Muhammad Ayaz Anwar

Mechanism of Cisplatin-Induced Cytotoxicity Is Correlated to Impaired Metabolism Due to Mitochondrial ROS Generation

The chemotherapeutic use of cisplatin is limited by its severe side effects. In this study, by conducting different omics data analyses, we demonstrated that cisplatin induces cell death in a proximal tubular cell line by suppressing glycolysis- and tricarboxylic acid (TCA)/mitochondria-related genes. Furthermore, analysis of the urine from cisplatin-treated rats revealed the lower expression levels of enzymes involved in glycolysis, TCA cycle, and genes related to mitochondrial stability and confirmed the cisplatin-related metabolic abnormalities. Additionally, an increase in the level of p53, which directly inhibits glycolysis, has been observed. Inhibition of p53 restored glycolysis and significantly reduced the rate of cell death at 24 h and 48 h due to p53 inhibition. The foremost reason of cisplatin-related cytotoxicity has been correlated to the generation of mitochondrial reactive oxygen species (ROS) that influence multiple pathways. Abnormalities in these pathways resulted in the collapse of mitochondrial energy production, which in turn sensitized the cells to death. The quenching of ROS led to the amelioration of the affected pathways. Considering these observations, it can be concluded that there is a significant correlation between cisplatin and metabolic dysfunctions involving mROS as the major player.

Insights into the species-specific TLR4 signaling mechanism in response to Rhodobacter sphaeroides lipid A detection

TLR4 in complex with MD2 senses the presence of lipid A (LA) and initiates a signaling cascade that curb the infection. This complex is evolutionarily conserved and can initiate the immune system in response to a variety of LAs. In this study, molecular dynamics simulation (25 ns) was performed to elucidate the differential behavior of TLR4/MD2 complex in response to Rhodobacter sphaeroides lipid A (RsLA). Penta-acyl chain-containing RsLA is at the verge of agonist (6 acyl-chains) and antagonist (4 acyl-chains) structure, and activates the TLR4 pathway in horses and hamsters, while inhibiting in humans and murine. In the time-evolved coordinates, the promising factors that dictated the differential response included the local and global mobility pattern of complexes, solvent-accessible surface area of ligand, and surface charge distributions of TLR4 and MD2. We showed that the GlcN1-GlcN2 backbone acquires agonist (3FXI)-like configurations in horses and hamsters, while acquiring antagonist (2E59)-like configurations in humans and murine systems. Moreover, analysis of F126 behavior in the MD2 F126 loop (amino acids 123–129) and loop EF (81–89) suggested that certain sequence variations also contribute to species-specific response. This study underlines the TLR4 signaling mechanism and provides new therapeutic opportunities.

Multiple roles of toll-like receptor 4 in colorectal cancer

Toll-like receptor (TLR) signaling has been implicated in the inflammatory responses in intestinal epithelial cells (IECs). Such inflammatory signals mediate complex interactions between commensal bacteria and TLRs and are required for IEC proliferation, immune response, repair, and homeostasis. The upregulation of certain TLRs in colorectal cancer (CRC) tissues suggests that TLRs may play an essential role in the prognosis of chronic and inflammatory diseases that ultimately culminate in CRC. Here, we provide a comprehensive review of the literature on the involvement of the TLR pathway in the initiation, progression, and metastasis of CRC, as well as inherited genetic variation and epigenetic regulation. The differential expression of TLRs in epithelial cells has also been discussed. In particular, we emphasize the physiological role of TLR4 in CRC development and pathogenesis, and propose novel and promising approaches for CRC therapeutics with the aid of TLR ligands.

Activating transcription factor 3 represses inflammatory responses by binding to the p65 subunit of NF-κB

Activating transcription factor 3 (ATF3) is induced by inflammatory responses, cell death, cytokines, and oxidative stress conditions. ATF3 is a negative regulator in the Toll-like receptor 4 signalling pathway. The principal molecule in this pathway is nuclear factor κB (NF-κB) that translocates into the nucleus to initiate the transcription of inflammatory mediators. However, scarce data are available regarding the interaction of ATF3 and p65, a part of the NF-κB dimer. Therefore, we studied the mechanism of regulation of p65 by ATF3 in RAW 264.7 cells. First, LPS-mediated NF-κB activation was confirmed, and then the direct interaction of ATF3 and p65 was observed through immunoprecipitation (IP). The presence of histone deacetylase 1 (HDAC1) was also detected in the complex. In ATF3 deficient cells, NF-κB activity was up-regulated and HDAC1 was not detected by IP. These observations suggest that p65 is attenuated by ATF3 such that ATF3 recruits HDAC1 to the ATF3/p65 complex and facilitates the deacetylation of p65. Likewise, inflammatory response genes were induced by translocated NF-κB in ATF3-deficient cells. Cumulatively, we uncovered a novel mechanism for the negative regulation of NF-κB by ATF3 via direct interaction with p65.

Etoposide induced cytotoxicity mediated by ROS and ERK in human kidney proximal tubule cells.

Etoposide (ETO) is a commonly used chemotherapeutic drug that inhibits topoisomerase II activity, thereby leading to genotoxicity and cytotoxicity. However, ETO has limited application due to its side effects on normal organs, especially the kidney. Here, we report the mechanism of ETO-induced cytotoxicity progression in human kidney proximal tubule (HK-2) cells. Our results show that ETO perpetuates DNA damage, activates mitogen-activated protein kinase (MAPK), and triggers morphological changes, such as cell and nuclear swelling. When NAC, a well-known reactive oxygen species (ROS) scavenger, is co-treated with ETO, it inhibits an ETO-induced increase in mitochondrial mass, mitochondrial DNA (ND1 and ND4) copy number, intracellular ATP level, and mitochondrial biogenesis activators (TFAM, PGC-1α and PGC-1β). Moreover, co-treatment with ETO and NAC inhibits ETO-induced necrosis and cell swelling, but not apoptosis. Studies using MAPK inhibitors reveal that inhibition of extracellular signal regulated kinase (ERK) protects ETO-induced cytotoxicity by inhibiting DNA damage and caspase 3/7 activity. Eventually, ERK inhibitor treated cells are protected from ETO-induced nuclear envelope (NE) rupture and DNA leakage through inhibition of caspase activity. Taken together, these data suggest that ETO mediates cytotoxicity in HK-2 cells through ROS and ERK pathways, which highlight the preventive avenues in ETO-induced cytotoxicity in kidney.

Gram-Negative Marine Bacteria: Structural Features of Lipopolysaccharides and Their Relevance for Economically Important Diseases

Gram-negative marine bacteria can thrive in harsh oceanic conditions, partly because of the structural diversity of the cell wall and its components, particularly lipopolysaccharide (LPS). LPS is composed of three main parts, an O-antigen, lipid A, and a core region, all of which display immense structural variations among different bacterial species. These components not only provide cell integrity but also elicit an immune response in the host, which ranges from other marine organisms to humans. Toll-like receptor 4 and its homologs are the dedicated receptors that detect LPS and trigger the immune system to respond, often causing a wide variety of inflammatory diseases and even death. This review describes the structural organization of selected LPSes and their association with economically important diseases in marine organisms. In addition, the potential therapeutic use of LPS as an immune adjuvant in different diseases is highlighted.

In silico mechanistic analysis of IRF3 inactivation and high-risk HPV E6 species-dependent drug response

The high-risk human papillomavirus E6 (hrHPV E6) protein has been widely studied due to its implication in cervical cancer. In response to viral threat, activated kinases phosphorylate the IRF3 autoinhibitory domain, inducing type1 interferon production. HPV circumvents the antiviral response through the possible E6 interaction with IRF3 and abrogates p53’s apoptotic activity by recruiting E6-associated protein. However, the molecular mechanism of IRF3 inactivation by hrHPV E6 has not yet been delineated. Therefore, we explored this mechanism through in silico examination of protein-protein and protein-ligand docking, binding energy differences, and computational alanine mutagenesis. Our results suggested that the LxxLL motifs of IRF3 binds within the hydrophobic pocket of E6, precluding Ser-patch phosphorylation, necessary for IRF3 activation and interferon induction. This model was further supported by molecular dynamics simulation. Furthermore, protein-ligand docking and drug resistance modeling revealed that the polar patches in the pocket of E6, which are crucial for complex stability and ligand binding, are inconsistent among hrHPV species. Such variabilities pose a risk of treatment failure owing to point mutations that might render drugs ineffective, and allude to multi-drug therapy. Overall, this study reveals a novel perspective of innate immune suppression in HPV infections and suggests a plausible therapeutic intervention.

A structural insight into the negative effects of opioids in analgesia by modulating the TLR4 signaling: An in silico approach

Opioids are considered the gold standard therapy for pain. However, TLR-dependent negative effects in analgesia have highlighted the complexities in the pharmacodynamics of opioids. While successive studies have reported that morphine and Morphine-3-glucuronide (M3G) activate the TLR4 pathway, the structural details of this mechanism are lacking. Here, we have utilized various computational tools to reveal the structural dynamics of the opioid-bound TLR4/MD2 complex, and have proposed a potential TLR4 activation mechanism. Our results support previous findings, and include the novel insight that the stable binding of morphine and naloxone, but not M3G, in the MD2 cavity, is TLR4 dependent. Morphine interacts with MD2 near its Phe126 loop to induce the active conformation (MD2C); however, this binding is likely reversible, and the complex gains stability upon interaction with TLR4. M3G also induces the MD2C state, with both the Phe126 loop and the H1 loop being involved in MD2-M3G complex stability. Remarkably, naloxone, which requires TLR4 interaction for complex stability, switches the conformation of the gating loop to the inactive state (MD2°). Cumulatively, our findings suggest that ligand binding and receptor clustering occur successively in opioid-induced TLR4 signaling, and that MD2 plasticity and pocket hydrophobicity are crucial for the recognition and accommodation of ligands.

A Combined Molecular Docking/Dynamics Approach to Probe the Binding Mode of Cancer Drugs with Cytochrome P450 3A4

Cytarabine, daunorubicin, doxorubicin and vincristine are clinically used for combinatorial therapies of cancers in different combinations. However, the knowledge about the interaction of these drugs with the metabolizing enzyme cytochrome P450 is limited. Therefore, we utilized computational methods to predict and assess the drug-binding modes. In this study, we performed docking, MD simulations and free energy landscape analysis to understand the drug-enzyme interactions, protein domain motions and the most populated free energy minimum conformations of the docked protein-drug complexes, respectively. The outcome of docking and MD simulations predicted the productive, as well as the non-productive binding modes of the selected drugs. Based on these interaction studies, we observed that S119, R212 and R372 are the major drug-binding residues in CYP3A4. The molecular mechanics Poisson–Boltzmann surface area analysis revealed the dominance of hydrophobic forces in the CYP3A4-drug association. Further analyses predicted the residues that may contain favorable drug-specific interactions. The probable binding modes of the cancer drugs from this study may extend the knowledge of the protein-drug interaction and pave the way to design analogs with reduced toxicity. In addition, they also provide valuable insights into the metabolism of the cancer drugs.

ATF3 attenuates cyclosporin A-induced nephrotoxicity by downregulating CHOP in HK-2 cells.

Calcineurin inhibitors such as cyclosporin A (CsA) are widely used to treat organ transplantation-associated complications. However, CsA use is limited due to renal dysfunction. This study attempts to characterize the mechanism of CsA-induced nephrotoxicity using a human embryonic kidney cell line (HK-2). We performed microarray-based whole-genome expression analysis in HK-2 cells. CsA treatment induced the expression of endoplasmic reticulum (ER) stress-related and apoptosis-inducing genes at 6 and 24h, respectively, indicating that ER-stress predisposed the cells to apoptosis. G1 phase cell-cycle arrest was also observed via ER stress in CsA-treated cells. Furthermore, we found an inverse relationship between activating transcription factor 3 (ATF3), a stress-inducible protein, and C/EBP homologous protein (CHOP), an apoptosis-inducing protein. Moreover, when ATF3 knockdown cells were exposed to CsA, a prompt induction of CHOP was observed, which stimulated ROS production and induced cell death-related genes as compared to wild type. Taken together, our data demonstrate that ATF3 plays a pivotal role in the attenuation of CsA-induced nephrotoxicity by downregulating CHOP and ROS production mediated by ER stress.