Molecular dynamics simulation studies for DNA sequence recognition by reactive metabolites of anticancer compounds

The discovery of novel anticancer molecules 5F‐203 (NSC703786) and 5‐aminoflavone (5‐AMF, NSC686288) has addressed the issues of toxicity and reduced efficacy by targeting over expressed Cytochrome P450 1A1 (CYP1A1) in cancer cells. CYP1A1 metabolizes these compounds into their reactive metabolites, which are proven to mediate their anticancer effect through DNA adduct formation. However, the drug metabolite–DNA binding has not been explored so far. Hence, understanding the binding characteristics and molecular recognition for drug metabolites with DNA is of practical and fundamental interest. The present study is aimed to model binding preference shown by reactive metabolites of 5F‐203 and 5‐AMF with DNA in forming DNA adducts. To perform this, three different DNA crystal structures covering sequence diversity were selected, and 12 DNA‐reactive metabolite complexes were generated. Molecular dynamics simulations for all complexes were performed using AMBER 11 software after development of protocol for DNA‐reactive metabolite system. Furthermore, the MM‐PBSA/GBSA energy calculation, per‐nucleotide energy decomposition, and Molecular Electrostatic Surface Potential analysis were performed. The results obtained from present study clearly indicate that minor groove in DNA is preferable for binding of reactive metabolites of anticancer compounds. The binding preferences shown by reactive metabolites were also governed by specific nucleotide sequence and distribution of electrostatic charges in major and minor groove of DNA structure. Overall, our study provides useful insights into the initial step of mechanism of reactive metabolite binding to the DNA and the guidelines for designing of sequence specific DNA interacting anticancer agents. Copyright © 2014 John Wiley & Sons, Ltd.     

p87 and p101 Subunits Are Distinct Regulators Determining Class IB Phosphoinositide 3-Kinase (PI3K) Specificity

Class I_B phosphoinositide 3-kinase γ (PI3Kγ) comprises a single catalytic p110γ subunit, which binds to two non-catalytic subunits, p87 or p101, and controls a plethora of fundamental cellular responses. The non-catalytic subunits are assumed to be redundant adaptors for Gβγ enabling G-protein-coupled receptor-mediated regulation of PI3Kγ. Growing experimental data provide contradictory evidence. To elucidate the roles of the non-catalytic subunits in determining the specificity of PI3Kγ, we tested the impact of p87 and p101 in heterodimeric p87-p110γ and p101-p110γ complexes on the modulation of PI3Kγ activity in vitro and in living cells. RT-PCR, biochemical, and imaging data provide four lines of evidence: (i) specific expression patterns of p87 and p101, (ii) up-regulation of p101, providing the basis to consider p87 as a protein forming a constitutively and p101 as a protein forming an inducibly expressed PI3Kγ, (iii) differences in basal and stimulated enzymatic activities, and (iv) differences in complex stability, all indicating apparent diversity within class I_B PI3Kγ. In conclusion, expression and activities of PI3Kγ are modified differently by p87 and p101 in vitro and in living cells, arguing for specific regulatory roles of the non-catalytic subunits in the differentiation of PI3Kγ signaling pathways.     

An ecofriendly synthesis and DNA binding interaction study of some pyrazolo [1,5-a]pyrimidines derivatives

The DNA molecule is a target for plethora of anticancer and antiviral drugs that forms covalent and non-covalent adducts with major or minor groove of DNA. In present study we synthesized series of novel Pyrazolo [1,5-a]pyrimidine derivatives. The newly synthesized compounds were characterized by elemental analysis, IR, ¹H NMR, and mass spectral data. The selected compounds were studied for interaction with Calf thymus DNA (CT-DNA) using electronic spectra, viscosity measurement and thermal denaturation studies. Further, molecular interactions were revealed for compound IIIa and IVa by computational methodologies. The preferred mode of ligand binding with double helical DNA as well as preferable DNA groove were explored by molecular docking in different DNA models.     

Immune unresponsiveness to secondary heterologous bacterial infection after sepsis induction is TRAIL dependent

Sepsis is the leading cause of death in most intensive care units, and patients who survive the hyperinflammation that develops early during sepsis later display severely compromised immunity. Not only is there apoptosis of lymphoid and myeloid cells during sepsis that depletes these critical cellular components of the immune system, but also the remaining immune cells show decreased function. Using a cecal-ligation and puncture (CLP) model to induce intra-abdominal polymicrobial peritonitis, we recently established a link between the apoptotic cells generated during sepsis and induction of sepsis-induced suppression of delayed-type hypersensitivity. The present study extends this earlier work to include a secondary heterologous bacterial infection (OVA(257)-expressing Listeria monocytogenes [LM-OVA]) subsequent to sepsis initiation to investigate sepsis-induced alterations in the control of this secondary infection and the associated naive Ag-specific CD8 T cell response. We found that CLP-treated wild-type (WT) mice had a reduced ability to control the LM-OVA infection, which was paralleled by suppressed T cell responses, versus sham-treated WT mice. In contrast, CLP-treated Trail(-/-) and Dr5(-/-) mice were better able to control the secondary bacterial infection, and the Ag-specific CD8 T cell response was similar to that seen in sham-treated mice. Importantly, administration of a blocking anti-TRAIL mAb to CLP-treated WT mice was able to restore the ability to control the LM-OVA infection and generate Ag-specific CD8 T cell responses like those seen in sham-treated mice. These data further implicate TRAIL-dependent immune suppression during sepsis and suggest TRAIL neutralization may be a potential therapeutic target to restore cellular immunity in septic patients.     

The magnitude of the T cell response to a clinically significant dose of influenza virus is regulated by TRAIL

An immune response of appropriate magnitude should be robust enough to control pathogen spread but not simultaneously lead to immunopathology. Primary infection with influenza A virus (IAV) results in a localized pulmonary infection and inflammation and elicits an IAV-specific CD8 T cell immune response necessary for viral clearance. Clearance of IAV-infected cells, and recovery from infection, is mediated by perforin/granzyme B- and Fas/FasL-mediated mechanisms. We recently reported that TRAIL is another means by which IAV-specific CD8 T cells can kill IAV-infected cells. The current study examined the role of TRAIL in the pulmonary CD8 T cell response to a clinically significant IAV [A/PR/8/34 (PR8; H1N1)] infection (i.e., leads to observable, but limited, morbidity and mortality in wild-type [WT] mice). Compared with WT mice, IAV-infected Trail(-/-) mice experienced increased morbidity and mortality despite similar rates of viral clearance from the lungs. The increased morbidity and mortality in Trail(-/-) mice correlated with increased pulmonary pathology and inflammatory chemokine production. Analysis of lung-infiltrating lymphocytes revealed increased numbers of IAV-specific CD8 T cells in infected Trail(-/-) mice, which correlated with increased pulmonary cytotoxic activity and increased pulmonary expression of MIG and MIP-1α. In addition, there was decreased apoptosis and increased proliferation of IAV-specific CD8 T cells in the lungs of Trail(-/-) mice compared with WT mice. Together, these data suggest that TRAIL regulates the magnitude of the IAV-specific CD8 T cell response during a clinically significant IAV infection to decrease the chance for infection-induced immunopathology.     

Role of inflammasomes in host defense against Citrobacter rodentium infection

Citrobacter rodentium is an enteric bacterial pathogen of the mouse intestinal tract that triggers inflammatory responses resembling those of humans infected with enteropathogenic and enterohemorrhagic Escherichia coli. Inflammasome signaling is emerging as a central regulator of inflammatory and host responses to several pathogens, but the in vivo role of inflammasome signaling in host defense against C. rodentium has not been characterized. Here, we show that mice lacking the inflammasome components Nlrp3, Nlrc4, and caspase-1 were hypersusceptible to C. rodentium-induced gastrointestinal inflammation. This was due to defective interleukin (IL)-1β and IL-18 production given that il-1β(-/-) and il-18(-/-) mice also suffered from increased bacterial burdens and exacerbated histopathology. C. rodentium specifically activated the Nlrp3 inflammasome in in vitro-infected macrophages independently of a functional bacterial type III secretion system. Thus, production of IL-1β and IL-18 downstream of the Nlrp3 and Nlrc4 inflammasomes plays a critical role in host defense against enteric infections caused by C. rodentium.     

Comparative Proteomics Among Cytochrome P450 Family 1 for Differential Substrate Specificity

Apart from playing key roles in drug metabolism and adverse drug–drug interactions, CYPs are potential drug targets to treat a variety of diseases. The intervention of over expression of P450 1A1 (CYP1A1) in tumor cells is identified as a novel strategy for anticancer therapy. We investigated three isoforms of CYP1 family (CYP1A1, CYP1A2, and CYP1B1) for their substrate specificity. The understanding of macromolecular features that govern substrate specificity is required to understand the interplay between the protein function and dynamics. This can help in design of new antitumor molecule specifically metabolized by CYP1A1 to mediate their antitumor activity. In the present study, we carried out the comparative protein structure analysis of the three isoforms. Sequence alignment, root mean square deviation (RMSD) analysis, B-factor analysis was performed to give a better understanding of the macromolecular features involved in substrate specificity and to understand the interplay between protein dynamics and functions which will have important implications on rational design of anticancer drugs. We identified the differences in amino acid residues among the three isoforms of CYP1 family, which may account for differential substrate specificity. Six putative substrate recognition sequences are characterized along with the regions they form in the protein structure. Further the RMSD and B-factor analysis provides the information about the identified residues having the maximum RMSD and B-factor deviations.     

Ensemble docking and molecular dynamics identify knoevenagel curcumin derivatives with potent anti-EGFR activity

Epidermal growth factor receptor tyrosine kinase (EGFR-TK) is an attractive target for cancer therapy. Despite a number of effective EGFR inhibitors that are constantly expanding and different methods being employed to obtain novel compounds, the search for newer EGFR inhibitors is still a major scientific challenge. In the present study, a molecular docking and molecular dynamics investigation has been carried out with an ensemble of EGFR-TK structures against a synthetically feasible library of curcumin analogs to discover potent EGFR inhibitors. To resolve protein flexibility issue we have utilized 5 EGFR wild type crystal structures during docking as this gives improved possibility of identifying an active compound as compared to using a single crystal structure. We then identified five curcumin analogs representing different scaffolds that can serve as lead molecules. Finally, the 5 ns molecular dynamics simulation shows that knoevenagel condensate of curcumin specifically C29 and C30 can be used as starting blocks for developing effective leads capable of inhibiting EGFR.