Synthesis, characterization and biological evaluation of thiadiazole amide derivatives as nucleoside triphosphate diphosphohydrolases (NTPDases) inhibitors.

Importance of extracellular nucleotides is widely understood. These nucleotides act as ligand for P2X and P2Y receptors and modulate a variety of biological functions. However, their extracellular concentration is maintained by a chain of enzymes termed as ecto-nucleotidases. Amongst them, nucleoside triphosphate diphosphohydrolases (NTPDases) is an important enzyme family responsible for the dephosphorylation of these nucleotides. Overexpression of NTPDases leads to many pathological conditions such as cancer and thrombosis. So far, only a few NTPDase inhibitors have been reported. Considering this scarcity of (NTPDase) inhibitors, a number of thiadiazole amide derivatives were synthesized and screened against human (h)-NTPDases. Several compounds showed promising inhibitory activity; compound 5a (IC50 (µM); 0.05 ± 0.008) and 5g (IC50 (µM); 0.04 ± 0.006) appeared to be the most distinguished molecules corresponding to h-NTPDase1 and -2. However, h-NTPDase3 was the least susceptible isozyme and only three compounds (5d, 5e, 5j) strongly inhibited h-NTPDase3. Interestingly, compound 5e was recognized as the most active compound that showed dual inhibition against h-NTPDase3 as well as against h-NTPDase8. For better comprehension of binding mode of these inhibitors, most potent inhibitors were docked with their respective isozyme.

Development of coumarin-thiosemicarbazone hybrids as aldose reductase inhibitors: Biological assays, molecular docking, simulation studies and ADME evaluation.

The over expression of aldose reductase (ALR2) in the state of hyperglycemia causes the conversion of glucose into sorbitol and initiates polyol pathway. Accumulation of sorbitol in insulin insensitive tissue like peripheral nerves, glomerulus and eyes, induces diabetic complications like neuropathy, nephropathy and retinopathy. For the treatment of diabetic complications, the inhibition of aldose reductase (ALR2) is a promising approach. A series of coumarin-based thiosemicarbazone derivatives was synthesized as potential inhibitor of aldose reductase. Compound N-(2-fluorophenyl)-2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)hydrazinecarbiothioamide (3n) was found to be the most promising inhibitor of ALR2 with an IC50 in micromolar range (2.07 µM) and high selectivity, relative to ALR1. The crystal structure of ALR2 complexed with 3n explored the types of interaction pattern which further demonstrated its high affinity. Compound 3n has excellent lead-likeness, underlined by its physicochemical parameters, and can be considered as a likely prospect for further structural optimization to get a drugable molecule.

Hydrazine clubbed 1,3-thiazoles as potent urease inhibitors: design, synthesis and molecular docking studies.

Synthesis of a novel series of hydrazine clubbed 1,3-thiazoles (5a-m) has been described by reacting hydrazine-1-carbothioamides (3a-k) with α-chloro- or bromo-acetophenones (4a-d) in refluxing ethanol in good to excellent yields (65-86%). Structural confirmation was based upon spectroscopic techniques such as 1H-NMR, 13C-NMR, FT-IR and mass spectrometry. The biological application of these motifs has been demonstrated in terms of their strong urease inhibition activity. The results of in vitro study revealed that all the compounds are the potent inhibitors of urease. The IC50 (ranging in between 110 and 440 nM) values were higher as compared to that of standard, i.e., thiourea (IC50 = 490 ± 10 nM). The synthesized compounds were docked at the active sites of the Jack bean urease enzyme in order to explore the possible binding interactions of enzyme-ligand complexes; the results reinforced the in vitro biological activity results.

First Record of Chaetomium globosum Causing Leaf Spot of Pomegranate in Pakistan.

Pomegranate (Punica granatum L.) is a non-climacteric and a favorite fruit of tropical, sub-tropical and arid regions of the world. During a survey in autumn 2019, leaf lesions were observed on plants (cv. Kandhari) in different orchards of Muzaffargarh (30°4'27.7572″ N, 71°11'4.7544″ E), a major pomegranate-producing region in Punjab Province. Disease incidence ranged from 17 to 20%. Leaf lesions were initially small (1 to 3 mm in diameter), round, purple or reddish-brown, scattered spots. At later stages, spots increased in size and the centers of mature lesions became dark red or black with fungal sporulation. To isolate the pathogen, samples of leaf (5 × 5 mm) were cut from the junction of diseased and healthy tissue, surface disinfected in 75% alcohol for 30 s, sterilized with 6% sodium hypochlorite for 3 min, washed with sterile distilled water three times, air dried in laminar flow hood, and cultured on potato dextrose agar (PDA). After one week of incubation at 25 ± 2°C with a 12-h photoperiod, fungal colonies developed, which were initially white and became pale yellow with olivaceous green mycelium after 20 days. On PDA, ascomata were olivaceous green, with a papillate ostiole, globose or ovoidal to obovoidal (155 to 220 × 120 to 240 µm, n=50). Terminal and lateral setae were abundant, brown, and tapering toward the tips (4 to 6 µm, n=50). Asci were greenish and lemon-shaped (6 to 8 × 9 to 13.5 µm, n=50). Ascospores were limoniform and olivaceous gray-brown (10 to 11.5 × 7 to 9 µm, n=50). These morphological characteristics were consistent with the morphology of Chaetomium globosum (Lan et al. 2011; Wang et al. 2016). Genomic DNA was extracted from two isolates and identification of the pathogen was confirmed by amplification and sequencing of the internal transcribed spacer region (ITS) and the partial translation elongation factor 1-α (TEF1) gene using ITS1/ITS4 (White et al. 1999) and EF1-983F/EF1-2218R primers (Wang et al. 2016), respectively. The sequences of the PCR products were deposited in GenBank with accession numbers MW522514, MW522352 (ITS), and MW530423, MW530424 (TEF1). BLAST results of the obtained sequences of the ITS and TEF1 genes revealed 100% (513/513 bp) and 99.78% (927/929 bp) similarity with those of C. globosum in GenBank (ITS: KX834823 and KT898637, and TEF1: MG812564 and KC485028). To confirm pathogenicity, inoculum was prepared by harvesting conidia from 10-day-old culture grown in PDA. The surface-disinfected (70% ethyl alcohol, 30 s) leaves of ten 1-year-old seedlings (cv. Kandhari) were sprayed with a spore suspension (1×106 conidia/ml). Leaves of ten seedlings sprayed with sterile distilled water served as controls. All seedlings were covered with plastic bags and placed in a greenhouse at 26°C with 12 h photoperiod. After eight days, symptoms on inoculated leaves were similar to those observed in the orchards; no symptoms were observed on controls. The fungus was reisolated from all symptomatic tissues. C. globosum has been reported on Punica granatum (Guo et al. 2015), Cannabis sativa (Chaffin et al. 2020) and Brassica oleracea (Zhu et al. 2020). This is the first report of C. globosum causing leaf spot on pomegranate in Pakistan. This finding suggests a potential threat to pomegranate production in Pakistan and further studies should focus on effective prevention and control practices of this disease.

First Record of Colletotrichum gloeosporioides Causing Anthracnose of Banana in Pakistan.

Banana (Musa spp.) is one of the most widely grown and consumed fruits in Pakistan and all around the world due to their distinct aroma and taste. In 2018, anthracnose symptoms were observed on banana fruit harvested from different plantations of Sindh- a major banana producing Province of Pakistan. Approximately, 25% of banana fruit collected from different plantations were infected. The symptoms consisted of small brown to reddish-brown spots on the fruit surface and then became sunken lesions as the disease progressed. To identify the pathogen, infected tissues (5 mm in diameter) from the margin of the lesions were surface sterilized by dipping in 1% sodium hypochlorite (NaOCl) for 2 min, 70% ethanol for 30 s, and then rinsed twice with sterile distilled water, plated onto potato dextrose agar (PDA), and incubated at 27°C for 5 days with 12 h light and darkness cycle. Colonies with a similar pattern were consistently isolated and all colonies were sub-cultured using the single-spore method. Colonies first appeared with white colored mycelium and later turned to dark gray. Conidia produced in acervuli were cylindric, hyaline, straight, and aseptate, with both ends rounded. Conidia measured 14.0 ± 0.5 × 3.4 ± 0.6 µm. Conidiomata were dark brown and spherical. On the basis of morphological characterization, the pathogen was identified as Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. (Weir et al. 2012). Two independent isolates (PDL2031 and PDL2032) were used for further genetic analysis. The internal transcribed spacer (ITS) region and chitin synthase 1 (CHS-1) gene were amplified from genomic DNA using primer pairs of ITS1/ITS4 and CHS-79F/CHS-345R, respectively (White et al. 1990; Damm et al. 2012). The GenBank accession numbers (MW493198, MW504711 for ITS and MW530421, MW530422 for CHS-1) of the sequences exhibited 99% to 100% identity to multiple sequences of C. gloeosporioides. To conduct a pathogenicity test, 10 healthy fruits were selected and surface sterilized with 70% ethanol followed by a wash of sterilized water. The fruits were stabbed with a sterile needle and a drop of 20 µl of spore suspension (106 spores/ml) was placed on each wound independently. Meanwhile 10 fruits inoculated with sterile water were treated as controls. The fruits were incubated at 27°C with 90% relative humidity for 10 days. Inoculated fruits exhibited symptoms similar to the original infection. No visible lesions appeared on control fruit. C. gloeosporioides was successfully reisolated from the inoculated fruit, confirming Koch's postulates. Anthracnose of banana is known to be caused by C. musae, C. gloeosporioides, C. siamense, C. tropicale, C. chrysophilum, C. theobromicola, and C. scovillei (Kumar et al. 2017; Peres et al. 2001; Vieira et al. 2017; Zakaria et al. 2009; Zhou et al. 2017). To our knowledge, this is first report of anthracnose of banana caused by C. gloeosporioides in Pakistan. The new disease primarily reduces the quality and yield of Banana. Effective measures should be taken to manage this disease.