RESUMEN
Alkaline phosphatases (ALPs) are membrane bound metalloenzymes, distributed all over the body. Recent studies have revealed that by targeting ALPs can lead towards the treatment of many deadliest diseases including cardiac, cancerous and brain diseases. Thioureas and their derivatives are of considerable significance and are privileged scaffolds in medicinal chemistry. They show a wide range of pharmacological activities such as antibacterial, antiparasitic, anti-inflammatory and antioxidants etc. On the other hand, salicylic acid and its derivatives are known for its broad spectrum of activities. The work presented comprises of synthesis of N-acyl-N'-aryl substituted bisthioureas of pimelic acid (1-7) and 3,5-dimethyl pyrazole (11), 1-aroyl-3-aryl thiourea (12) and 1,3,4-oxadiazole (13) derivatives of 4-methyl salicylic acid. Structures of all the synthesized compounds were characterized by FT-IR and 1H NMR spectroscopic analysis. Synthesized compounds were evaluated for their alkaline phosphatases inhibition potential and exhibited high potency as well as selectivity towards h-TNAP and h-IAP. Compound 7 and 12 which were the bisthiourea derivative of pimmelic acid and thiourea derivative of 4-methyl salicylic acid, respectively, showed excellent selectivity against h-TNAP and h-IAP, respectively.
Asunto(s)
Fosfatasa Alcalina/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Intestinos/enzimología , Ácidos Pimélicos/química , Salicilatos/química , Tiourea/farmacología , Inhibidores Enzimáticos/síntesis química , Enlace de Hidrógeno , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Tiourea/químicaRESUMEN
Substituted phenyl[(5-benzyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetates/acetamides 9a-j were synthesized as alkaline phosphatase inhibitors. Phenyl acetic acid 1 through a series of reactions was converted into 5-benzyl-1,3,4-oxadiazole-2-thione 4. The intermediate oxadiazole 4 was then reacted with chloroacetyl derivatives of phenols 6a-f and anilines derivatives 8a-d to afford the title oxadiazole derivatives 9a-j. All of the title compounds 9a-j were evaluated for their inhibitory activity against human alkaline phosphatise (ALP). It was found that compounds 9a-j exhibited good to excellent alkaline phosphatase inhibitory activity especially 9h displayed potent activity with IC50 value 0.420⯱â¯0.012⯵M while IC50 value of standard (KH2PO4) was 2.80⯵M. The enzyme inhibitory kinetics of most potent inhibitor 9h was determined by Line-weaever Burk plots showing non-competitive mode of binding with enzyme. Molecular docking studies were performed against alkaline phosphatase enzyme (1EW2) to check the binding affinity of the synthesized compounds 9a-j against target protein. The compound 9h exhibited excellent binding affinity having binding energy value (-7.90â¯kcal/mol) compared to other derivatives. The brine shrimp viability assay results proved that derivative 9h was non-toxic at concentration used for enzyme assay. The lead compound 9h showed LD50 106.71⯵M while the standard potassium dichromate showed LD50 0.891⯵M. The DNA binding interactions of the synthesized compound 9h was also determined experimentally by spectrophotometric and electrochemical methods. The compound 9h was found to bind with grooves of DNA as depicted by both UV-Vis spectroscopy and cyclic voltammetry with binding constant values 7.83â¯×â¯103 and 7.95â¯×â¯103â¯M-1 respectively revealing significant strength of 9h-DNA complex. As dry lab and wet lab results concise each other it was concluded that synthesized compounds, especially compound 9h may serve as lead compound to design most potent inhibitors of human ALP.
Asunto(s)
Acetamidas/química , Fosfatasa Alcalina/antagonistas & inhibidores , Artemia/crecimiento & desarrollo , ADN/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Oxadiazoles/química , Animales , Artemia/efectos de los fármacos , Artemia/enzimología , Supervivencia Celular , Biología Computacional , ADN/química , Cinética , Simulación del Acoplamiento Molecular , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Owing to the biological importance of cyclic sulfonamides (sultams), herein we report a new, facile and cost-effective method for the synthesis of sultams that makes use of a reaction between dansyl amide and easily accessible benzaldehydes under mildly acidic conditions. All compounds were obtained in good yields (69-96%). Consequently a series of cyclic sulfonamides (7a-7n) was synthesized and characterized using FTIR, MS and NMR spectroscopy, crystal structure of compound 7b has also been determined. All compounds were evaluated for their potential to inhibit alkaline phosphatase (bTNAP and bIAP). All compounds were found to be excellent inhibitors of bTNAP with IC50 values in lower micro-molar range (0.11-6.63µM). Most of the compounds were selective inhibitors of bTNAP over bIAP. Only six compounds were found to be active against bIAP (IC50 values in the range 0.38-3.48µM). Molecular docking studies were carried out to identify and rationalize the structural elements necessary for efficient AP inhibition.
Asunto(s)
Fosfatasa Alcalina/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Animales , Bovinos , Simulación del Acoplamiento Molecular , Relación Estructura-ActividadRESUMEN
This work describes the development of a novel fluorescent biosensor based on the inhibition of alkaline phosphatase (ALP). The biosensor is composed of the enzyme ALP and the conjugated cationic polyfluorene HTMA-PFP. The working principle of the biosensor is based on the fluorescence quenching of this polyelectrolyte by p-nitrophenol (PNP), a product of the hydrolysis reaction of p-nitrophenyl phosphate (PNPP) catalyzed by ALP. Because HTMA-PFP forms unstable aggregates in buffer, with low fluorescence efficiency, previous stabilization of the polyelectrolyte was required before the development of the biosensor. HTMA-PFP was stabilized through its interaction with lipid vesicles to obtain stable blue-emitting nanoparticles (NPs). Fluorescent NPs were characterized, and the ability to be quenched by PNP was evaluated. These nanoparticles were coupled to ALP and entrapped in a sol-gel matrix to produce a biosensor that can serve as a screening platform to identify ALP inhibitors. The components of the biosensor were examined before and after sol-gel entrapment, and the biosensor was optimized to allow the determination of phosphate ion in aqueous medium.
Asunto(s)
Técnicas Biosensibles , Fosfatasa Alcalina , Fluorenos , Liposomas , Nanopartículas , Nitrofenoles , FosfatosRESUMEN
The effect of bioisosteric replacement of carboxamide linking group with sulfonamide linking group, on alkaline phosphatase (AP) and carbonic anhydrase (CA) inhibition activity of aromatic benzenesulfonamides was investigated. A series of carboxamide linked aromatic benzenesulfonamides 1a-1c, 2a-2d and their sulfonamide linked bioisosteres 3a-3d, 4a-4d was synthesized and evaluated for inhibitory activity against bovine tissue non-specific alkaline phosphatase (TNAP), intestinal alkaline phosphatase (IAP) and bCA II. A significant increase in CA inhibition activity was observed upon bioisosteric replacement of carboxamide linking group with a sulfonamide group. Some of these compounds were identified as highly potent and selective AP inhibitors. Compounds 1b, 2b, 3d, 4d 5b and 5c were found to be selective bTNAP inhibitors, whereas compounds 1a, 1c, 2a, 2c, 2d, 3a, 3c, 4a, 4b, 4c, 5a were found to be selective bIAP inhibitors. For most active AP inhibitor 3b, detailed kinetic studies indicated a competitive mode of inhibition against tissue non-specific alkaline phosphatase (TNAP) and non-competitive mode of inhibition against intestinal alkaline phosphatase (IAP). Molecular docking studies were carried out to rationalize important binding site interactions.
Asunto(s)
Fosfatasa Alcalina/antagonistas & inhibidores , Derivados del Benceno/síntesis química , Anhidrasa Carbónica II/química , Inhibidores Enzimáticos/síntesis química , Sulfonamidas/síntesis química , Fosfatasa Alcalina/química , Animales , Derivados del Benceno/química , Sitios de Unión , Bovinos , Pruebas de Enzimas , Inhibidores Enzimáticos/química , Ésteres , Cinética , Simulación del Acoplamiento Molecular , Especificidad de Órganos , Unión Proteica , Relación Estructura-Actividad , Sulfonamidas/química , TermodinámicaRESUMEN
A new series of structurally diverse chromone containing sulfonamides has been developed. Crystal structures of three representative compounds (2a, 3a and 4a) in the series are reported. All compounds were screened for their inhibitory potential against alkaline phosphatases (ALPs). Two main classes of ALP isozymes were selected for this study, the tissue non-specific alkaline phosphatase (TNALP) from bovine and porcine source and the tissue-specific intestinal alkaline phosphatases (IALPs) from bovine source. All sulfonamide compounds had a marked preference for IALP (K(i), up to 0.01 ± 0.001 µM) over TNALPs. Kinetics studies of the compounds showed competitive mode of inhibition. Molecular docking studies were carried out in order to characterize the selective inhibition of the compounds. An additional interesting aspect of these chromone sulfonamides is their inhibitory activity against ecto-5'-nucleotidase enzyme.
Asunto(s)
Fosfatasa Alcalina/antagonistas & inhibidores , Cromonas/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Animales , Bovinos , Inhibidores Enzimáticos/metabolismo , Simulación del Acoplamiento Molecular , Conformación Proteica , Relación Estructura-Actividad , Especificidad por Sustrato , Sulfonamidas/metabolismoRESUMEN
Our objective was to characterize the modulation of the activity of Saccharomyces cerevisiae alkaline phosphatases (ALPs) by classic inhibitors of ALP activity, cholesterol and steroid hormones, in order to identify catalytic similarities between yeast and mammalian ALPs. S. cerevisiae expresses two ALPs, coded for by the PHO8 and PHO13 genes. The product of the PHO8 gene is repressible by Pi in the medium. ALP activity from yeast (grown in low or high phosphate medium) homogenates was determined with p-nitrophenylphosphate as substrate, pH 10.4 (lPiALP or hPiALP, respectively). Activation of hPiALP was observed with 5 mM L-amino acids (L-homoarginine _ 186 percent, L-leucine _ 155 percent and L-phenylalanine - 168 percent) and with 1 mM levamisole (122 percent; percentage values, in comparison to control, of recovered activity). EDTA (5 mM) and vanadate (1 mM) distinctly inhibited hPiALP (2 and 20 percent, respectively). L-homoarginine (5 mM) had a lower activating effect on lPiALP (166 percent) and was the strongest hPiALP activator. Corticosterone (5 mM) inhibited hPiALP to 90 percent, but no effect was observed in low phosphate medium. Cholesterol, ß-estradiol and progesterone also had different effects on lPiALP and hPiALP. A concentration-dependent activation of lPiALP minus hPiALP was evident with all three compounds, most especially with ß-estradiol and cholesterol. These results do not allow us to identify similarities of the behavior of S. cerevisiae ALPs and any of the mammalian ALPs but allow us to raise the hypothesis of differential regulation of S. cerevisiae ALPs by L-homoarginine, ß-estradiol and cholesterol and of using these compounds to discriminate between S. cerevisiae lPiALP and hPiALP.