Historical Roots of Radicalization in Pashtun’s Society

This research article aims to trace the history of radical movements in the North-West frontier of sub-continent. Historically, radical movements have long roots in Pakhtun Society.  People recruited in different epochs from Pakhtun society branch into various freedom movements before the partition of sub-continent. Freedom movements against the Sikh, Hindu and the British lifted radical impact on Pakhtun Society before the partition of sub-continent.  Radical movements after the partition of sub-continent also established their roots in the North-West region of Pakistan. These radical movements engineered the pluralistic cultural values of Pakhtun Society. These movements have lifted radical trends in the North-West frontier of sub-continent. Pakhtuns and their cultural values were not only exposed to violence but the evolution of their culture had been disturbed.

Identification and Characterization of Pharmacological Inhibitors of Alkaline Phosphatase Isozymes & Nucleotide Pyrophosphatase Isozymes

Ecto–nucleotidases are nucleotide metabolizing enzymes that are categorized into four different families; Alkaline Phosphatases (APs), Nucleotide Pyrophosphatase/phosphodiesterases (NPPs), Nucleoside Triphosphate Diphosphohydrolases (NTPDases) and Ecto–5′–Nucleotidase (e5′NT). These enzymes are responsible for the hydrolysis of extracellular nucleotides, i.e., adenosine–5′–triphosphate (ATP), adenosine–5′–diphosphate (ADP), adenosine–5′– monophosphate (AMP), uridine–5′–triphosphate (UTP) and uridine–5΄–diphosphate (UDP) into nucleosides, i.e., ADP, AMP, UDP, UMP and adenosine, respectively. The structural and functional role of these ecto–nucleotidases in purinergic signaling varies considerably between enzyme classes. Each member possesses different enzymatic and cellular expression properties. Among the different ecto–nucleotidase families, APs and NPPs synergize and overlap in their functions, particularly during skeletal mineralization. Among different isozymes of APs and NPPs, tissue non– specific alkaline phosphatase (TNAP) and Nucleotide Pyrophosphatase/phosphodiesterases-1 (NPP1) play an essential role in maintaining extracellular levels of pyrophosphate (PPi) and inorganic phosphate (Pi), an important factor to control mineralization process. This balance is highly conserved by opposing actions of NPP1 that produces PPi and TNAP which generates Pi by catalyzing PPi. In this way, PPi/Pi ratio remains constant inside and outside the cell membrane. An overexpression of these isozymes is implicated in a variety of pathophysiological processes, including chondrocalcinosis, immunological diseases, osteoarthritis, type 2 diabetes, neurodegenerative diseases, bone mineralization, cell adhesion, activation, proliferation, vascular calcification and cancer, and thus they represent an emerging drug targets. Therefore, potent and selective inhibitors of h-TNAP and h-NPP1 might be useful candidates for the treatment or prevention of some diseases. In this study, different derivatives of amides, chromones, quinolones and pyrazoles were tested for their potential to inhibit membrane–bound isozymes. The obtained results suggested that amide derivatives 3b, 4d, 2b (diarylsulphonamides), 4i, 4f, 4b (1H–pyrazol–4–yl benzamides), 2i, 2e and 2a (thiazol–2–ylidene–benzamides) were found highly potent inhibitors of h-TNAP Among the tested compounds, 3b, 4i and 2e showed the maximum inhibitory potential with an IC50 values of 0.21 ± 0.02, 0.34 ± 0.08 and 0.079 ± 0.002 µM, respectively. In the chromone derivatives, 1f, 1d, 1c (3,3′– carbonyl–bis(chromones), 7c, 7h, 7i (3–(5–(benzylideneamino)thiozol–3–yl)–2H– chromen–2–ones), 10a and 10g (triazolothiadiazin–3–yl 2–H–chromone) were found potent inhibitors of h-TNAP. Among the chromone derivatives 1d, 7h and 10a exhibited maximum inhibition with an IC50 values of (IC50±SEM) 2.47 ± 0.03, 0.21 ± 0.04 and 0.31 ± 0.09 µM, respectively. From the quinolone and quinoline derivatives, 3j, 3b (quinoline–4–carboxylic acid), 3a, 2b and 5a (4–quinolone) were found to be potent inhibitors against h-TNAP and among these compound 3j and 2b showed maximum inhibitory potential with an IC50±SEM values of 0.11 ± 0.07 and 1.34 ± 0.11 µM, respectively. The isoquinoline derivatives; 4p, 4l and 4i were identified as potent inhibitors of NPPs, where 4i was found to be the most potent inhibitor with an IC50 value of 0.11 ± 0.01 µM. The last group of compounds, i.e., Pyrazoles derivatives, 6i, 6e, 5e (2–arylated thiadiazolopyrimidones) were identified as the selective inhibitors of NPPs, and the most potent derivative was 6e (IC50±SEM= 0.31±0.01 µM). Compounds 4i, 4m and 4n (5–perfluoroalkylpyrazoles) were found as the selective inhibitors of APs with 4i (IC50±SEM= 0.45±0.01 µM) as the most potent inhibitor of the series. Compound 6a and 6b (pyrazole pyrimidones) were identified as the dual inhibitors of both APs and h-NPP-1. Kinetics experiments of the most potent derivatives were carried out to find the mechanism of inhibition on the respective isozyme by these derivatives. To determine the plausible binding modes and binding energies, docking studies were performed that supported the in–vitro inhibitory activity of potent and selective inhibitors. The cytotoxic results obtained from MTT assay confirmed that the selected compounds library had anticancer potential against MCF–7, K–562 and HeLa cell lines in comparison to normal cell line, i.e., BHK–21. Compounds 3b (diarylsulphonamides), 4i (1H–pyrazol–4–yl benzamides), 2i (thiazol–2–ylidene–benzamides), 1f (3,3′–carbonyl–bis(chromones), 7c (thiozol–3–yl–2H–chromen–2–ones), 10a (triazolothiadiazin–3–yl 2H–chromen– 2–ones), 4p (isoquinolones), 3j (quinoline–4–carboxylic acid), 3a (4–quinolone), 6i (2–arylated thiadiazolopyrimidones), 4i (5–perfluoroalkylpyrazoles) and 6b (pyrazole pyrimidones) induced maximum growth inhibition of MCF–7 cells and exhibited GI50 values 5.75 ± 0.12, 8.59 ± 0.16, 4.16 ± 0.17, 10.2± 1.07, 8.99 ± 1.24, 8.51 ± 0.62, 8.21 ± 0.31, 5.49 ± 0.32, 10.4 ± 2.05, 5.61 ± 0.72, 5.52 ± 0.92, 5.65 ± 0.75 and 13.5 ± 1.03 µM, respectively. Compounds 4d (diarylsulphonamides), 4f (1H–pyrazol–4–yl benzamides), 2e (thiazol–2–ylidene–benzamides), 1d (3,3′–carbonyl–bis(chromones), 7h (thiozol–3–yl–2H–chromen–2–ones), 10a (triazolothiadiazin–3–yl 2–H– chromone), 4l (isoquinolones), 3j (quinoline–4–carboxylic acid), 2b (4–quinolone), 6e (2–arylated thiadiazolopyrimidones), 4m (5–perfluoroalkylpyrazoles) and 6a (pyrazole pyrimidones) induced maximum growth inhibition of K–562 cells and exhibited GI50 values: 12.2 ± 1.09, 7.27 ± 0.48, 5.86 ± 0.15, 5.53 ± 0.35, 25.4 ± 1.09, 8.37 ± 0.14, 10.9 ± 1.04, 25.8 ± 2.79, 7.91 ± 0.92, 16.3 ± 1.25, 22.4 ± 1.88 and 16.6 ± 0.04 µM. Compounds 2b (diarylsulphonamides), 4b (1H–pyrazol–4–yl benzamides), 2a (thiazol–2–ylidene–benzamides), 1c (3,3′–carbonyl–bis(chromones), 7i (thiozol– 3–yl–2H–chromen–2–ones), 10g (triazolothiadiazin–3–yl 2H–chromen–2–ones), 4i (isoquinolones), 3b (quinoline–4–carboxylic acid), 2b (4–quinolone), 5e (2–arylated thiadiazolopyrimidones), 4n (5–perfluoroalkylpyrazoles) and 6c (pyrazole pyrimidones) caused significant growth inhibition of HeLa cells and exhibited GI50 values: 4.64 ± 0.34, 8.22± 0.78, 11.5 ± 0.15, 10.1 ± 0.73, 8.37 ± 0.45, 12.9 ± 0.13, 14.3 ± 1.26, 11.5 ± 1.05, 7.65 ± 0.97, 6.13 ± 0.92, 5.79 ± 0.56 and 12.4 ± 0.94 µM, respectively. Cell cycle arrest and apoptosis was confirmed by following the estimation of apoptosis by fluorescence microscopy using two nucleus staining dyes, i.e., DAPI and PI. The compounds exhibiting maximum anticancer potential also induced maximum apoptosis in the respective cell lines. Moreover, the obtained results suggested that untreated cells exhibited the homogenous staining of the nuclei, while the cells treated with different derivatives exhibited nuclear condensation and cell shrinkage along with the membrane blebbing which showed that the treated compounds have induced the cell death of respective cell lines. Furthermore, the mechanism of cytotoxic compound was determined by DNA interaction studies and it was found that the most potent inhibitors exhibited the non–covalent mode of interaction with the herring sperm–DNA (HS–DNA). The mechanism of action of the cytotoxic derivatives against MCF–7 cells suggested that the compound 3b (diarylsulphonamides), 1f (3,3′–carbonyl–bis(chromones), 3a (4–quinolone) and 6i (2–arylated thiadiazolopyrimidones) exhibited maximum inhibitory potential towards MCF–7, also depicted higher DNA interactions having Gibbs free energy Δ–17.48, Δ–17.50, Δ–18.19 and Δ–17.51 KJ/mol. Against the K–562 cells , compounds 4f (1H–pyrazol–4–yl benzamides), 1d (3,3′–carbonyl–bis(chromones), 2b (4–quinolone) and 6a (pyrazole pyrimidones) showed the maximum DNA interactions having Gibbs free energy Δ–17.88, Δ–17.86, Δ–18.09 and Δ–18.31 KJ/mol, respectively. Similarly, against HeLa, 4b (1H–pyrazol–4–yl benzamides), 10g (triazolothiadiazin–3–yl 2H– chromen–2–ones) and 3b (quinoline–4–carboxylic acid) exhibited maximum DNA interactions with Gibbs free energy Δ–17.21, Δ–18.36 and Δ–18.20 KJ/mol, respectively. Results obtained through the present studies revealed that the many of the compounds were potent and selective inhibitors of APs and NPPs with strong anticancer potential can be used as potential leads to synthesize more derivatives that can be beneficial for the treatment of health disorders associated with the over-expression of APs and NPPs. It was further concluded that due to strong inhibitory potential and lower effective concentration against enzymes and cancer cell lines these compounds must be further exploited to explore molecular basis of underlying anticancer mechanisms through in vivo studies for pharmaceutical point of view. Knowledge thus generated will be helpful for the development of future novel drugs." xml:lang="en_US