochrome b is also a druggable target. Our results demonstrating a synergistic relationship between oxidation and reduction site inhibitors suggests a future strategy for new combination therapies in the treatment of malaria.


Assuntos
Antimaláricos/farmacologia , Citocromos b/antagonistas & inibidores , Descoberta de Drogas/métodos , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Antimaláricos/síntese química , Antimaláricos/química , Sequência de Bases , Domínio Catalítico , Citocromos b/química , Citocromos b/genética , Resistência a Medicamentos , Sinergismo Farmacológico , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Sequenciamento de Nucleotídeos em Larga Escala , Ensaios de Triagem em Larga Escala , Humanos , Lactamas Macrocíclicas/síntese química , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/farmacologia , Malária Falciparum/parasitologia , Dados de Sequência Molecular , Oxirredução , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacologia , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Bibliotecas de Moléculas Pequenas , Ubiquinona/metabolismo
4.
J Am Chem Soc ; 137(24): 7929-34, 2015 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-26042473

RESUMO

Phenotypic cell-based screening is a powerful approach to small-molecule discovery, but a major challenge of this strategy lies in determining the intracellular target and mechanism of action (MoA) for validated hits. Here, we show that the small-molecule BRD0476, a novel suppressor of pancreatic ß-cell apoptosis, inhibits interferon-gamma (IFN-γ)-induced Janus kinase 2 (JAK2) and signal transducer and activation of transcription 1 (STAT1) signaling to promote ß-cell survival. However, unlike common JAK-STAT pathway inhibitors, BRD0476 inhibits JAK-STAT signaling without suppressing the kinase activity of any JAK. Rather, we identified the deubiquitinase ubiquitin-specific peptidase 9X (USP9X) as an intracellular target, using a quantitative proteomic analysis in rat ß cells. RNAi-mediated and CRISPR/Cas9 knockdown mimicked the effects of BRD0476, and reverse chemical genetics using a known inhibitor of USP9X blocked JAK-STAT signaling without suppressing JAK activity. Site-directed mutagenesis of a putative ubiquitination site on JAK2 mitigated BRD0476 activity, suggesting a competition between phosphorylation and ubiquitination to explain small-molecule MoA. These results demonstrate that phenotypic screening, followed by comprehensive MoA efforts, can provide novel mechanistic insights into ostensibly well-understood cell signaling pathways. Furthermore, these results uncover USP9X as a potential target for regulating JAK2 activity in cellular inflammation.


Assuntos
Células Secretoras de Insulina/efeitos dos fármacos , Interferon gama/imunologia , Janus Quinase 2/imunologia , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia , Fator de Transcrição STAT1/imunologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/imunologia , Fosforilação/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina Tiolesterase/imunologia , Ubiquitinação/efeitos dos fármacos
5.
Proc Natl Acad Sci U S A ; 108(17): 6751-6, 2011 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-21383141

RESUMO

Complexity and the presence of stereogenic centers have been correlated with success as compounds transition from discovery through the clinic. Here we describe the synthesis of a library of pyran-containing macrocycles with a high degree of structural complexity and up to five stereogenic centers. A key feature of the design strategy was to use a modular synthetic route with three fragments that can be readily interchanged or "shuffled" to produce subtly different variants with distinct molecular shapes. A total of 352 macrocycles were synthesized ranging in size from 14- to 16-membered rings. In order to facilitate the generation of stereostructure-activity relationships, the complete matrix of stereoisomers was prepared for each macrocycle. Solid-phase assisted parallel solution-phase techniques were employed to allow for rapid analogue generation. An intramolecular nitrile-activated nucleophilic aromatic substitution reaction was used for the key macrocyclization step.


Assuntos
Compostos Macrocíclicos/química , Compostos Macrocíclicos/síntese química , Piranos/química , Estrutura Molecular
6.
bioRxiv ; 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38464220

RESUMO

Previous studies have shown that bicyclic azetidines are potent and selective inhibitors of apicomplexan phenylalanine tRNA synthetase (PheRS), leading to parasite growth inhibition in vitro and in vivo, including in models of Toxoplasma infection. Despite these useful properties, additional optimization is required for the development of efficacious treatments of toxoplasmosis from this inhibitor series, in particular to achieve sufficient exposure in the brain. Here, we describe a series of PheRS inhibitors built on a new bicyclic pyrrolidine core scaffold designed to retain the exit-vector geometry of the isomeric bicyclic azetidine core scaffold while offering avenues to sample diverse chemical space. Relative to the parent series, bicyclic pyrrolidines retain reasonable potency and target selectivity for parasite PheRS vs. host. Further structure-activity relationship studies revealed that the introduction of aliphatic groups improved potency, ADME and PK properties, including brain exposure. The identification of this new scaffold provides potential opportunities to extend the analog series to further improve selectivity and potency and ultimately deliver a novel, efficacious treatment of toxoplasmosis.

7.
ACS Infect Dis ; 10(6): 2212-2221, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38743643

RESUMO

Previous studies have shown that bicyclic azetidines are potent and selective inhibitors of apicomplexan phenylalanine tRNA synthetase (PheRS), leading to parasite growth inhibition in vitro and in vivo, including in models of Toxoplasma infection. Despite these useful properties, additional optimization is required for the development of efficacious treatments of toxoplasmosis from this inhibitor series, in particular, to achieve optimal exposure in the brain. Here, we describe a series of PheRS inhibitors built on a new bicyclic pyrrolidine core scaffold designed to retain the exit-vector geometry of the isomeric bicyclic azetidine core scaffold while offering avenues to sample diverse chemical space. Relative to the parent series, bicyclic pyrrolidines retain reasonable potency and target selectivity for parasite PheRS vs host. Further structure-activity relationship studies revealed that the introduction of aliphatic groups improved potency and ADME and PK properties, including brain exposure. The identification of this new scaffold provides potential opportunities to extend the analogue series to further improve selectivity and potency and ultimately deliver a novel, efficacious treatment of toxoplasmosis.


Assuntos
Encéfalo , Fenilalanina-tRNA Ligase , Pirrolidinas , Toxoplasma , Toxoplasma/efeitos dos fármacos , Toxoplasma/enzimologia , Pirrolidinas/farmacologia , Pirrolidinas/química , Animais , Encéfalo/parasitologia , Relação Estrutura-Atividade , Fenilalanina-tRNA Ligase/antagonistas & inibidores , Fenilalanina-tRNA Ligase/química , Antiparasitários/farmacologia , Antiparasitários/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Camundongos , Toxoplasmose/tratamento farmacológico , Humanos , Azetidinas/farmacologia , Azetidinas/química
8.
Structure ; 30(7): 962-972.e3, 2022 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-35460612

RESUMO

Bicyclic azetidine compounds possess antimalarial activity via targeting of the cytoplasmic Plasmodium falciparum (Pf) protein translation enzyme phenylalanine-tRNA synthetase (cFRS). These drugs kill parasites both in vitro and in vivo, including the blood, liver, and transmission developmental stages. Here we present the co-crystal structure of PfcFRS with a potent inhibitor, the bicyclic azetidine BRD7929. Our studies reveal high-affinity binding of BRD7929 with PfcFRS along with exquisite specificity compared with the human enzyme, leading in turn to potent and selective inhibition of the parasite enzyme. Our co-crystal structure shows that BRD7929 binds in the active site in the α subunit of PfcFRS, where it occupies the amino acid site, an auxiliary site, and partially the ATP site. This structural snapshot of inhibitor-bound PfcFRS thus provides a platform for the structure-guided optimization of novel antimalarial compounds.


Assuntos
Aminoacil-tRNA Sintetases , Antimaláricos , Azetidinas , Antimaláricos/química , Antimaláricos/farmacologia , Humanos , Fenilalanina , Plasmodium falciparum/genética
9.
Nat Commun ; 13(1): 459, 2022 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-35075105

RESUMO

Toxoplasma gondii commonly infects humans and while most infections are controlled by the immune response, currently approved drugs are not capable of clearing chronic infection in humans. Hence, approximately one third of the world's human population is at risk of reactivation, potentially leading to severe sequelae. To identify new candidates for treating chronic infection, we investigated a series of compounds derived from diversity-oriented synthesis. Bicyclic azetidines are potent low nanomolar inhibitors of phenylalanine tRNA synthetase (PheRS) in T. gondii, with excellent selectivity. Biochemical and genetic studies validate PheRS as the primary target of bicyclic azetidines in T. gondii, providing a structural basis for rational design of improved analogs. Favorable pharmacokinetic properties of a lead compound provide excellent protection from acute infection and partial protection from chronic infection in an immunocompromised mouse model of toxoplasmosis. Collectively, PheRS inhibitors of the bicyclic azetidine series offer promise for treatment of chronic toxoplasmosis.


Assuntos
Antiprotozoários/administração & dosagem , Azetidinas/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Fenilalanina-tRNA Ligase/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Toxoplasma/efeitos dos fármacos , Toxoplasma/enzimologia , Toxoplasmose/tratamento farmacológico , Animais , Antiprotozoários/química , Azetidinas/química , Inibidores Enzimáticos/química , Feminino , Humanos , Cinética , Masculino , Camundongos , Camundongos Endogâmicos CBA , Fenilalanina-tRNA Ligase/química , Fenilalanina-tRNA Ligase/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Toxoplasma/genética , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose/parasitologia
10.
J Org Chem ; 76(19): 8042-8, 2011 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-21875084

RESUMO

An efficient build/couple/pair approach to diversity-oriented synthesis was employed to access several structurally complex macrolactams. In this paper, we describe the successful evaluation of ring-closing metathesis toward the systematic generation of skeletal diversity. By appropriately varying the nature and chain length of the alkenol fragment, a diverse collection of 13- to 18-membered macrolactams were obtained.


Assuntos
Técnicas de Química Sintética/métodos , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/síntese química , Aldeídos/química , Estereoisomerismo
11.
Nat Commun ; 12(1): 343, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436639

RESUMO

The inhibition of Plasmodium cytosolic phenylalanine tRNA-synthetase (cFRS) by a novel series of bicyclic azetidines has shown the potential to prevent malaria transmission, provide prophylaxis, and offer single-dose cure in animal models of malaria. To date, however, the molecular basis of Plasmodium cFRS inhibition by bicyclic azetidines has remained unknown. Here, we present structural and biochemical evidence that bicyclic azetidines are competitive inhibitors of L-Phe, one of three substrates required for the cFRS-catalyzed aminoacylation reaction that underpins protein synthesis in the parasite. Critically, our co-crystal structure of a PvcFRS-BRD1389 complex shows that the bicyclic azetidine ligand binds to two distinct sub-sites within the PvcFRS catalytic site. The ligand occupies the L-Phe site along with an auxiliary cavity and traverses past the ATP binding site. Given that BRD1389 recognition residues are conserved amongst apicomplexan FRSs, this work lays a structural framework for the development of drugs against both Plasmodium and related apicomplexans.


Assuntos
Azetidinas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Malária/enzimologia , Parasitos/enzimologia , Fenilalanina-tRNA Ligase/antagonistas & inibidores , Fenilalanina-tRNA Ligase/química , Plasmodium falciparum/enzimologia , Sequência de Aminoácidos , Aminoacilação , Animais , Domínio Catalítico , Citosol/enzimologia , Resistência a Medicamentos/genética , Modelos Moleculares , Mutação/genética , Fenilalanina/metabolismo , Fenilalanina-tRNA Ligase/metabolismo , Plasmodium falciparum/efeitos dos fármacos
12.
J Am Chem Soc ; 132(47): 16962-76, 2010 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21067169

RESUMO

An aldol-based build/couple/pair (B/C/P) strategy was applied to generate a collection of stereochemically and skeletally diverse small molecules. In the build phase, a series of asymmetric syn- and anti-aldol reactions were performed to produce four stereoisomers of a Boc-protected γ-amino acid. In addition, both stereoisomers of O-PMB-protected alaninol were generated to provide a chiral amine coupling partner. In the couple step, eight stereoisomeric amides were synthesized by coupling the chiral acid and amine building blocks. The amides were subsequently reduced to generate the corresponding secondary amines. In the pair phase, three different reactions were employed to enable intramolecular ring-forming processes: nucleophilic aromatic substitution (S(N)Ar), Huisgen [3+2] cycloaddition, and ring-closing metathesis (RCM). Despite some stereochemical dependencies, the ring-forming reactions were optimized to proceed with good to excellent yields, providing a variety of skeletons ranging in size from 8- to 14-membered rings. Scaffolds resulting from the RCM pairing reaction were diversified on the solid phase to yield a 14 400-membered library of macrolactams. Screening of this library led to the discovery of a novel class of histone deacetylase inhibitors, which display mixed enzyme inhibition, and led to increased levels of acetylation in a primary mouse neuron culture. The development of stereo-structure/activity relationships was made possible by screening all 16 stereoisomers of the macrolactams produced through the aldol-based B/C/P strategy.


Assuntos
Aldeídos/química , Descoberta de Drogas/métodos , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/farmacologia , Animais , Produtos Biológicos/síntese química , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Inibidores de Histona Desacetilases/química , Compostos Macrocíclicos/química , Camundongos , Modelos Moleculares , Conformação Molecular , Estereoisomerismo , Especificidade por Substrato
13.
Sci Transl Med ; 12(563)2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32998973

RESUMO

Cryptosporidium is a protozoan parasite and a leading cause of diarrheal disease and mortality in young children. Currently, there are no fully effective treatments available to cure infection with this diarrheal pathogen. In this study, we report a broad drug repositioning effort that led to the identification of bicyclic azetidines as a new anticryptosporidial series. Members of this series blocked growth in in vitro culture of three Cryptosporidium parvum isolates with EC50 's in 1% serum of <0.4 to 96 nM, had comparable potencies against Cryptosporidium hominis and C. parvum, and was effective in three of four highly susceptible immunosuppressed mice with once-daily dosing administered for 4 days beginning 2 weeks after infection. Comprehensive genetic, biochemical, and chemical studies demonstrated inhibition of C. parvum phenylalanyl-tRNA synthetase (CpPheRS) as the mode of action of this new lead series. Introduction of mutations directly into the C. parvum pheRS gene by CRISPR-Cas9 genome editing resulted in parasites showing high degrees of compound resistance. In vitro, bicyclic azetidines potently inhibited the aminoacylation activity of recombinant ChPheRS. Medicinal chemistry optimization led to the identification of an optimal pharmacokinetic/pharmacodynamic profile for this series. Collectively, these data demonstrate that bicyclic azetidines are a promising series for anticryptosporidial drug development and establish a broad framework to enable target-based drug discovery for this infectious disease.


Assuntos
Azetidinas , Criptosporidiose , Cryptosporidium , Parasitos , Fenilalanina-tRNA Ligase , Animais , Azetidinas/farmacologia , Criptosporidiose/tratamento farmacológico , Diarreia , Camundongos
14.
Cell Chem Biol ; 25(12): 1506-1518.e13, 2018 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-30318461

RESUMO

Apart from their antimicrobial properties, tetracyclines demonstrate clinically validated effects in the amelioration of pathological inflammation and human cancer. Delineation of the target(s) and mechanism(s) responsible for these effects, however, has remained elusive. Here, employing quantitative mass spectrometry-based proteomics, we identified human 80S ribosomes as targets of the tetracyclines Col-3 and doxycycline. We then developed in-cell click selective crosslinking with RNA sequence profiling (icCL-seq) to map binding sites for these tetracyclines on key human rRNA substructures at nucleotide resolution. Importantly, we found that structurally and phenotypically variant tetracycline analogs could chemically discriminate these rRNA binding sites. We also found that tetracyclines both subtly modify human ribosomal translation and selectively activate the cellular integrated stress response (ISR). Together, the data reveal that targeting of specific rRNA substructures, activation of the ISR, and inhibition of translation are correlated with the anti-proliferative properties of tetracyclines in human cancer cell lines.


Assuntos
Biossíntese de Proteínas/efeitos dos fármacos , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Tetraciclinas/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , RNA Ribossômico/genética , Relação Estrutura-Atividade , Tetraciclinas/química
15.
ACS Infect Dis ; 4(10): 1499-1507, 2018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30058798

RESUMO

Toxoplasma gondii is an obligate intracellular parasite capable of causing severe disease due to congenital infection and in patients with compromised immune systems. Control of infection is dependent on a robust Th1 type immune response including production of interferon gamma (IFN-γ), which is essential for control. IFN-γ activates a variety of antimicrobial mechanisms in host cells, which are then able to control intracellular parasites such as T. gondii. Despite the effectiveness of these pathways in controlling acute infection, the immune system is unable to eradicate chronic infections that can persist for life. Similarly, while antibiotic treatment can control acute infection, it is unable to eliminate chronic infection. To identify compounds that would act synergistically with IFN-γ, we performed a high-throughput screen of diverse small molecule libraries to identify inhibitors of T. gondii. We identified a number of compounds that inhibited parasite growth in vitro at low µM concentrations and that demonstrated enhanced potency in the presence of a low level of IFN-γ. A subset of these compounds act by enhancing the recruitment of light chain 3 (LC3) to the parasite-containing vacuole, suggesting they work by an autophagy-related process, while others were independent of this pathway. The pattern of IFN-γ dependence was shared among the majority of analogs from 6 priority scaffolds, and analysis of structure activity relationships for one such class revealed specific stereochemistry associated with this feature. Identification of these IFN-γ-dependent leads may lead to development of improved therapeutics due to their synergistic interactions with immune responses.


Assuntos
Inibidores do Crescimento/análise , Inibidores do Crescimento/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Interferon gama/metabolismo , Toxoplasma/crescimento & desenvolvimento , Autofagia/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Inibidores do Crescimento/química , Células HeLa , Humanos , Imunidade Inata , Modelos Lineares , Luciferases/análise , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , Bibliotecas de Moléculas Pequenas , Estereoisomerismo , Células Th1/imunologia , Vacúolos/metabolismo
16.
Medchemcomm ; 9(11): 1831-1842, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30542533

RESUMO

Increased expression of the Tribbles pseudokinase 1 gene (TRIB1) is associated with lower plasma levels of LDL cholesterol and triglycerides, higher levels of HDL cholesterol and decreased risk of coronary artery disease and myocardial infarction. We identified a class of tricyclic glycal core-based compounds that upregulate TRIB1 expression in human HepG2 cells and phenocopy the effects of genetic TRIB1 overexpression as they inhibit expression of triglyceride synthesis genes and ApoB secretion in cells. In addition to predicted effects related to downregulation of VLDL assembly and secretion these compounds also have unexpected effects as they upregulate expression of LDLR and stimulate LDL uptake. This activity profile is unique and favorably differs from profiles produced by statins or other lipoprotein targeting therapies. BRD8518, the initial lead compound from the tricyclic glycal class, exhibited stereochemically dependent activity and the potency far exceeding previously described benzofuran BRD0418. Gene expression profiling of cells treated with BRD8518 demonstrated the anticipated changes in lipid metabolic genes and revealed a broad stimulation of early response genes. Consistently, we found that BRD8518 activity is MEK1/2 dependent and the treatment of HepG2 cells with BRD8518 stimulates ERK1/2 phosphorylation. In agreement with down-regulation of genes controlling triglyceride synthesis and assembly of lipoprotein particles, the mass spectrometry analysis of cell extracts showed reduced rate of incorporation of stable isotope labeled glycerol into triglycerides in BRD8518 treated cells. Furthermore, we describe medicinal chemistry efforts that led to identification of BRD8518 analogs with enhanced potency and pharmacokinetic properties suitable for in vivo studies.

17.
Org Lett ; 9(11): 2123-6, 2007 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-17472394

RESUMO

Diversification of enantioenriched Michael adducts through functional group pairing to gain access to a range of five- to ten-membered complex fused and bridged ring systems is described.


Assuntos
Hidrocarbonetos Cíclicos/síntese química , Modelos Moleculares , Estrutura Molecular
18.
Cell Host Microbe ; 19(1): 114-26, 2016 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-26749441

RESUMO

Preventing transmission is an important element of malaria control. However, most of the current available methods to assay for malaria transmission blocking are relatively low throughput and cannot be applied to large chemical libraries. We have developed a high-throughput and cost-effective assay, the Saponin-lysis Sexual Stage Assay (SaLSSA), for identifying small molecules with transmission-blocking capacity. SaLSSA analysis of 13,983 unique compounds uncovered that >90% of well-characterized antimalarials, including endoperoxides and 4-aminoquinolines, as well as compounds active against asexual blood stages, lost most of their killing activity when parasites developed into metabolically quiescent stage V gametocytes. On the other hand, we identified compounds with consistent low nanomolar transmission-blocking activity, some of which showed cross-reactivity against asexual blood and liver stages. The data clearly emphasize substantial physiological differences between sexual and asexual parasites and provide a tool and starting points for the discovery and development of transmission-blocking drugs.


Assuntos
Antimaláricos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Malária/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Humanos , Malária/transmissão , Plasmodium falciparum/fisiologia
19.
ACS Infect Dis ; 2(4): 281-293, 2016 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-27275010

RESUMO

In order to identify the most attractive starting points for drugs that can be used to prevent malaria, a diverse chemical space comprising tens of thousands to millions of small molecules may need to be examined. Achieving this throughput necessitates the development of efficient ultra-high-throughput screening methods. Here, we report the development and evaluation of a luciferase-based phenotypic screen of malaria exoerythrocytic-stage parasites optimized for a 1536-well format. This assay uses the exoerythrocytic stage of the rodent malaria parasite, Plasmodium berghei, and a human hepatoma cell line. We use this assay to evaluate several biased and unbiased compound libraries, including two small sets of molecules (400 and 89 compounds, respectively) with known activity against malaria erythrocytic-stage parasites and a set of 9886 diversity-oriented synthesis (DOS)-derived compounds. Of the compounds screened, we obtain hit rates of 12-13 and 0.6% in preselected and naïve libraries, respectively, and identify 52 compounds with exoerythrocytic-stage activity less than 1 µM and having minimal host cell toxicity. Our data demonstrate the ability of this method to identify compounds known to have causal prophylactic activity in both human and animal models of malaria, as well as novel compounds, including some exclusively active against parasite exoerythrocytic stages.

20.
PLoS Negl Trop Dis ; 9(9): e0004094, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26407168

RESUMO

Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery.


Assuntos
Antiprotozoários/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Leishmania donovani/efeitos dos fármacos , Cultura Axênica , Humanos , Leishmania donovani/crescimento & desenvolvimento , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Valor Preditivo dos Testes
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