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1.
Bioorg Chem ; 151: 107689, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39111119

RESUMO

Immune-mediated inflammatory diseases (IMIDs) comprise a broad spectrum of conditions characterized by systemic inflammation affecting various organs and tissues, for which there is no known cure. The isoform-specific inhibition of phosphodiesterase-4B (PDE4B) over PDE4D constitutes an effective therapeutic strategy for the treatment of IMIDs that minimizes the adverse effects associated with non-selective PDE4 inhibitors. Thus, we report a new class of isoquinolone derivatives as next-generation PDE4 inhibitors for effective management of rheumatoid arthritis (RA) and psoriasis. Among the series, 8 compounds i.e. 1e, 1l, 1m, 1n, 1o, 2m, 2o and 3o showed promising PDE4B inhibition (>80 %) in vitro with IC50 ∼ 1.4-6.2 µM. The compound 1l was identified as an initial hit and was pursued for further studies. According to structure-activity relationship (SAR), an allyl group at C-4 position improved PDE4B inhibition. The correlation between in vitro activity data and binding affinities obtained via molecular docking suggested that the high-affinity binding to PDE4B is a prerequisite for the effective inhibition of PDE4B. Notably, the hit 1l showed selectivity towards PDE4B over PDE4D in vitro. Furthermore, 1l treatment (30 mg/kg) in the adjuvant-induced arthritis (AIA) rat model induced by complete Freund's adjuvant (CFA) demonstrated anti-arthritic potential via ameliorating paw swelling and body weight, narrowing joint space, reducing excessive immune cells infiltration and pannus formation in addition to reducing mRNA expression of pro-inflammatory cytokines such as TNF-α and IL-6 in synovial tissues of experimental rats. Additionally, 1l reduced the hyper-proliferative state and colony forming potential of IMQ-induced psoriatic keratinocytes. The treatment of these cells with 1l markedly reduced the protein levels of Ki67 and mRNA levels of pro-inflammatory cytokines e.g. IL-17A and TNF-α suggesting its potent anti-psoriatic potential. Furthermore, 1l did not show any significant adverse effects when evaluated in a systematic toxicity (e.g. teratogenicity, hepatotoxicity and cardiotoxicity) studies in zebrafish at the tested concentrations (1-100 µM) and the NOAEL (no-observed-adverse-effect level) was found to be 100 µM. Thus, with promising anti-inflammatory effects both in vitro and in vivo along with PDE4B selectivity with an acceptable safety margin, 1l emerged as a new and promising inhibitor for further studies.


Assuntos
Artrite Reumatoide , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Inibidores da Fosfodiesterase 4 , Psoríase , Inibidores da Fosfodiesterase 4/farmacologia , Inibidores da Fosfodiesterase 4/química , Inibidores da Fosfodiesterase 4/síntese química , Inibidores da Fosfodiesterase 4/uso terapêutico , Psoríase/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Animais , Relação Estrutura-Atividade , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Humanos , Ratos , Estrutura Molecular , Relação Dose-Resposta a Droga , Simulação de Acoplamento Molecular , Peixe-Zebra , Artrite Experimental/tratamento farmacológico , Artrite Experimental/induzido quimicamente , Artrite Experimental/patologia , Masculino
2.
Dev Cell ; 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38870942

RESUMO

Retrograde transport of WLS (Wntless) from endosomes to trans-Golgi network (TGN) is required for efficient Wnt secretion during development. However, the molecular players connecting endosomes to TGN during WLS trafficking are limited. Here, we identified a role for Eyes Absent (EYA) proteins during retrograde trafficking of WLS to TGN in human cell lines. By using worm, fly, and zebrafish models, we found that the EYA-secretory carrier-associated membrane protein 3 (SCAMP3) axis is evolved in vertebrates. EYAs form a complex and interact with retromer on early endosomes. Retromer-bound EYA complex recruits SCAMP3 to endosomes, which is necessary for the fusion of WLS-containing endosomes to TGN. Loss of EYA complex or SCAMP3 leads to defective transport of WLS to TGN and failed Wnt secretion. EYA mutations found in patients with hearing loss form a dysfunctional EYA-retromer complex that fails to activate Wnt signaling. These findings identify the EYA complex as a component of retrograde trafficking of WLS from the endosome to TGN.

3.
Hum Mol Genet ; 33(16): 1391-1405, 2024 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-38710511

RESUMO

Fragile X syndrome (FXS) is an inherited neurodevelopmental disorder and the leading genetic cause of autism spectrum disorders. FXS is caused by loss of function mutations in Fragile X mental retardation protein (FMRP), an RNA binding protein that is known to regulate translation of its target mRNAs, predominantly in the brain and gonads. The molecular mechanisms connecting FMRP function to neurodevelopmental phenotypes are well understood. However, neither the full extent of reproductive phenotypes, nor the underlying molecular mechanisms have been as yet determined. Here, we developed new fmr1 knockout zebrafish lines and show that they mimic key aspects of FXS neuronal phenotypes across both larval and adult stages. Results from the fmr1 knockout females also showed that altered gene expression in the brain, via the neuroendocrine pathway contribute to distinct abnormal phenotypes during ovarian development and oocyte maturation. We identified at least three mechanisms underpinning these defects, including altered neuroendocrine signaling in sexually mature females resulting in accelerated ovarian development, altered expression of germ cell and meiosis promoting genes at various stages during oocyte maturation, and finally a strong mitochondrial impairment in late stage oocytes from knockout females. Our findings have implications beyond FXS in the study of reproductive function and female infertility. Dissection of the translation control pathways during ovarian development using models like the knockout lines reported here may reveal novel approaches and targets for fertility treatments.


Assuntos
Modelos Animais de Doenças , Proteína do X Frágil da Deficiência Intelectual , Síndrome do Cromossomo X Frágil , Ovário , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Feminino , Ovário/metabolismo , Ovário/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Oócitos/metabolismo , Oócitos/crescimento & desenvolvimento , Encéfalo/metabolismo , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Fenótipo , Humanos , Proteínas de Ligação a RNA
4.
J Biochem Mol Toxicol ; 37(9): e23413, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37335823

RESUMO

Tyrosine kinase inhibitors (TKIs) are a major class of targeted cancer therapy drugs. Overcoming the limitations of approved TKIs and the development of new TKIs continues to be an important need. The adoption of higher throughput and accessible animal models to evaluate TKI adverse effects will help in this regard. We exposed zebrafish larvae to a set of 22 Food and Drug Administration-approved TKIs and assessed mortality, early developmental abnormalities, and gross morphological abnormalities posthatching. We found edema posthatching as a consistent and prominent consequence of VEGFR inhibitors, and of cabozantinib in particular. The edema occurred at concentrations that did not cause lethality or any other abnormality, and was independent of the developmental stage. Further experiments identified loss of blood and lymphatic vasculature, and suppression of renal function in larvae exposed to 10 µM cabozantinib. Molecular analysis showed downregulation of the vasculature marker genes vegfr, prox1a, sox18, and the renal function markers nephrin and podocin as the potential molecular basis for the above defects, implicating them in the mechanism of cabozantinib-induced edema. Our findings reveal edema as a previously unreported phenotypic effect of cabozantinib and identify the likely mechanistic basis. These findings also highlight the need for studies investigating edema due to vascular and renal dysfunction as a potential clinical adverse effect of cabozantinib, and possibly other VEGFR inhibitors.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Animais , Carcinoma de Células Renais/tratamento farmacológico , Peixe-Zebra , Inibidores de Proteínas Quinases , Neoplasias Renais/tratamento farmacológico , Rim/fisiologia
5.
Bioorg Chem ; 138: 106606, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37210826

RESUMO

The 12R-lipoxygenase (12R-LOX), a (non-heme) iron-containing metalloenzyme belonging to the lipoxygenase (LOX) family catalyzes the conversion of arachidonic acid (AA) to its key metabolites. Studies suggested that 12R-LOX plays a critical role in immune modulation for the maintenance of skin homeostasis and therefore can be considered as a potential drug target for psoriasis and other skin related inflammatory diseases. However, unlike 12-LOX (or 12S-LOX) the enzyme 12R-LOX did not receive much attention till date. In our effort, the 2-aryl quinoline derivatives were designed, synthesized and evaluated for the identification of potential inhibitors of 12R-hLOX. The merit of selection of 2-aryl quinolines was assessed by in silico docking studies of a representative compound (4a) using the homology model of 12R-LOX. Indeed, in addition to participating in H-bonding with THR628 and LEU635 the molecule formed a hydrophobic interaction with VAL631. The desired 2-aryl quinolines were synthesized either via the Claisen-Schmidt condensation followed by one-pot reduction-cyclization or via the AlCl3 induced heteroarylation or via the O-alkylation approach in good to high (82-95%) yield. When screened against human 12R-LOX (12R-hLOX) in vitro four compounds (e.g. 4a, 4d, 4e and 7b) showed encouraging (>45%) inhibition at 100 µM among which 7b and 4a emerged as the initial hits. Both the compounds showed selectivity towards 12R-hLOX over 12S-hLOX, 15-hLOX and 15-hLOXB and concentration dependent inhibition of 12R-hLOX with IC50 = 12.48 ± 2.06 and 28.25 ± 1.63 µM, respectively. The selectivity of 4a and 7b towards 12R-LOX over 12S-LOX was rationalized with the help of molecular dynamics simulations. The SAR (Structure-Activity Relationship) within the present series of compounds suggested the need of a o-hydroxyl group on the C-2 phenyl ring for the activity. The compound 4a and 7b (at 10 and 20 µM) reduced the hyper-proliferative state and colony forming potential of IMQ-induced psoriatic keratinocytes in a concentration dependent manner. Further, both compounds decreased the protein levels of Ki67 and the mRNA expression of IL-17A in the IMQ-induced psoriatic-like keratinocytes. Notably, 4a but not 7b inhibited the production of IL-6 and TNF-α in the keratinocyte cells. In the preliminary toxicity studies (i.e. teratogenicity, hepatotoxicity and heart rate assays) in zebrafish both the compounds showed low safety (<30 µM) margin. Overall, being the first identified inhibitors of 12R-LOX both 4a and 7b deserve further investigations.


Assuntos
Quinolinas , Peixe-Zebra , Animais , Humanos , Peixe-Zebra/metabolismo , Araquidonato 12-Lipoxigenase/metabolismo , Pele/metabolismo , Quinolinas/farmacologia , Relação Estrutura-Atividade , Inibidores de Lipoxigenase/farmacologia , Simulação de Acoplamento Molecular
6.
Bioorg Chem ; 134: 106452, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36889201

RESUMO

The enzyme chorismate mutase (or CM that is vital for the survival of bacteria) is an interesting pharmacological target for the identification of new anti-tubercular agents. The 5,5-disibstituted pyrazolo[4,3-d]pyrimidinone derivatives containing the fragment based on 4-amino-1-methyl-3-propyl-1H-pyrazole-5-carboxamide were designed and explored as the potential inhibitors of chorismate mutase. Based on encouraging docking results of two representative molecules evaluated in silico against MtbCM (PDB: 2FP2) the Wang resin catalysed sonochemical synthesis of target N-heteroarenes were undertaken. The methodology involved the reaction of 4-amino-1-methyl-3-propyl-1H-pyrazole-5-carboxamide with the appropriate cyclic/acyclic ketones to afford the desired products in acceptable (51-94%) yields. The methodology was also extended successfully towards the synthesis of 2,2-disubstituted 2,3-dihydroquinazolin-4(1H)-ones in excellent (85-90%) yields. In vitro MTT assay against the RAW 264.7 cell line followed by enzymatic assay against MtbCM identified 3b and 3c as active compounds that showed two H-bonding via their NH (at position 6) and CO group with MtbCM in silico and encouraging (54-57%) inhibition at 30 µM in vitro. Notably, none of the 2,2-disubstituted 2,3-dihydroquinazolin-4(1H)-ones showed any significant inhibition of MtbCM suggesting the favourable role of the pyrazole moiety in case of pyrazolo[4,3-d]pyrimidinones. The favourable role of cyclopentyl ring attached to the pyrazolo[4,3-d]pyrimidinone moiety and that of two methyl groups in place of cyclopentyl ring was also indicated by the SAR study. Besides showing effects against MtbCM in the concentration response study, 3b and 3c showed little or no effects on mammalian cell viability up to 100 µM in an MTT assay but decreased the % Mtb cell viability at 10-30 µM with > 20% decrease at 30 µM in an Alamar Blue Assay. Moreover, no adverse effects were noted for these compounds when tested for teratogenicity and hepatotoxicity in zebrafish at various concentrations. Overall, being the only example of MtbCM inhibitors that showed effects on Mtb cell viability the compound 3b and 3c are of further interest form the view point of discovery and development of new anti-tubercular agents.


Assuntos
Mycobacterium tuberculosis , Animais , Estrutura Molecular , Pirimidinonas/química , Relação Estrutura-Atividade , Corismato Mutase , Sobrevivência Celular , Peixe-Zebra/metabolismo , Mamíferos/metabolismo
7.
STAR Protoc ; 3(4): 101819, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36386866

RESUMO

In this report, we describe an approach to generate a zebrafish larval model of lipid accumulation that can be used as an in vivo system to study hyperlipidemic conditions such as atherosclerosis. Furthermore, we detail steps on staining techniques, lipid estimation assays, RNA isolation, and utilization of ImageJ to evaluate larval dimensions and to explore the model in the context of hyperlipidemia. Researchers should be aware of context specificity of the proposed protocols and interpret results accordingly. For complete details on the use and execution of this protocol, please refer to Balamurugan et al., (2022).


Assuntos
Hipercolesterolemia , Hiperlipidemias , Animais , Peixe-Zebra , Larva , Hiperlipidemias/diagnóstico , Hipercolesterolemia/genética , Lipídeos
8.
STAR Protoc ; 3(4): 101779, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36317180

RESUMO

The application of CRISPR has greatly facilitated genotype-phenotype studies of human disease models. In this protocol, we describe CRISPR-Cas9-induced gene knockout in zebrafish, utilizing purified Cas9 protein and in vitro-transcribed sgRNA. This protocol targets the PHLPP1 gene in an Indian wild-caught strain, but is broadly applicable. Major factors influencing protocol success include zebrafish health and fecundity, sgRNA efficiency and specificity, germline transmission, and mutant viability. For complete details on the use and execution of this protocol, please refer to Balamurugan et al. (2022).


Assuntos
Sistemas CRISPR-Cas , Peixe-Zebra , Animais , Humanos , Técnicas de Inativação de Genes , Peixe-Zebra/genética , Sistemas CRISPR-Cas/genética , Proteína 9 Associada à CRISPR/genética , Células Germinativas
9.
Bioorg Chem ; 124: 105857, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35594765

RESUMO

Addressing the increasing incidences of cancer worldwide along with the multifaceted problem of drug resistance via development of new anticancer agents has become an essential goal. Due to the known cytotoxic effects and reported Akt inhibitory potential of azaindoles we designed a new framework incorporating the structural features of rosuvastatin and 5- or 7-azaindole. The framework was used to construct a library of small molecules for further pharmacological evaluation. The design was supported by the docking studies of two representative molecules in silico. A one-pot sonochemical approach was established for the synthesis of these rosuvastatin based azaindoles that involved the coupling-cyclization of a rosuvastatin derived terminal alkyne with appropriate 3-iodopyridine derivatives under Pd/Cu-catalysis. When tested using an MTT assay, some of the synthesized compounds showed desirable cytotoxic effects against three cancer cell lines e.g. HCT 116, Hep G2 and PA-1 but no significant effects against the non-cancerous HEK cell line. According to the SAR the 5-azaindole ring appeared to be marginally better than the 7-azaindole whereas the activity was varied with the variation of sulfonamide moiety attached to the N-1 atom of the azaindole ring. Among all the groups present in the sulfonamide moiety the p-MeC6H4 group appeared to be most effective in terms of activity. While 3b and 5b were identified as initial hit molecules the compound 5b (in addition to 3b) also showed significant inhibition of Akt1 in vitro that was reflected by its strong interaction with Akt1 in silico (with the docking score -11.7 kcal/mol) involving two H-bonding interactions with Ser7 and Asp439 residues. Further, a reasonable ADME was predicted for 5bin silico. Being a potent inhibitor (MIA Paca-2 IC50 = 18.79 ± 0.17 nM) and with NOAEL (No Observed Adverse Effect Level) > 100 µM in Zebrafish, 5b emerged as a promising compound.


Assuntos
Antineoplásicos , Neoplasias , Animais , Antineoplásicos/química , Catálise , Linhagem Celular Tumoral , Proliferação de Células , Ciclização , Citotoxinas/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Simulação de Acoplamento Molecular , Estrutura Molecular , Rosuvastatina Cálcica/farmacologia , Relação Estrutura-Atividade , Sulfonamidas/farmacologia , Peixe-Zebra
10.
iScience ; 25(2): 103766, 2022 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-35141506

RESUMO

Infiltration of arterial intima by foamy macrophages is a hallmark of early atherosclerotic lesions. Here, we investigated the potential role of Ser/Thr phosphatase PHLPP1 in foam cell development. PHLPP1 levels were elevated in OxLDL-exposed macrophages and high-fat diet (HFD)-fed zebrafish larvae. Using overexpression and knockdown approaches, we show that PHLPP1 promotes the accumulation of neutral lipids, and augments cellular total cholesterol and free fatty acid (FFA) levels. RNA-Seq analysis uncovered PHLPP1 role in lipid metabolism pathways. PHLPP1 interacted with and modestly increased ChREBP recruitment to Fasn promoter. PHLPP1-mediated lipid accumulation was attenuated by AMPK activation. Pharmacological inhibition or CRISPR/Cas9-mediated disruption of PHLPP1 resulted in lower lipid accumulation in the intersegmental vessels of HFD-fed zebrafish larvae along with a reduction in total cholesterol and triglyceride levels. Deficiency of phlp-2, C. elegans PHLPP1/2 ortholog, abolished lipid accumulation in high cholesterol-fed worms. We conclude that PHLPP1 exerts a significant effect on lipid buildup.

11.
Bioorg Chem ; 121: 105667, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35182886

RESUMO

In search of potent and new anti-inflammatory agents, we explored a new class of isocoumarin derivatives possessing the 3-oxoalkyl moiety at C-4 position. These compounds were synthesized via the FeCl3 catalyzed construction of isocoumarin ring. The methodology involved coupling of 2-alkynyl benzamides with alkyl vinyl ketone and proceeded via a regioselective cyclization to give the desired compound as a result of formation of CO and CC bonds. A large number of isocoumarins were synthesized and assessed against PDE4B in vitro. While isocoumarins containing an aminosulfonyl moiety attached to the C-3 aryl ring showed encouraging inhibition of PDE4B, some of the derivatives devoid of aminosulfonyl moiety also showed considerable inhibition. According to the SAR analysis the C6H4NHSO2R2-m moiety at C-3 position of the isocoumarin ring was favorable when the R2 was chosen as an aryl or 2-thienyl group whereas the presence of F or OMe substituent at C-7 of the isocoumarin ring was found to be beneficial. The compound 5f with IC50 values 0.125 ± 0.032 and 0.43 ± 0.013 µM against PDE4B and 4D, respectively was identified as an initial hit. It showed in silico interaction with the PHE678 residue in the CR3 region of PDE4B and relatively less number of interactions with PDE4D. Besides showing the PDE4 selectivity over other PDEs and TNF-α inhibition in vitro the compound 5f at an intraperitoneal dose of 30 mg/kg demonstrated the protective effects against the development of arthritis and potent immunomodulatory activity in adjuvant induced arthritic (AIA) rats. Furthermore, no significant adverse effects were observed for this compound when evaluated in a systematic toxicity (e.g. teratogenicity, hepatotoxicity and cardiotoxicity) studies in zebrafish at various concentrations. Collectively, being a new, potent, moderately selective and safe inhibitor of PDE4B the isocoumarin 5f can be progressed into further pharmacological studies.


Assuntos
Compostos Férricos , Isocumarinas , Animais , Catálise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Isocumarinas/química , Ratos , Relação Estrutura-Atividade , Peixe-Zebra/metabolismo
12.
Bioorg Chem ; 116: 105380, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34670330

RESUMO

A series of indole based novel Schiff bases was designed as potential agonists of 5-HT2C receptor that was supported by docking studies in silico. These compounds were synthesized via Amberlyst-15 catalysed condensation of an appropriate pyrazole based primary amine with the corresponding indole-3-aldehyde under ultrasound irradiation at ambient temperature. A number of target Schiff bases were obtained in good yields (77-87%) under mild conditions within 1 h. Notably, the methodology afforded the corresponding pyrazolo[4,3-d]pyrimidin-7(4H)-one derivatives when the primary amine was replaced by a secondary amine. Several Schiff bases showed agonist activity when tested against human 5-HT2C using luciferase assay in HEK293T cells in vitro. The SAR (Structure-Activity-Relationship) studies suggested that the imine moiety was more favorable over its cyclic form i.e. the corresponding pyrazolopyrimidinone ring. The Schiff bases 3b (EC50 1.8 nM) and 3i (EC50 5.7 nM) were identified as the most active compounds and were comparable with Lorcaserin (EC50 8.5 nM). Also like Lorcaserin, none of these compounds were found to be PAM of 5-HT2C. With ∼24 and ∼150 fold selectivity towards 5-HT2C over 5-HT2A and 5-HT2B respectively the compound 3i that reduced locomotor activity in zebrafish (Danio rerio) larvae model emerged as a promising hit molecule for further study.


Assuntos
Indóis/farmacologia , Receptor 5-HT2C de Serotonina/metabolismo , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Estirenos/química , Ondas Ultrassônicas , Catálise , Relação Dose-Resposta a Droga , Humanos , Indóis/síntese química , Indóis/química , Estrutura Molecular , Agonistas do Receptor 5-HT2 de Serotonina/síntese química , Agonistas do Receptor 5-HT2 de Serotonina/química , Relação Estrutura-Atividade
13.
Bioorg Chem ; 115: 105265, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34426160

RESUMO

In spite of possessing a wide range of pharmacological properties the anti-inflammatory activities of isoquinolin-1(2H)-ones were rarely known or explored earlier. PDE4 inhibitors on the other hand in addition to their usefulness in treating inflammatory diseases have been suggested to attenuate the cytokine storm in COVID-19 especially TNF-α. In our effort, a new class of isoquinolin-1(2H)-ones derivatives containing an aminosulfonyl moiety were designed and explored as potential inhibitors of PDE4. Accordingly, for the first time a CuCl2-catalyzed inexpensive, faster and ligand/additive free approach has been developed for the synthesis of these predesigned isoquinolin-1(2H)-one derivatives via the coupling-cyclization strategy. Thus, the CuCl2-catalyzed reaction of 2-iodobenzamides with appropriate terminal alkynes proceeded with high chemo and regioselectivity affording the desired compounds in 77-84% yield within 1-1.5 h. The methodology also afforded simpler isoquinolin-1(2H)-ones devoid of aminosulfonyl moiety showing a broader generality and scope of this approach. Several of the synthesized compounds especially 3c, 3k and 3s showed impressive inhibition (83-90%) of PDE4B when tested at 10 µM in vitro whereas compounds devoid of aminosulfonyl moiety was found to be less active. In spite of high inhibition showed at 10 µM these compounds did not show proper concertation dependent inhibition below 1 µM that was reflected in their IC50 values e.g. 2.43 ± 0.32, 3.26 ± 0.24 and 3.63 ± 0.80 µM for 3k, 3o and 3s respectively. The anti-inflammatory potential of these compounds was indicated by their TNF-α inhibition (60-50% at 10 µM). The in silico docking studies of these molecules suggested good interactions with PDE4B and selective inhibition of PDE4B by 3k over PDE4D that was supported by in vitro assay results. These observations together with the favorable ADME and safety predicted for 3kin silico not only suggested 3k as an interesting hit molecule for further studies but also reveal the first example of isoquinolin-1(2H)-one based inhibitor of PDE4B.


Assuntos
Anti-Inflamatórios/química , Cobre/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Isoquinolinas/química , Inibidores da Fosfodiesterase 4/química , Animais , Anti-Inflamatórios/síntese química , Catálise , Ciclização , Ensaios Enzimáticos , Humanos , Isoquinolinas/síntese química , Camundongos , Estrutura Molecular , Inibidores da Fosfodiesterase 4/síntese química , Células RAW 264.7 , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/antagonistas & inibidores
14.
Eur J Med Chem ; 221: 113514, 2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-33992926

RESUMO

While anti-inflammatory properties of isocoumarins are known their PDE4 inhibitory potential was not explored previously. In our effort the non-PDE4 inhibitor isocoumarins were transformed into the promising inhibitors via introducing an aminosulfonyl/aminocarboxamide moiety to the C-3 benzene ring attached to the isocoumarin framework. This new class of isocoumarins were synthesized via a PdCl2-catalyzed construction of the 4-allyl substituted 3-aryl isocoumarin ring starting from the appropriate 2-alkynyl benzamide derivative. Several compounds showed good inhibition of PDE4B in vitro and the SAR indicated superiority of aminosulfonamide moiety over aminocarboxamide in terms of PDE4B inhibition. Two compounds 3q and 3u with PDE4B IC50 = 0.43 ± 0.11 and 0.54 ± 0.19 µM and ≥ 2-fold selectivity over PDE4D emerged as initial hits. The participation of aminosulfonamide moiety in PDE4B inhibition and the reason for selectivity though moderate shown by 3q and 3u was revealed by the in silico docking studies. In view of potential usefulness of moderately selective PDE4B inhibitors the compound 3u (that showed PDE4 selectivity over other PDEs) was further evaluated in adjuvant induced arthritic rats. At an intraperitoneal dose of 30 mg/kg the compound showed a significant reduction in paw swelling (in a dose dependent manner), inflammation and pannus formation (in the knee joints) as well as pro-inflammatory gene expression/mRNA levels and increase in body weight. Moreover, besides its TNF-α inhibition and no significant toxicity in an MTT assay the compound did not show any adverse effects in a thorough toxicity studies e.g. teratogenicity, hepatotoxicity, cardiotoxicity and apoptosis in zebrafish. Thus, the isocoumarin 3u emerged as a new, safe and moderately selective PDE4B inhibitor could be useful for inflammatory diseases possibly including COVID-19.


Assuntos
Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Isocumarinas/uso terapêutico , Inibidores da Fosfodiesterase 4/uso terapêutico , Sulfonamidas/uso terapêutico , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/toxicidade , Artrite Experimental/patologia , Catálise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Feminino , Isocumarinas/síntese química , Isocumarinas/metabolismo , Isocumarinas/toxicidade , Articulação do Joelho/efeitos dos fármacos , Articulação do Joelho/patologia , Masculino , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Paládio/química , Inibidores da Fosfodiesterase 4/síntese química , Inibidores da Fosfodiesterase 4/metabolismo , Inibidores da Fosfodiesterase 4/toxicidade , Ligação Proteica , Células RAW 264.7 , Ratos Wistar , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/metabolismo , Sulfonamidas/toxicidade , Peixe-Zebra
15.
Eur J Med Chem ; 201: 112335, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32599323

RESUMO

A new class of 3-substituted isocoumarin/3-alkylidenephthalide based novel small molecules derived from rosuvastatin were designed and synthesized via the ultrasound assisted Cu-mediated coupling-cyclization in a single pot with remarkable regioselectivity. The phthalides were generally obtained at lower temperature whereas the use of elevated temperature afforded isocoumarins. Two compounds e.g. 3n and 4d showed promising cytotoxic effects when tested against HCT 116, HepG2 and PA-1 cell lines at 10 µM. Indeed, 4d was found to be a potent cytotoxic agent (IC50 ∼ 0.76-4.51 µM). Both 3n and 4d were tested for their effects on PANC-1 cells. Considerable decrease in p-Akt substrates shown by 4d and 3n at 50 µM (western blot analysis) indicated their ability to inhibit p-Akt signal transduction pathway and arresting growth of PANC-1 cells in vitro. This was further supported by the cytotoxic effect of 4d on PANC-1 cells (MTT assay) that was better than rosuvastatin. While none of 3n and 4d showed any significant effect on non-cancerous HEK cell line (indicating their potential selectivity towards cancer cells) these compounds were further evaluated for their toxicities in Zebrafish embryo. The NOAEL (No Observed Adverse Effect Level) for teratogenicity, hepatotoxicity and cardiotoxicity was found to be 100 µM for both compound. Thus, 4d as a novel and potent but safer cytotoxic agent with potential to treat colorectal/ovarian and pancreatic cancer is of further medicinal interest.


Assuntos
Antineoplásicos/farmacologia , Benzofuranos/farmacologia , Isocumarinas/farmacologia , Rosuvastatina Cálcica/análogos & derivados , Rosuvastatina Cálcica/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Benzofuranos/síntese química , Benzofuranos/toxicidade , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Embrião não Mamífero/efeitos dos fármacos , Humanos , Isocumarinas/síntese química , Isocumarinas/toxicidade , Estrutura Molecular , Rosuvastatina Cálcica/toxicidade , Relação Estrutura-Atividade , Ondas Ultrassônicas , Peixe-Zebra
16.
J Pharmacol Toxicol Methods ; 101: 106656, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31734279

RESUMO

INTRODUCTION: FXS is the leading cause of intellectual disabilities in males and a major monogenic cause of ASD (Autism spectrum disorders). It occurs due to the loss of FMRP, whose role in early development is not well understood. In this study, we have used a novel DNAzyme based approach to create a larval model of FXS in zebrafish with specific focus on the early developmental window. METHODS: Fmr1specific DNAzymes were electroporated into embryos to create the knockdown. Changes in RNA and protein levels of FMRP and relevant biomarkers were measured in the 0-7dpf window. Behavioral tests to measure anxiety, cognitive impairments and irritability in the larvae were conducted at the 7dpf stage. Drug treatment was carried out at various time points in the 0-7dpf window to identify the critical window for pharmacological intervention. RESULTS: The DNAzyme based knockdown approach led to a significant knockdown of FMRP in the zebrafish embryos, accompanied by increased anxiety, irritability and cognitive impairments at 7dpf, thus creating a robust larval model of FXS. Treatment with the Mavoglurant was able to rescue the behavioral phenotypes in the FXS larvae, and found to be more efficacious in the 0-3dpf window. DISCUSSION: The results from this study have revealed that a) a DNAzyme based knockdown approach can be used to create robust larval zebrafish model of disease, in a high-throughput manner and b) optimal window for therapeutic intervention for FXS as well as other pediatric diseases with a monogenic cause can be identified using such a model.


Assuntos
DNA Catalítico/genética , Síndrome do Cromossomo X Frágil/metabolismo , Técnicas de Silenciamento de Genes , Animais , Escala de Avaliação Comportamental , Modelos Animais de Doenças , Larva , Masculino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
17.
Eur J Med Chem ; 174: 198-215, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31035240

RESUMO

A new class of PDE4 inhibitors were designed and synthesized via the InCl3 mediated heteroarylation of indoles and their further derivatization through the Pd(II)-catalyzed CH activation strategy. This effort allowed us to discover a series of 2-(1H-indol-3-yl)-quinoxaline based inhibitors possessing PDE4B selectivity over PDE4D and PDE4C. One of these compounds i.e. 3b (PDE4B IC50 = 0.39 ±â€¯0.13 µM with ∼27 and > 250 fold selectivity for PDE4B over PDE4D and C, respectively) showed effects in Zebrafish experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis when dosed at 3, 10 and 30 mg/kg intraperitoneally. Indeed, it halted the progression of the disease across all these doses tested. At an intraperitoneal dose of 30 mg/kg the compound 3b showed promising effects in adjuvant induced arthritic rats. The compound reduced paw volume, inflammation and pannus formation (in the knee joints) as well as pro-inflammatory gene expression/mRNA levels significantly in arthritic rats. Moreover, this compound was found to be selective towards PDE4 over other families of PDEs in vitro and safe when tested for its probable toxicity (e.g. teratogenicity, hepatotoxicity and cardiotoxicity) in Zebrafish.


Assuntos
Artrite/tratamento farmacológico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Indóis/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Inibidores da Fosfodiesterase 4/uso terapêutico , Quinoxalinas/uso terapêutico , Animais , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/tratamento farmacológico , Adjuvante de Freund , Índio , Indóis/síntese química , Indóis/química , Indóis/toxicidade , Estrutura Molecular , Esclerose Múltipla/induzido quimicamente , Glicoproteína Oligodendrócito-Mielina , Inibidores da Fosfodiesterase 4/síntese química , Inibidores da Fosfodiesterase 4/química , Inibidores da Fosfodiesterase 4/toxicidade , Quinoxalinas/síntese química , Quinoxalinas/química , Quinoxalinas/toxicidade , Ratos , Relação Estrutura-Atividade , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
18.
Artigo em Inglês | MEDLINE | ID: mdl-30500431

RESUMO

INTRODUCTION: Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder of early onset, characterized by impaired sociability, cognitive function and stereotypies. The etiology of ASD involves a multidimensional link between genetic, neurobiological and environmental factors. Since existing, comprehensive animal models for ASD are time consuming and laborious, the need for simple, quick approaches to study subsets of ASD-associated characteristics has always been in demand for better understanding of disease. The aim of the present study was to develop a cost and time effective zebrafish model with quantifiable parameters to facilitate mechanistic studies as well as high-throughput screening of new molecules for autism. METHODS: Zebrafish embryos were treated with valproic acid (75 µM) beginning at 4-h post fertilization to 5-days post fertilization. A series of behavioral tests (anxiety, inattentive behavior and circling behavior) and molecular studies were performed as surrogate parameters of ASD-like characteristic on the larvae at 7-dayspost fertilization for a quick screen. The study was followed by validation of model by screening positive control and negative control drugs. The social interaction test was performed on 21-days post fertilization to confirm that the surrogate phenotypes were indicative of social deficit (a core symptom of ASD). RESULTS: The model showed a significant behavioral impairment (2-4fold difference) in valproic acid treated larvae compared to control larvae, which was further supported by alterations in select high-risk genes and proteins, implicated in human ASD. Reversal of behavioral impairments using standard drugs marketed for symptomatic treatment in ASD and no effect on behaviors when treated with paracetamol (negative control) signifies the role of model in preliminary drug screening. CONCLUSION: The model shows robust parameters to study behavior, molecular mechanism and drug screening approach in a single frame. Thus, we postulate that our 7-day larval model could be a useful preliminary screening tool to identify novel targets as well as potential drugs for autism and also can be applied to develop a high-throughput screening approach.


Assuntos
Transtorno do Espectro Autista/induzido quimicamente , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Larva/efeitos dos fármacos , Ácido Valproico/toxicidade , Animais , Transtorno do Espectro Autista/tratamento farmacológico , Comportamento Animal/fisiologia , Avaliação Pré-Clínica de Medicamentos/métodos , Larva/fisiologia , Ácido Valproico/administração & dosagem , Peixe-Zebra
19.
Drug Deliv Transl Res ; 8(5): 1450-1459, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29717474

RESUMO

Lung cancer is the single largest cause of cancer related deaths in the world. Current treatments include surgery, radiation therapy, chemotherapy using cytotoxic drugs, and monoclonal antibodies. Such treatments have limited efficacy due to diverse nature of lung cells involved and lack of tissue penetration. Cytotoxic drugs, while potent, have the enormous drawback of limited entry into the lung selectively, thus causing collateral damage to other tissues. To overcome these shortcomings, we report here the development of new magnetic irinotecan containing nanoparticles (NPs), which target the lung over other tissues by over 5-fold. Selective targeting of lungs is achieved by deliberately incorporating a facilitated transport mechanism into the NPs. The iron containing NPs can be further exploited to retain the drug into the lung for maximum efficacy using an external magnet. This irinotecan nanoformulation can be used as mono therapy or combination therapy and offers a cost-effective and efficacious therapy for lung cancers.


Assuntos
Portadores de Fármacos/química , Irinotecano/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Nanopartículas de Magnetita/química , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Irinotecano/química , Irinotecano/farmacocinética , Camundongos , Coelhos , Ensaios Antitumorais Modelo de Xenoenxerto
20.
J Pharmacol Toxicol Methods ; 88(Pt 2): 147-152, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28974368

RESUMO

INTRODUCTION: Adult zebrafish pharmacology is evolving rapidly for creating efficacy and safety models for drug discovery. However, there is very limited research in understanding pharmacokinetics (PK) in adult zebrafish. Methods for understanding PK will help in conducting pharmacokinetic - pharmacodynamic (PK-PD) correlations and improving the quality and applicability of data obtained using zebrafish. METHODS: We conducted adult zebrafish PK and brain penetration studies on two known compounds (irinotecan and lorcaserin) with distinct PK and brain penetration properties using validated LCMS/MS method. Irinotecan was studied at a dose of 100mg/kg i.p. and levels of the parent drug and active metabolite SN-38 were measured. Loracserin was studies at a dose of 10mg/kg by two routes i.p. and p.o. RESULTS: Zebrafish PK and brain penetration profiles for both compounds were very similar to that of higher mammals including humans. Irinotecan was metabolised to SN-38 in ratios similar to ratios seen in other species and the compound had long half life with very low brain penetration in our studies. Loracasin was highly permeable in brain as compared to the exposure in blood, with long half life and high relative bioavailability, similar to other mammalian species including humans. DISCUSSION: Adult zebrafish PK studies are relatively an unexplored area of zebrafish research. The zebrafish data for key parameters of irinotecan and loracserin shows a high correlation to the data from higher species, including human. This report explores and discusses the use of adult zebrafish as a predictive PK tool for higher animal studies.


Assuntos
Benzazepinas/farmacocinética , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Camptotecina/análogos & derivados , Fatores Etários , Animais , Benzazepinas/administração & dosagem , Disponibilidade Biológica , Camptotecina/administração & dosagem , Camptotecina/farmacocinética , Humanos , Injeções Intraperitoneais , Irinotecano , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologia , Peixe-Zebra
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