Dynamics of Physical Interaction between HIV-1 Nef and ASK1: Identifying the Interacting Motif(S)

<div><p>FasL mediated preferential apoptosis of bystander CTLs while protection of infected CD4⁺T cells remains one of the hallmarks of immune evasion during HIV infection. The property of infected host cells to evade cell-autonomous apoptosis emanates from ability of HIV-1Nef -protein to physically interact with ASK-1 and thereby inhibit its enzymatic activity. The specific domains of HIV-1Nef through which it may interact with ASK1 and thereby impair the ASK1 activity remain unidentified so far and represent a major challenge towards developing clear understanding about the dynamics of this interaction. Using mammalian two hybrid screen in association with site directed mutagenesis and competitive inhibitor peptides, we identified constituent minimal essential domain (152 DEVGEANN 159) through which HIV-1Nef interacts with ASK1 and inhibits its function. Furthermore our study also unravels a novel alternate mechanism underlying HIV-1 Nef mediated ASK1 functional modulation, wherein by potentiating the inhibitory ser⁹⁶⁷ phosphorylation of ASK1, HIV-1Nef negatively modulated ASK1function.</p> </div

Mutation with in 152DEVGEANN159 region of Nef impairs its ability to interact with and restore ASK1 functions.

<div><p>(<b>A</b>) HEK-293 cell was transfected with pYFP-N1Nef (152AAAAAAAA159) plasmid and vector back bone. Cells were harvested after 48 hrs and western blotting was done by anti-Nef antibody. Showing almost equal level of protein expression. (<b>B</b>) Mammalian two hybrid assay was done for ASK1 (1-1051 aa), Nef and mutated HIV-1Nef (152-AAAAA159). Plasmid encoding GAL4-ASK1 was cloned in pBIND vector and Vp-16-Nef was cloned in pACT vector. HEK-293 cells co-transfected with ASK1 (1-1051 aa), Nef and pG5LUC vector exhibited a 156 fold increase in luciferase/renilla expression as compared to pACT-pBIND vector control. GAL4-ASK1 (1-1051 aa) and Nef (152AAAAA159) co-transfected cell in combination with pG5LUC showed only 8.75 fold increase in luciferase/renilla expression over from pACT-pBIND. The data are means <u>+</u>SEM from three distinct experiments. <i>P</i> values of <0.05 were obtained from two-tailed paired <i>t</i> test. (<b>C</b>) Effect of mutated HIV-1Nef (152-AAAAA159) on ASK1 (1-1051 aa) mediated apoptosis was evaluated using flow cytometric analysis of Annexin-V/PI labeling assay. Mutation with in critical regions of HIV-1Nef markedly affected anti-apoptotic potential of Nef as indicated by much elevated apoptotic cell count viz. 44.6% in case of HIV-1Nef (152-AAAAA159) as opposed to 15.16% in case of Nef. The data are means <u>+</u>SEM from three distinct experiments. <i>P</i> values of <0.05 were obtained from two-tailed paired <i>t</i> test.</p> <p>(<b>D</b>) Effect of mutated HIV-1Nef (152-AAAAA159) on ASK1 (1-1051 aa) mediated p54/46JNK 1pathway activation was studied using western blot /densitometry analysis of HEK-293 cells that were transfected with ASK1 (1-1051 aa) with/without Nef or mutated HIV-1Nef (152-AAAAA159). Mutation with in critical regions of HIV-1Nef altered p54/46JNK1 activation profile markedly as indicated by higher phospho-p54/46JNK1 levels. Western blotting was done three times showing consistent result.</p></div

Identification of Nef region interacting with ASK1.

<p>(<b>A</b>) Using Nef core domain (57-207 aa) sequences, two distinct Nef truncations were designed for mammalian two hybrid studies by successively deleting 33 aa form C-terminal and subsequent cloning in pACT vector so as to obtain Nef (57-174 aa), Nef (57-141 aa) fragments. The Nef core domain (57-207 aa) served as positive control. (<b>B</b>) The HEK-293 cells when co-transfected with ASK1 (1-1051aa) fragment and either of Nef fragments viz. Nef (57-207 aa), Nef (57-174 aa), Nef (57-141 aa) and pG5<i>LUC</i> vector showed 152.56 ,81.68 ,and 1.9 fold increase in expression of luciferase/renilla over pACT-pBIND negative control vector respectively. The data are means <u>+</u>SEM from three distinct experiments. <i>P</i> values of <0.05 were obtained (two-tailed paired <i>t</i> test) when luciferase/renilla expression from ASK1 fragments and Nef co-transfected cell were compared with negative control pACT-pBIND.</p

Identification of Nef interacting region within ASK1.

<p>(<b>A</b>) ASK1 overlapping fragments casing full length of ASK1 were designed to generate multiple ASK1 truncations viz. ASK1 (1-345 aa), ASK1 (319-670 aa), ASK1 (607-904 aa), ASK1 (861-1051 aa), ASK1 (1059-1182 aa), ASK1 (1152-1374 aa). The HEK-293 cells were co-transfected with ASK1 truncations and Nef along with pG5<i>LUC</i> reported vector. Different truncations exhibited varying degrees of interaction as measured by fold increase in luciferase/renilla expression over pACT-pBIND negative control vector viz. 2.05, 1.08, 0.59, 2.93, 0.71, 0.79 respectively. ASK1(1-345 aa) and ASK1(861-1051 aa) fragment showing significant interaction while other fragments are not significantly (ns) interact with Nef (<b>B</b>) A distinct ASK1 truncation that contained only C-terminal Nef interacting domain and lacked N-terminal Nef interacting domain i.e. ASK1 (319-1051 aa) was designed. Similarly, another distinct ASK1 truncation that contained only N-terminal Nef interacting domain and lacked C-terminal Nef interacting domain i.e. ASK1 (1-904 aa) was also designed. Using co-transfection studies in HEK-293 cells, both the truncations were individually evaluated for their interaction ability with Nef vis a vis ASK1 (1-1051 aa) truncation that harbored both N and C terminal domains and ASK1 (319-904 aa) truncation that lacked both N and C terminal domains. Co-transfection with ASK1 (1-1051 aa) caused 156.56 fold increase in luciferase/renilla expression compared to negative control pACT-pBIND while other ASK1 fragment viz. ASK1 (319-1051 aa), ASK1 (1-904 aa), ASK1 (319-904 aa) caused only 2.83, 8.75, 0.59 fold increases respectively. The data are means <u>+</u>SEM from three distinct experiments. <i>P</i> values of <0.05 were obtained (two-tailed paired <i>t</i> test) when luciferase/renilla expression from ASK1 fragments and Nef co-transfected cell were compared with negative control pACT-pBIND.</p

Synthetic peptide N-DEVGEANN-C successfully inhibited Nef from interacting with and there by restore the functions of endogenous wild type ASK1 activity.

<p>(<b>A</b>) HEK-293 cells were transfected with Nef plasmid or vector backbone. 24 hrs post transfection, cells were stimulated to progress through ASK1 mediated apoptotic progression using classic ligand viz. TNF-α (100ng/ml) either in absence or presence of synthetic peptide N-DEVGEANN-C (10ng/ml). The extent of interaction of endogenous wild type ASK1 with Nef and ability of peptide N-DEVGEANN-C to impair the interaction was evaluated using <i>In situ</i> proximity ligation assay. (<b>B</b>) Synthetic peptide N-DEVGEANN-C prevented HIV-1Nef from offering protection against TNF -α induced, endogenous wild type ASK1 mediated apoptosis as evaluated using Annexin-V/PI assay. (<b>C</b>) Endogenous wild type ASK1 activity was rendered active by synthetic peptide N-DEVGEANN-C even in presence of HIV-1Nef as indicated by markedly decreased inhibitory Ser⁹⁶⁷ phosphorylation of ASK1 with a corresponding increase in p54/46JNK1 activation profile as indicated by higher phospho-p54/46JNK1 levels in western blot analysis. All western blotting was done three times and integrated density was determined by densitometry analysis using imageJ software.</p

Inhibition of ASK1 (1-1051 aa) induced apoptosis by Nef.

<p>(<b>A</b>) <b>Expression of Nef:</b> HEK-293 cell was transfected with pEYFP-N1-Nef plasmid and vector back bone. Cells were harvested after 48 hrs and western bolting was done by anti Nef antibody. Showing stable expression of Nef protein. (<b>B</b>) HEK-293 cell was transfected with ASK1(1-1051 aa) along with/without Nef showing ASK1-Ser⁹⁶⁷ phosphorylation. ASK1-Ser⁹⁶⁷ was very low in vector control and ASK1 transfected HEK-293 cells, co-transfection with Nef significantly potentiated ASK1-Ser⁹⁶⁷ phosphorylation. (<b>C</b>) HEK-293 cells transfected with ASK1 (1-1051 aa) exhibited apoptotic cell count of 45.65% while HEK-293 cell that were co-transfected with ASK1 (1-1051 aa) and Nef (full length) exhibited 23.61% apoptotic cell count. pYFP-N1 vector control exhibited an apoptotic cell count of 11.78%. Cells that harbored vector backbone served as control. The data are means <u>+</u>SEM from three distinct experiments. <i>P</i> values of <0.05 were obtained from two-tailed paired <i>t</i> test. (<b>D</b>) p54/46JNK1 pathway activation was studied using western blot /densitometry analysis of HEK-293 cells that were transfected with ASK1 (1-1051 aa) with/without Nef. (<b>E</b>) Annexin-V/PI labeling followed by flow cytometric analysis of ASK1(1-1051 aa) transfected Jurkat T cell revealed 32.85% Annexin-V positive apoptotic cell count, While Jurkat T cell that were co-transfected with ASK1(1-1051 aa) and Nef (full length) exhibited 12.9% apoptotic cell count. Vector control (pYFP-N1) showed 12.79% Annexin-V positive apoptotic cells. (<b>F</b>) Jurkat T cell were transfected with plasmid encoding ASK1 (1-1051 aa) with and without plasmid encoding HIV-1Nef. Cells were harvested after 40 hrs of transfection and western blot analysis for JNK1 activation was done using anti phospho-p54/46JNK1 and p54/46JNK1 antibodies showing relative levels of activated p54/46JNK1 i.e. ratio between integrated density of phospho-p54/46JNK1 and p54/46JNK1 bands. All western bolting was done three times and integrated density was determined by densitometry analysis using imageJ software.</p

Synthetic peptide N-DEVGEANN-C and Nef(152AAAAAAAA159) restore endogenous ASK1 activity in SupT1 and Jurkat T cell.

<p>(<b>A</b>) Jurkat T cells were transfected with Nef plasmid, Nef (152AAAAAAAA159) plasmid or vector backbone. 24 hrs post transfection, cells were stimulated to progress through ASK1 mediated apoptotic progression using usual ligand viz. TNF-α (100ng/ml) either in absence or presence of synthetic peptide N-DEVGEANN-C (10ng/ml). Cells treated with TNF-α showing higher phospho-p54/46JNK1 levels and Nef, Nef (152AAAAAAAA159) transfected cell show p54/46JNK1 activation profile as indicated by in western blot analysis lower and high respectively. (<b>B</b>) SupT1 cells were transfected with Nef plasmid, Nef (152AAAAAAAA159) plasmid or vector backbone. 24 hrs post transfection, cells were stimulated to progress through ASK1 mediated apoptotic progression using usual ligand viz. TNF-α (100ng/ml) either in absence or presence of synthetic peptide N-DEVGEANN-C (10ng/ml). Cells treated with TNF-α showing higher phospho-p54/46JNK1 levels and Nef, Nef (152AAAAAAAA159) transfected cell show p54/46JNK1 activation profile as indicated by in western blot analysis lower and high respectively.</p

BRD9 Inhibition by Natural Polyphenols Targets DNA Damage/Repair and Apoptosis in Human Colon Cancer Cells

Epigenetic mechanisms play an important role in the etiology of colorectal cancer (CRC) and other malignancies due, in part, to deregulated bromodomain (BRD) functions. Inhibitors of the bromodomain and extraterminal (BET) family have entered into clinical trials as anticancer agents, and interest has grown in other acetyl &lsquo;reader&rsquo; proteins as therapeutic targets, including non-BET member bromodomain-containing protein 9 (BRD9). We report here that overexpression of BRD9 is associated with poor prognosis in CRC patients, and that siRNA-mediated knockdown of BRD9 decreased cell viability and activated apoptosis in human colon cancer cells, coincident with increased DNA damage. Seeking natural compounds as BRD9 antagonists, molecular docking in silico identified several polyphenols such as Epigallocatechin-3-gallate (EGCG), Equol, Quercetin, and Aspalathin, with favorable binding energies, supported by BROMOscan&reg; (DiscoverX) and isothermal titration calorimetry experiments. Polyphenols mimicked BRD9 knockdown and iBRD9 treatment in reducing colon cancer cell viability, inhibiting colony formation, and enhancing DNA damage and apoptosis. Normal colonic epithelial cells were unaffected, signifying cancer-specific effects. These findings suggest that natural polyphenols recognize and target BRD9 for inhibition, and might serve as useful lead compounds for bromodomain therapeutics in the clinical setting

Docking and mutagenesis studies lead to improved inhibitor development of ML355 for human platelet 12-lipoxygenase

Human platelet 12-(S)-Lipoxygenase (12-LOX) is a fatty acid metabolizing oxygenase that plays an important role in platelet activation and cardiometabolic disease. ML355 is a specific 12-LOX inhibitor that has been shown to decrease thrombosis without prolonging hemostasis and protect human pancreatic islets from inflammatory injury. It has an amenable drug-like scaffold with nM potency and encouraging ADME and PK profiles, but its binding mode to the active site of 12-LOX remains unclear. In the current work, we combined computational modeling and experimental mutagenesis to propose a model in which ML355 conforms to the "U" shape of the 12-LOX active site, with the phenyl linker region wrapping around L407. The benzothiazole of ML355 extends into the bottom of the active site cavity, pointing towards residues A417 and V418. However, reducing the active site depth alone did not affect ML355 potency. In order to lower the potency of ML355, the cavity needed to be reduced in both length and width. In addition, H596 appears to position ML355 in the active site through an interaction with the 2-methoxy phenol moiety of ML355. Combined, this binding model suggested that the benzothiazole of ML355 could be enlarged. Therefore, a naphthyl-benzothiazole derivative of ML355, Lox12Slug001, was synthesized and shown to have 7.2-fold greater potency than ML355. This greater potency is proposed to be due to additional van der Waals interactions and pi-pi stacking with F414 and F352. Lox12Slug001 was also shown to be highly selective against 12-LOX relative to the other LOX isozymes and more importantly, it showed activity in rescuing human islets exposed to inflammatory cytokines with comparable potency to ML355. Further studies are currently being pursued to derivatize ML355 in order to optimize the additional space in the active site, while maintaining acceptable drug-like properties

Docking and Mutagenesis Studies Lead to Improved Inhibitor Development of ML355 for Human Platelet 12-Lipoxygenase

Human platelet 12-(S)-Lipoxygenase (12-LOX) is a fatty acid metabolizing oxygenase that plays an important role in platelet activation and cardiometabolic disease. ML355 is a specific 12-LOX inhibitor that has been shown to decrease thrombosis without prolonging hemostasis and protect human pancreatic islets from inflammatory injury. It has an amenable drug-like scaffold with nM potency and encouraging ADME and PK profiles, but its binding mode to the active site of 12-LOX remains unclear. In the current work, we combined computational modeling and experimental mutagenesis to propose a model in which ML355 conforms to the U shape of the 12-LOX active site, with the phenyl linker region wrapping around L407. The benzothiazole of ML355 extends into the bottom of the active site cavity, pointing towards residues A417 and V418. However, reducing the active site depth alone did not affect ML355 potency. In order to lower the potency of ML355, the cavity needed to be reduced in both length and width. In addition, H596 appears to position ML355 in the active site through an interaction with the 2-methoxy phenol moiety of ML355. Combined, this binding model suggested that the benzothiazole of ML355 could be enlarged. Therefore, a naphthyl-benzothiazole derivative of ML355, Lox12Slug001, was synthesized and shown to have 7.2-fold greater potency than ML355. This greater potency is proposed to be due to additional van der Waals interactions and pi-pi stacking with F414 and F352. Lox12Slug001 was also shown to be highly selective against 12-LOX relative to the other LOX isozymes and more importantly, it showed activity in rescuing human islets exposed to inflammatory cytokines with comparable potency to ML355. Further studies are currently being pursued to derivatize ML355 in order to optimize the additional space in the active site, while maintaining acceptable drug-like properties