Integrated analysis of single cell-RNA sequencing and Mendelian randomization identifies lactate dehydrogenase B as a target of melatonin in ischemic stroke.

AIMS: Despite the success of single-cell RNA sequencing in identifying cellular heterogeneity in ischemic stroke, clarifying the mechanisms underlying these associations of differently expressed genes remains challenging. Several studies that integrate gene expression and gene expression quantitative trait loci (eQTLs) with genome wide-association study (GWAS) data to determine their causal role have been proposed. METHODS: Here, we combined Mendelian randomization (MR) framework and single cell (sc) RNA sequencing to study how differently expressed genes (DEGs) mediating the effect of gene expression on ischemic stroke. The hub gene was further validated in the in vitro model. RESULTS: We identified 2339 DEGs in 10 cell clusters. Among these DEGs, 58 genes were associated with the risk of ischemic stroke. After external validation with eQTL dataset, lactate dehydrogenase B (LDHB) is identified to be positively associated with ischemic stroke. The expression of LDHB has also been validated in sc RNA-seq with dominant expression in microglia and astrocytes, and melatonin is able to reduce the LDHB expression and activity in vitro ischemic models. CONCLUSION: Our study identifies LDHB as a novel biomarker for ischemic stroke via combining the sc RNA-seq and MR analysis.

Epigenetic silencing of LDHB promotes hepatocellular carcinoma by remodeling the tumor microenvironment.

Lactate dehydrogenase B (LDHB) reversibly catalyzes the conversion of pyruvate to lactate or lactate to pyruvate and expressed in various malignancies. However, the role of LDHB in modulating immune responses against hepatocellular carcinoma (HCC) remains largely unknown. Here, we found that down-regulation of lactate dehydrogenase B (LDHB) was coupled with the promoter hypermethylation and knocking down the DNA methyltransferase 3A (DNMT 3A) restored LDHB expression levels in HCC cell lines. Bioinformatics analysis of the HCC cohort from The Cancer Genome Atlas revealed a significant positive correlation between LDHB expression and immune regulatory signaling pathways and immune cell infiltrations. Moreover, immune checkpoint inhibitors (ICIs) have shown considerable promise for HCC treatment and patients with higher LDHB expression responded better to ICIs. Finally, we found that overexpression of LDHB suppressed HCC growth in immunocompetent but not in immunodeficient mice, suggesting that the host immune system was involved in the LDHB-medicated tumor suppression. Our findings indicate that DNMT3A-mediated epigenetic silencing of LDHB may contribute to HCC progression through remodeling the tumor immune microenvironment, and LDHB may become a potential prognostic biomarker and therapeutic target for HCC immunotherapy.

The Impact of Terminal Peptide Extensions of Retinal Inosine 5´Monophosphate Dehydrogenase 1 Isoforms on their DNA-binding Activities.

The main structural difference between the mutation-susceptible retinal isoforms of inosine 5´-monophosphate dehydrogenase-1 (IMPDH-1) with the canonical form resides in the C- and N-terminal peptide extensions with unknown structural/functional impacts. In this report, we aimed to experimentally evaluate the functional impact of these extensions on the specific/non-specific single-stranded DNA (ssDNA)-binding activities relative to those of the canonical form. Our in silico findings indicated the possible contribution of the C-terminal segment to the reduced flexibility of the Bateman domain of the enzyme. In addition, the in silico data indicated that the N-terminal tail acts by altering the distance between the tetramers in the concave octamer complex (the native form) of the enzyme. The overall impact of these predicted structural variations became evident, first, through higher Km values with respect to either of the substrates relative to the canonical isoform, as reported previously (Andashti et al. in Mol Cell Biochem 465(1):155-164, 2020). Secondary, the binding of the recombinant mouse retinal isoform IMPDH1 (603) to its specific Rhodopsin target gene was significantly augmented while its binding to non-specific ssDNA was lower than that of the canonical isoform. The DNA-binding activity of the other mouse retinal isoform, IMPDH1(546), to specific and non-specific ssDNA was lower than that of the canonical form most probably due to the in silico predicted rigidity created in the Bateman domain by the C-terminal peptide extension. Furthermore, the DNA binding to the Rhodopsin target gene by each of the IMPDH isoforms influenced in the presence of GTP (Guanosine triphosphate) and ATP (Adenosine triphosphate).

Identification of UDP-glucuronosyltransferase (UGT) isoforms involved in the metabolism of Chlorophenols (CPs).

Chlorophenols (CPs) are a group of pollutants that pose a great threat to the environment, they are widely used in industrial and agricultural wastes, pesticides, herbicides, textiles, pharmaceuticals and plastics. Among CPs, pentachlorophenol was listed as one of the persistent organic pollutants (POPs) by the Stockholm convention. This study aims to identify the UDP-glucosyltransferase (UGT) isoforms involved in the metabolic elimination of CPs. CPs' mono-glucuronide was detected in the human liver microsomes (HLMs) incubation mixture with co-factor uridine-diphosphate glucuronic acid (UDPGA). HLMs-catalyzed glucuronidation metabolism reaction equations followed Michaelis-Menten or substrate inhibition type. Recombinant enzymes and chemical reagents inhibition experiments were utilized to phenotype the main UGT isoforms involved in the glucuronidation of CPs. UGT1A6 might be the major enzyme in the glucuronidation of mono-chlorophenol isomer. UGT1A1, UGT1A6, UGT1A9, UGT2B4 and UGT2B7 were the most important five UGT isoforms for metabolizing the di-chlorophenol and tri-chlorophenol isomers. UGT1A1 and UGT1A3 were the most important UGT isoforms in the catalysis of tetra-chlorophenol and pentachlorophenol isomers. Species differences were investigated using rat liver microsomes (RLMs), pig liver microsomes (PLMs), dog liver microsomes (DLMs), and monkey liver microsomes (MyLMs). All these results were helpful for elucidating the metabolic elimination and toxicity of CPs.

Comparison of the Backbone Dynamics of Dehaloperoxidase-Hemoglobin Isoenzymes.

Dehaloperoxidase (DHP) is a multifunctional hemeprotein with a functional switch generally regulated by the chemical class of the substrate. Its two isoforms, DHP-A and DHP-B, differ by only five amino acids and have an almost identical protein fold. However, the catalytic efficiency of DHP-B for oxidation by a peroxidase mechanism ranges from 2- to 6-fold greater than that of DHP-A depending on the conditions. X-ray crystallography has shown that many substrates and ligands have nearly identical binding in the two isoenzymes, suggesting that the difference in catalytic efficiency could be due to differences in the conformational dynamics. We compared the backbone dynamics of the DHP isoenzymes at pH 7 through heteronuclear relaxation dynamics at 11.75, 16.45, and 19.97 T in combination with four 300 ns MD simulations. While the overall dynamics of the isoenzymes are similar, there are specific local differences in functional regions of each protein. In DHP-A, Phe35 undergoes a slow chemical exchange between two conformational states likely coupled to a swinging motion of Tyr34. Moreover, Asn37 undergoes fast chemical exchange in DHP-A. Given that Phe35 and Asn37 are adjacent to Tyr34 and Tyr38, it is possible that their dynamics modulate the formation and migration of the active tyrosyl radicals in DHP-A at pH 7. Another significant difference is that both distal and proximal histidines have a 15-18% smaller S2 value in DHP-B, thus their greater flexibility could account for the higher catalytic activity. The distal histidine grants substrate access to the distal pocket. The greater flexibility of the proximal histidine could also accelerate H2O2 activation at the heme Fe by increased coupling of an amino acid charge relay to stabilize the ferryl Fe(IV) oxidation state in a Poulos-Kraut "push-pull"-type peroxidase mechanism.

LDHB contributes to the regulation of lactate levels and basal insulin secretion in human pancreatic ß cells.

Using 13C6 glucose labeling coupled to gas chromatography-mass spectrometry and 2D 1H-13C heteronuclear single quantum coherence NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning ß cell function. In both mouse and human islets, the contribution of glucose to the tricarboxylic acid (TCA) cycle is similar. Pyruvate fueling of the TCA cycle is primarily mediated by the activity of pyruvate dehydrogenase, with lower flux through pyruvate carboxylase. While the conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in islets of both species, lactate accumulation is 6-fold higher in human islets. Human islets express LDH, with low-moderate LDHA expression and ß cell-specific LDHB expression. LDHB inhibition amplifies LDHA-dependent lactate generation in mouse and human ß cells and increases basal insulin release. Lastly, cis-instrument Mendelian randomization shows that low LDHB expression levels correlate with elevated fasting insulin in humans. Thus, LDHB limits lactate generation in ß cells to maintain appropriate insulin release.

The Expression and Secretion Profile of TRAP5 Isoforms in Gaucher Disease.

BACKGROUND: Gaucher disease (GD) is caused by glucocerebrosidase (GCase) enzyme deficiency, leading to glycosylceramide (Gb-1) and glucosylsphingosine (Lyso-Gb-1) accumulation. The pathological hallmark for GD is an accumulation of large macrophages called Gaucher cells (GCs) in the liver, spleen, and bone marrow, which are associated with chronic organ enlargement, bone manifestations, and inflammation. Tartrate-resistant acid phosphatase type 5 (TRAP5 protein, ACP5 gene) has long been a nonspecific biomarker of macrophage/GCs activation; however, the discovery of two isoforms of TRAP5 has expanded its significance. The discovery of TRAP5's two isoforms revealed that it is more than just a biomarker of macrophage activity. While TRAP5a is highly expressed in macrophages, TRAP5b is secreted by osteoclasts. Recently, we have shown that the elevation of TRAP5b in plasma is associated with osteoporosis in GD. However, the role of TRAP isoforms in GD and how the accumulation of Gb-1 and Lyso-Gb-1 affects TRAP expression is unknown. METHODS: 39 patients with GD were categorized into cohorts based on bone mineral density (BMD). TRAP5a and TRAP5b plasma levels were quantified by ELISA. ACP5 mRNA was estimated using RT-PCR. RESULTS: An increase in TRAP5b was associated with reduced BMD and correlated with Lyso-Gb-1 and immune activator chemokine ligand 18 (CCL18). In contrast, the elevation of TRAP5a correlated with chitotriosidase activity in GD. Lyso-Gb-1 and plasma seemed to influence the expression of ACP5 in macrophages. CONCLUSIONS: As an early indicator of BMD alteration, measurement of circulating TRAP5b is a valuable tool for assessing osteopenia-osteoporosis in GD, while TRAP5a serves as a biomarker of macrophage activation in GD. Understanding the distinct expression pattern of TRAP5 isoforms offers valuable insight into both bone disease and the broader implications for immune system activation in GD.

KAT7 enhances the proliferation and metastasis of head and neck squamous carcinoma by promoting the acetylation level of LDHA.

Lysine acetyltransferase 7 (KAT7), a histone acetyltransferase, has recently been identified as an oncoprotein and has been implicated in the development of various malignancies. However, its specific role in head and neck squamous carcinoma (HNSCC) has not been fully elucidated. Our study revealed that high expression of KAT7 in HNSCC patients is associated with poor survival prognosis and silencing KAT7 inhibits the Warburg effect, leading to reduced proliferation, invasion, and metastatic potential of HNSCC. Further investigation uncovered a link between the high expression of KAT7 in HNSCC and tumor-specific glycolytic metabolism. Notably, KAT7 positively regulates Lactate dehydrogenase A (LDHA), a key enzyme in metabolism, to promote lactate production and create a conducive environment for tumor proliferation and metastasis. Additionally, KAT7 enhances LDHA activity and upregulates LDHA protein expression by acetylating the lysine 118 site of LDHA. Treatment with WM3835, a KAT7 inhibitor, effectively suppressed the growth of subcutaneously implanted HNSCC cells in mice. In conclusion, our findings suggest that KAT7 exerts pro-cancer effects in HNSCC by acetylating LDHA and may serve as a potential therapeutic target. Inhibiting KAT7 or LDHA expression holds promise as a therapeutic strategy to suppress the growth and progression of HNSCC.

The role of genetic polymorphisms in the sulfation of pregnenolone by human cytosolic sulfotransferase SULT2B1a.

Pregnenolone is a key intermediate in the biosynthesis of many steroid hormones and neuroprotective steroids. Sulfotransferase family cytosolic 2B member 1 (SULT2B1a) has been reported to be highly selective to sulfate pregnenolone. This study aimed to clarify the effect of missense single nucleotide polymorphisms (SNPs) of the human SULT2B1 gene on the sulfating activity of coded SULT2B1a allozymes toward Pregnenolone. To investigate the effects of single nucleotide polymorphisms of the SULT2B1 gene on the sulfation of pregnenolone by SULT2B1a allozymes, 13 recombinant SULT2B1a allozymes were generated, expressed, and purified using established procedures. Human SULT2B1a SNPs were identified by a comprehensive database search. 13 SULT2B1a nonsynonymous missense coding SNPs (cSNPs) were selected, and site-directed mutagenesis was used to generate the corresponding cDNAs, packaged in pGEX-2TK expression vector, encoding these 13 SULT2B1a allozymes, which were bacterially expressed in BL21 E. coli cells and purified by glutathione-Sepharose affinity chromatography. Purified SULT2B1a allozymes were analyzed for sulfating activities towards pregnenolone. In comparison with the wild-type SULT2B1a, of the 13 allozymes, 11 showed reduced activity toward pregnenolone at 0.1 µM. Specifically, P134L and R259Q allozymes, reported to be involved in autosomal-recessive congenital ichthyosis, displayed low activity (1-10%) toward pregnenolone. The findings of this study may demonstrate the impact of genetic polymorphism on the sulfation of pregnenolone in individuals with different SULT2B1 genotypes.

Substrate Specificities of Variants of Barley (1,3)- and (1,3;1,4)-ß-d-Glucanases Resulting from Mutagenesis and Segment Hybridization.

Barley (1,3;1,4)-ß-d-glucanase is believed to have evolved from an ancestral monocotyledon (1,3)-ß-d-glucanase, enabling the hydrolysis of (1,3;1,4)-ß-d-glucans in the cell walls of leaves and germinating grains. In the present study, we investigated the substrate specificities of variants of the barley enzymes (1,3;1,4)-ß-d-glucan endohydrolase [(1,3;1,4)-ß-d-glucanase] isoenzyme EII (HvEII) and (1,3)-ß-d-glucan endohydrolase [(1,3)-ß-d-glucanase] isoenzyme GII (HvGII) obtained by protein segment hybridization and site-directed mutagenesis. Using protein segment hybridization, we obtained three variants of HvEII in which the substrate specificity was that of a (1,3)-ß-d-glucanase and one variant that hydrolyzed both (1,3)-ß-d-glucans and (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3;1,4)-ß-d-glucans. Using substitutions of specific amino acid residues, we obtained one variant of HvEII that hydrolyzed both substrates. However, neither protein segment hybridization nor substitutions of specific amino acid residues gave variants of HvGII that could hydrolyze (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3)-ß-d-glucans. Other HvEII and HvGII variants showed changes in specific activity and their ability to degrade the (1,3;1,4)-ß-d-glucans or (1,3)-ß-d-glucans to larger oligosaccharides. We also used molecular dynamics simulations to identify amino-acid residues or structural regions of wild-type HvEII and HvGII that interact with (1,3;1,4)-ß-d-glucans and (1,3)-ß-d-glucans, respectively, and may be responsible for the substrate specificities of the two enzymes.

The significance of the two cytosolic glutamine synthetase enzymes, GLN1;3 and GLN1;5, in the context of seed development and germination in Arabidopsis thaliana.

Glutamine synthetase (GS), an initial enzyme in nitrogen (N) plant metabolism, exists as a group of isoenzymes found in both cytosolic (GS1) and plastids (GS2) and has gathered significant attention for enhancing N use efficiency and crop yield. This work focuses on the A. thaliana GLN1;3 and GLN1;5 genes, the two predicted most expressed genes in seeds, among the five isogenes encoding GS1 in this species. The expression patterns were studied using transgenic marker line plants and qPCR during seed development and germination. The observed patterns highlight distinct functions for the two genes and confirm GLN1;5 as the most highly expressed GS1 gene in seeds. The GLN1;5, expression, oriented towards hypocotyl and cotyledons, suggests a role in protein turnover during germination, while the radicle-oriented expression of GLN1;3 supports a function in early external N uptake. While the single mutants exhibited a normal phenotype, except for a decrease in seed parameters, the double gln1;3/gln1;5 mutant displayed a germination delay, substantial impairment in growth, nitrogen metabolism, and number and quality of the seeds, as well as a diminishing in flowering. Although seed and pollen-specific, GLN1;5 expression is upregulated in the meristems of the gln1;3 mutants, filling the lack of GLN1;3 and ensuring the normal functioning of the gln1;3 mutants. These findings validate earlier in silico data on the expression patterns of GLN1;3 and GL1;5 genes in seeds, explore their different functions, and underscore their essential role in plant growth, seed production, germination, and early stages of plant development.

Identification of Bromophenols' glucuronidation and its induction on UDP- glucuronosyltransferases isoforms.

Bromophenols (BPs) are prominent environmental pollutants extensively utilized in aquaculture, pharmaceuticals, and chemical manufacturing. This study aims to identify UDP- glucuronosyltransferases (UGTs) isoforms involved in the metabolic elimination of BPs. Mono-glucuronides of BPs were detected in human liver microsomes (HLMs) incubated with the co-factor uridine-diphosphate glucuronic acid (UDPGA). The glucuronidation metabolism reactions catalyzed by HLMs followed Michaelis-Menten or substrate inhibition kinetics. Recombinant enzymes and inhibition experiments with chemical reagents were employed to phenotype the principal UGT isoforms participating in BP glucuronidation. UGT1A6 emerged as the major enzyme in the glucuronidation of 4-Bromophenol (4-BP), while UGT1A1, UGT1A6, and UGT1A8 were identified as the most essential isoforms for metabolizing 2,4-dibromophenol (2,4-DBP). UGT1A1, UGT1A8, and UGT2B4 were deemed the most critical isoforms in the catalysis of 2,4,6-tribromophenol (2,4,6-TBP) glucuronidation. Species differences were investigated using the liver microsomes of pig (PLM), rat (RLM), monkey (MyLM), and dog (DLM). Additionally, 2,4,6-TBP effects on the expression of UGT1A1 and UGT2B7 in HepG2 cells were evaluated. The results demonstrated potential induction of UGT1A1 and UGT2B7 upon exposure to 2,4,6-TBP at a concentration of 50 µM. Collectively, these findings contribute to elucidating the metabolic elimination and toxicity of BPs.

Expression of neuronal NO synthase α- and ß-isoforms in skeletal muscle of mice.

Knowledge of the primary structure of neuronal NO synthase (nNOS) in skeletal muscle is still conflicting and needs further clarification. To elucidate the expression patterns of nNOS isoforms at both mRNA and protein level, systematic reverse transcription (RT)-PCR and epitope mapping by qualitative immunoblot analysis on skeletal muscle of C57/BL6 mice were performed. The ability of the nNOS isoforms to form aggregates was characterized by native low-temperature polyacrylamide electrophoresis (LT-PAGE). The molecular analysis was focused on the rectus femoris (RF) muscle, a skeletal muscle with a nearly balanced ratio of nNOS α- and ß-isoforms. RT-PCR amplificates from RF muscles showed exclusive exon-1d mRNA expression, either with or without exon-µ. Epitope mapping demonstrated the simultaneous expression of the nNOS splice variants α/µ, α/non-µ, ß/µ and ß/non-µ. Furthermore, immunoblotting suggests that the transition between nNOS α- and ß-isoforms lies within exon-3. In LT-PAGE, three protein nNOS associated aggregates were detected in homogenates of RF muscle and tibialis anterior muscle: a 320 kDa band containing nNOS α-isoforms, while 250 and 300 kDa bands consist of nNOS ß-isoforms that form homodimers or heterodimers with non-nNOS proteins.

A novel system to determine activity of individual uridine 5'-diphospho-glucuronosyltransferase (UGT) isoforms: Recombinant UGT-beads.

Previous work demonstrated that human liver microsomes (HLMs) can spontaneously bind to silica-coated magnetizable beads (HLM-beads) and that these HLM-beads retain uridine 5'-diphospho-glucuronosyltransferase (UGT) activity. However, the contributions of individual UGT isoforms are not directly assessable in this system except through use of model inhibitors. Thus, a preparation wherein recombinant UGT (rUGT) microsomes bound to these same beads to form rUGT-beads of individual UGT isoforms would provide a novel system for measuring the contribution of individual UGT isoforms in a direct manner. To this end, the enzyme activities and kinetic parameter estimates of various rUGT isoforms in rUGT-beads were investigated, as well as the impact of fatty acids (FAs) on enzyme activity. The catalytic efficiencies (Vmax/Km) of the tested rUGTs were twofold to sevenfold higher in rUGT-beads compared with rUGT microsomes, except for rUGT1A6, where Vmax is the maximum product formation rate normalized to milligram of microsomal protein (pmol/min/mg protein). Interestingly, in contrast to traditional rUGT preparations, the sequestration of UGT-inhibitory FA using bovine serum albumin did not alter the catalytic efficiency (Vmax/Km) of the rUGTs in rUGT-beads. Moreover, the increase in catalytic efficiency of rUGT-beads over rUGT microsomes was similar to increases in catalytic efficiency noted with rUGT microsomes (not bound to beads) incubated with bovine serum albumin, suggesting the beads in some way altered the potential for FAs to inhibit activity. The rUGT-bead system may serve as a useful albumin-free tool to determine kinetic constants for UGT substrates, particularly those that exhibit high binding to albumin.

Chemical Versatility in Catalysis and Inhibition of the Class IIb Histone Deacetylases.

ConspectusThe zinc-dependent histone deacetylases (HDACs 1-11) belong to the arginase-deacetylase superfamily of proteins, members of which share a common α/ß fold and catalytic metal binding site. While several HDACs play a role in epigenetic regulation by catalyzing acetyllysine hydrolysis in histone proteins, the biological activities of HDACs extend far beyond histones. HDACs also deacetylate nonhistone proteins in the nucleus as well as the cytosol to regulate myriad cellular processes. The substrate pool is even more diverse in that certain HDACs can hydrolyze other covalent modifications. For example, HDAC6 is also a lysine decrotonylase, and HDAC11 is a lysine-fatty acid deacylase. Surprisingly, HDAC10 is not a lysine deacetylase but instead is a polyamine deacetylase. Thus, the HDACs are biologically and chemically versatile catalysts as they regulate the function of diverse protein and nonprotein substrates throughout the cell.Owing to their critical regulatory functions, HDACs serve as prominent targets for drug design. At present, four HDAC inhibitors are FDA-approved for cancer chemotherapy. However, these inhibitors are active against multiple HDAC isozymes, and a lack of selectivity is thought to contribute to undesirable side effects. Current medicinal chemistry campaigns focus on the development of isozyme-selective inhibitors, and many such studies largely focus on HDAC6 and HDAC10. HDAC6 is a target for therapeutic intervention due to its cellular role as a tubulin deacetylase and tau deacetylase, and selective inhibitors are being studied in cancer chemotherapy and the treatment of peripheral neuropathy. Crystal structures of enzyme-inhibitor complexes reveal how various features of inhibitor design, such as zinc-coordinating groups, bifurcated capping groups, and aromatic fluorination patterns, contribute to affinity and isozyme selectivity. The polyamine deacetylase HDAC10 is also an emerging target for cancer chemotherapy. Crystal structures of intact substrates trapped in the HDAC10 active site reveal the molecular basis of strikingly narrow substrate specificity for N8-acetylspermidine hydrolysis. Active site features responsible for substrate specificity have been successfully exploited in the design of potent and selective inhibitors.In this Account, I review the structural chemistry and inhibition of HDACs, highlighting recent X-ray crystallographic and functional studies of HDAC6 and HDAC10 in my laboratory. These studies have yielded fascinating snapshots of catalysis as well as novel chemical transformations involving bound inhibitors. The zinc-bound water molecule in the HDAC active site is the catalytic nucleophile in the deacetylation reaction, but this activated water molecule can also react with inhibitor C═O or C═N groups to yield unanticipated reaction products that bind exceptionally tightly. Versatile active site chemistry unleashes the full inhibitory potential of such compounds, and X-ray crystallography allows us to view this chemistry in action.

Sulfonyl thiourea derivatives from 2-aminodiarylpyrimidines: In vitro and in silico evaluation as potential carbonic anhydrase I, II, IX, and XII inhibitors.

A series of synthesized sulfonyl thiourea derivatives (7a-o) of substituted 2-amino-4,6-diarylpyrimidines (4a-o) exhibited the remarkable inhibitory activity against some the human carbonic anhydrases (hCAs), including hCA I, II, IX, and XII isoforms. The inhibitory efficacy of synthesized sulfonyl thiourea derivatives were experimentally validated by in vitro enzymatic assays. 7a (KI = 46.14 nM), 7j (KI = 48.92 nM), and 7m (KI = 62.59 nM) (for isoform hCA I); 7f (KI = 42.72 nM), 7i (KI = 40.98 nM), and 7j (KI = 33.40 nM) (for isoform hCA II); 7j (KI = 228.5 nM), 7m (KI = 195.4 nM), and 7n (KI = 210.1 nM) (for isoform hCA IX); 7l (KI = 116.9 nM), 7m (KI = 118.8 nM), and 7n (KI = 147.2 nM) (for isoform hCA XII) in comparison with KI values of 452.1, 327.3, 437.2, and 338.9 nM, respectively, of the standard drug AAZ. These compounds also had significantly more potent inhibitory action against cytosolic isoform hCA I and tumor-associated isoforms hCA IX and hCA XII. Furthermore, the potential inhibitory compounds were subjected to in silico screening for molecular docking and molecular dynamics simulations. The results of in vitro and in silico studies revealed that compounds 7a, 7j, and 7m were the most promising derivatives in this series due to their significant effects on studied hCA I, II, IX, and XII isoforms, respectively. The results showed that the sulfonyl thiourea moiety was accommodated deeply in the active site and interacted with the zinc ion in the receptors.

Design, synthesis, and biological evaluation of 3-benzenesulfonamide-linked 3-hydrazinoisatin derivatives as carbonic anhydrase inhibitors.

A new series of isatin-linked benzenesulfonamide derivatives (9a-w) were synthesized using the tail approach and assayed for their inhibitory potency against four different human carbonic anhydrase (hCA) isoforms, hCA I, II, IX, and XII. Most of these synthesized compounds exhibited interesting inhibition potency against isoforms hCA I, IX, and XII in the nanomolar range and by taking the standard drug acetazolamide. The most potent compounds in the case of hCA I were 9c (435.8 nM) and 9s (956.4 nM), for hCA IX, 9a (60.5 nM), 9d (95.6 nM), 9g (92.1 nM), and 9k (75.4 nM), and for hCA XII, 9p (84.5 nM). However, these compounds showed more selectivity toward hCA IX over hCA I, II, and XII. Thus, these compounds can be further developed as potential lead molecules for the development of isoform-selective hCA IX inhibitors with further structural modifications.

Benzothiadiazinone-1,1-Dioxide Carbonic Anhydrase Inhibitors Suppress the Growth of Drug-Resistant Mycobacterium tuberculosis Strains.

2H-Benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (BTD) based carbonic anhydrase (CA) inhibitors are here explored as new anti-mycobacterial agents. The chemical features of BTD derivatives meet the criteria for a potent inhibition of ß-class CA isozymes. BTD derivatives show chemical features meeting the criteria for a potent inhibition of ß-class CA isozymes. Specifically, three ß-CAs (MtCA1, MtCA2, and MtCA3) were identified in Mycobacterium tuberculosis and their inhibition was shown to exert an antitubercular action. BTDs derivatives 2a-q effectively inhibited the mycobacterial CAs, especially MtCA2 and MtCA3, with Ki values up to a low nanomolar range (MtCA3, Ki = 15.1-2250 nM; MtCA2, Ki = 38.1-4480 nM) and with a significant selectivity ratio over the off-target human CAs I and II. A computational study was conducted to elucidate the compound structure-activity relationship. Importantly, the most potent MtCA inhibitors demonstrated efficacy in inhibiting the growth of M. tuberculosis strains resistant to both rifampicin and isoniazid-standard reference drugs for Tuberculosis treatment.

Physiological function and regulation of ascorbate peroxidase isoforms.

Ascorbate peroxidase (APX) reduces H2O2 to H2O by utilizing ascorbate as a specific electron donor and constitutes the ascorbate-glutathione cycle in organelles of plants including chloroplasts, cytosol, mitochondria, and peroxisomes. It has been almost 40 years since APX was discovered as an important plant-specific H2O2-scavenging enzyme, during which time many research groups have conducted molecular physiological analyses. It is now clear that APX isoforms function not only just as antioxidant enzymes but also as important factors in intracellular redox regulation through the metabolism of reactive oxygen species. The function of APX isoforms is regulated at multiple steps, from the transcriptional level to post-translational modifications of enzymes, thereby allowing them to respond flexibly to ever-changing environmental factors and physiological phenomena such as cell growth and signal transduction. In this review, we summarize the physiological functions and regulation mechanisms of expression of each APX isoform.

Isoenzyme characterization of Leishmania infantum toward checking the antioxidant activity of superoxide dismutase and glutathione peroxidase.

BACKGROUND: Leishmania infantum is the major causative agent of visceral leishmaniasis in Mediterranean regions. Isoenzyme electrophoresis (IE), as a biochemical technique, is applied in the characterization of Leishmania species. The current study attempted to investigate the isoenzyme patterns of logarithmic and stationary promastigotes and axenic amastigotes (amastigote-like) of L. infantum using IE. The antioxidant activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX) was also checked in the aforementioned forms. METHOD: After L. infantum cultivation and obtaining logarithmic and stationary promastigotes, axenic amastigotes were achieved by incubation of stationary promastigotes at 37 °C for 48 h. The lysate samples were prepared and examined for six enzymatic systems including glucose-6-phosphate dehydrogenase (G6PD), nucleoside hydrolase 1 (NH1), malate dehydrogenase (MDH), glucose-phosphate isomerase (GPI), malic enzyme (ME), and phosphoglucomutase (PGM). Additionally, the antioxidant activity of SOD and GPX was measured. RESULTS: GPI, MDH, NH1, and G6PD enzymatic systems represented different patterns in logarithmic and stationary promastigotes and axenic amastigotes of L. infantum. PGM and ME showed similar patterns in the aforementioned forms of parasite. The highest level of SOD activity was determined in the axenic amastigote form and GPX activity was not detected in different forms of L. infantum. CONCLUSION: The characterization of leishmanial-isoenzyme patterns and the measurement of antioxidant activity of crucial antioxidant enzymes, including SOD and GPX, might reveal more information in the biology, pathogenicity, and metabolic pathways of Leishmania parasites and consequently drive to designing novel therapeutic strategies in leishmaniasis treatment.