Discovery of potent antimycobacterial agents targeting lumazine synthase (RibH) of Mycobacterium tuberculosis.

Tuberculosis (TB) continues to be a global health crisis, necessitating urgent interventions to address drug resistance and improve treatment efficacy. In this study, we validate lumazine synthase (RibH), a vital enzyme in the riboflavin biosynthetic pathway, as a potential drug target against Mycobacterium tuberculosis (M. tb) using a CRISPRi-based conditional gene knockdown strategy. We employ a high-throughput molecular docking approach to screen ~ 600,000 compounds targeting RibH. Through in vitro screening of 55 shortlisted compounds, we discover 3 compounds that exhibit potent antimycobacterial activity. These compounds also reduce intracellular burden of M. tb during macrophage infection and prevent the resuscitation of the nutrient-starved persister bacteria. Moreover, these three compounds enhance the bactericidal effect of first-line anti-TB drugs, isoniazid and rifampicin. Corroborating with the in silico predicted high docking scores along with favourable ADME and toxicity profiles, all three compounds demonstrate binding affinity towards purified lumazine synthase enzyme in vitro, in addition these compounds exhibit riboflavin displacement in an in vitro assay with purified lumazine synthase indicative of specificity of these compounds to the active site. Further, treatment of M. tb with these compounds indicate reduced production of flavin adenine dinucleotide (FAD), the ultimate end product of the riboflavin biosynthetic pathway suggesting the action of these drugs on riboflavin biosynthesis. These compounds also show acceptable safety profile in mammalian cells, with a high selective index. Hence, our study validates RibH as an important drug target against M. tb and identifies potent antimycobacterial agents.

Unraveling the Molecular Nexus between GPCRs, ERS, and EMT.

G protein-coupled receptors (GPCRs) represent a large family of transmembrane proteins that transduce an external stimulus into a variety of cellular responses. They play a critical role in various pathological conditions in humans, including cancer, by regulating a number of key processes involved in tumor formation and progression. The epithelial-mesenchymal transition (EMT) is a fundamental process in promoting cancer cell invasion and tumor dissemination leading to metastasis, an often intractable state of the disease. Uncontrolled proliferation and persistent metabolism of cancer cells also induce oxidative stress, hypoxia, and depletion of growth factors and nutrients. These disturbances lead to the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and induce a cellular condition called ER stress (ERS) which is counteracted by activation of the unfolded protein response (UPR). Many GPCRs modulate ERS and UPR signaling via ERS sensors, IRE1α, PERK, and ATF6, to support cancer cell survival and inhibit cell death. By regulating downstream signaling pathways such as NF-κB, MAPK/ERK, PI3K/AKT, TGF-ß, and Wnt/ß-catenin, GPCRs also upregulate mesenchymal transcription factors including Snail, ZEB, and Twist superfamilies which regulate cell polarity, cytoskeleton remodeling, migration, and invasion. Likewise, ERS-induced UPR upregulates gene transcription and expression of proteins related to EMT enhancing tumor aggressiveness. Though GPCRs are attractive therapeutic targets in cancer biology, much less is known about their roles in regulating ERS and EMT. Here, we will discuss the interplay in GPCR-ERS linked to the EMT process of cancer cells, with a particular focus on oncogenes and molecular signaling pathways.

In vitro evidence of NLRP3 inflammasome regulation by histone demethylase LSD2 in renal cancer: a pilot study.

NLRP3 pathway plays a vital role in the pathogenesis of different human cancers but still the regulation of NLRP3 pathway largely unknown. Therefore, we examined the levels of NLRP3 and its downstream components (caspase-1 and IL-1ß) and its relationship with histone modifiers in renal cancer pathogenesis. Total 30 cases of clear cell renal cell carcinoma (ccRCC), were studied for NLRP3, caspase-1 and IL-1ß expression using real-time PCR, which showed the augmented levels of all the three components of NLRP3 inflammasome pathway in ccRCC. Next, role of the FAD dependent monoamine oxidases (LSD2) and jumonji C (JmjC)-domain-containing, iron-dependent dioxygenases (KDM5A) histone demethylases were evaluated in regulation of NLRP3 inflammasome pathway in-vitro using RCC cell line. It was observed that silencing of KDM5A didn't alter the levels of neither of the NLRP3 component but inhibition of LSD2 showed significant effect on NLRP3 expression while no change in caspase-1 and IL-1ß levels. This study suggests that rather LSD2 not KDM5A lysine demethylase family might be involved in the regulation of NLRP3 inflammasome in cancer cells which could be useful for deciphering the future therapeutic targets for the disease.

Expression profile of H3K4 demethylases with their clinical and pathological correlation in patients with clear cell renal cell carcinoma.

OBJECTIVE: Clear cell renal cell carcinoma (ccRCC) is one of the deadly diseases with poor metastatic disease prognosis. There is an urgent need to explore the potential molecular markers which can improve the prognosis of the disease. Histone demethylases have emerged as a powerful tool for cancer prognosis and therapeutics during the last decade. The implications of demethylases of histone 3 lysine 4 (H3K4) in ccRCC are however unrevealed. We therefore evaluated the expression of H3K4 demethylases in ccRCC, with emphasis on their clinical significance as a prognostic marker. METHODS: Total 50 histopathological confirmed cases of ccRCC were enrolled in the study. The expression of seven H3K4 demethylases was determined by Real-Time PCR using gene specific primers and correlated with tumor stage, grade and metastasis. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the prognostic significance of H3K4 demethylases. RESULTS: The median age of the patients was 54 years with predominance of male patients by 2.6-fold. Among seven genes viz FBXL10, LSD1, LSD2, KDM5A, KDM5B, KDM5C and KDM5D analyzed, LSD2 was found to be significantly associated with tumor stage and metastasis. The optimal cut-off value for LSD2 was 3.2 as calculated from ROC curve analysis for metastasis as well as TNM stage with area under curve of 0.74 and 0.78 respectively. In addition, LSD2 expression showed significant positive correlation with LSD1 expression in tumor metastasis. CONCLUSION: The expression of LSD2 was associated with higher TNM stage and metastasis of the tumor and thus, might serve as a useful marker for ccRCC progression.

Reduction in H3K4me patterns due to aberrant expression of methyltransferases and demethylases in renal cell carcinoma: prognostic and therapeutic implications.

Renal cell carcinoma (RCC) is the leading cause among cancer-related deaths due to urological cancers, which results in response to combination of genetic and epigenetic factors. Histone methylations have been implicated in renal tumorigenesis but their clinical significance and underlying pathology are unexplored. Here, we elucidated the histone 3 lysine 4 (H3K4) methylation patterns in clear cell RCC and its underlying pathology. Lower cellular levels of H3K4 mono-methylation, -dimethylation and -tri-methylation were fraternized with higher TNM staging and Fuhrman grading as well as tumor metastasis. Further, the expression profile of 20 H3K4 modifiers revealed the significant over-expression of histone demethylases compared to methyltransferases, indicating their role in the reduction of H3K4 methylation levels. In view of above facts, the role of LSD2 and KDM5A demethylases in RCC pathogenesis were explored using respective siRNAs. The RCC cells exhibited reduced cell viability after knockdown of LSD2 and KDM5A genes with concomitant induction of apoptosis. In addition, propidium iodide staining demonstrated an arrest of RCC cells at S-phase and sub-G1 phase of the cell cycle. Taken together, these observations provide new pathological insights behind the alterations of H3K4 methylation patterns in ccRCC with their prognostic and therapeutic implications.

Pathogenic and Therapeutic Role of H3K4 Family of Methylases and Demethylases in Cancers.

Histone modifications occupy an essential position in the epigenetic landscape of the cell, and their alterations have been linked to cancers. Histone 3 lysine 4 (H3K4) methylation has emerged as a critical epigenetic cue for the regulation of gene transcription through dynamic modulation by several H3K4 methyltransferases (writers) and demethylases (erasers). Any disturbance in the delicate balance of writers and erasers can result in the mis-regulation of H3K4 methylation, which has been demonstrated in several human cancers. Therefore, H3K4 methylation has been recognized as a putative therapeutic or prognostic tool and drug trials of different inhibitors of this process have demonstrated promising results. Henceforth, more detailed knowledge of H3K4 methylation is utmost important for elucidating the complex cellular processes, which might help in improving the disease outcome. The primary focus of this review will be directed on deciphering the role of H3K4 methylation along with its writers/erasers in different cancers.

In-silico analysis of novel p.(Gly14Ser) variant of ATOX1 gene: plausible role in modulating ATOX1-ATP7B interaction.

Clinical heterogeneity is commonly observed in Wilson disease (WD), including cases with identical ATP7B mutations. It is thought to be an outcome of impairment in other genes involved in cellular copper homeostasis in addition to the mutations in the ATP7B gene. ATOX1, a copper chaperone that delivers copper to ATP7B, is a potential genetic modifier of WD. In the present study, we analyzed the genetic variations in the ATOX1 gene in 50 WD patients and 60 controls. We identified four novel variants, of which, the coding region variant c.40G > A, p.(Gly14Ser) was observed in 2% alleles. Interestingly, p.(Gly14Ser) was seen with an early onset age, reduced serum ceruloplasmin level and manifestations of liver and brain in a WD patient unlike the other having identical ATP7B mutation but normal ATOX1 alleles. Further, computational analysis predicted that p.(Gly14Ser) substitution, in the critical copper binding motif (MXCXG14C) of the protein, affects the protein-protein interaction involved in copper sharing and transfer between ATOX1 and ATP7B-MBD4. Our findings suggest that p.(Gly14Ser) variant of ATOX1 might play a role as a genetic modifier leading to phenotypic variation in WD.

Characterization of mutation spectrum and identification of novel mutations in ATP7B gene from a cohort of Wilson disease patients: Functional and therapeutic implications.

Wilson disease (WD), a copper metabolism disorder, occurs due to the presence of mutations in the gene encoding ATP7B, a protein that primarily facilitates hepatic copper excretion. A better understanding of spectrum and functional significance of ATP7B variants is critical to formulating targeted and personalized therapies. Henceforth, we screened and sequenced 21 exons of ATP7B gene from 50 WD patients and 60 healthy subjects. We identified 28 variants comprising, seven novels in 20% alleles, while eight variations affecting 23% alleles were first time reported in Indian cohort. The c.813C>A, p.(Cys271*) (10%) was the most frequent mutation. Bioinformatics analysis revealed five of seven novel variants viz. c.1600C>A, p.(Pro534Thr); c.1616C>A, p.(Pro539His); c.1924G>T, p.(Asp642Tyr); c.2168G>C, p.(Arg723Thr); c.2174G>C, p.(Arg725Thr) resulted in protein misfolding. Sequence conservation analysis of ATP7B regions containing novel variants documented an evolutionarily conserved nature. Functional analysis of these novel variants in five different cell lines lacking inherent ATP7B expression demonstrated sensitivity to CuCl2 -treatment, experiencing augmented cellular copper retention and decreased copper excretion as well as ceruloplasmin secretion to that of wildtype-ATP7B expressing cells. Interestingly, pharmacological chaperone 4-phenylbutyrate, a clinically approved compound, partially restored protein function of ATP7B mutants. These findings might enable novel treatment strategies in WD by clinically enhancing the protein expression of mutant ATP7B with residual copper export activity.

Renal Cell Carcinoma: Molecular Aspects.

Renal cell carcinoma is the most common form of the kidney cancer accounting for more than 85% of the cases of which clear cell renal cell carcinoma (ccRCC) is the major histological subtype. The central molecular signature for ccRCC pathogenesis is the biallelic inactivation of VHL gene due to the presence of mutations/hyper-methylation/complete gene loss, which results in the downstream HIF activation. These events lead to increased tyrosine kinase receptor signalling pathways (RAS/MEK/ERK pathway, PI3K/AKT/mTOR pathway and NF-κB pathway), which through their downstream effector proteins causes the cell to proliferate and migrate. Recent studies have shown that VHL inactivation alone is not sufficient to induce the tumor. Mutations in numerous other genes that codes for chromatin modifiers (PBRM1, SETD2 and BAP1) and signalling proteins (PTEN and mTOR) have been identified along with activation of alternate signalling pathways like STAT and Sonic Hedgehog (SHH) pathway. It has also been shown that STAT pathway also works cooperatively with HIF to enhance the tumor progression. However, SHH pathway reactivation resulted in tumor regardless of the VHL status, indicating the complex nature of the tumor at the molecular level. Therefore, understanding the complete aetiology of ccRCC is important for future therapeutics.

Expression and clinical significance of COMPASS family of histone methyltransferases in clear cell renal cell carcinoma.

The regulation of histone 3 lysine 4 (H3K4) methylation code is indispensable for the cell as any disturbance in this has been associated with many pathologic conditions, like cancer. The SET domain-containing lysine methyltransferase 2 (KMT2) family of histone methyltransferases was the first to be identified as writers of H3K4 methylation. In mammals, seven members of the KMT2 family are responsible for carrying out the bulk of H3K4 methylation. Recent studies documented alterations in various histone methylases in human cancer, which were associated with therapeutic and prognostic potential. Notwithstanding, no report has documented the role of KMT2 family of histone methyltransferases in clear cell renal cell carcinoma (ccRCC). Therefore, we examined the expression profile of all the seven KMT2 family members of histone methyltransferases in ccRCC with the aim to establish their role as prognostic or therapeutic targets. Real-time PCR was employed to study the expression of KMT2 family genes using specific primers in 50 cases of ccRCC. The mRNA levels were correlated with stage, grade, and metastasis of the tumor. Among seven genes, KMT2G was significantly up-regulated in higher stages of the tumor as compared to low stage tumors (p = 0.017). Similarly, KMT2G levels were higher in the metastatic tumor (p = 0.027). Additionally, some of the KMT2G target genes were found to be up-regulated in metastatic tumors as compared to non-metastatic. ROC curve indicated KMT2G as a good marker for discriminating metastatic tumors from non-metastatic tumors (AUC = 0.738). Thus, we conclude that KMT2G histone methyltransferase could be a good prognostic marker for ccRCC. Additionally, KMT2G can also serve as a therapeutic target for ccRCC.