Patent Application: US-201314382428-A

Abstract:
the present invention relates to synthetic peptide inhibitors useful as anti - hiv therapeutics . the invention also relates to a novel screening method for screening anti - hiv molecules . the present invention relates to a synthetic peptide useful as anti - hiv therapeutic . the invention also relates to a novel screening method for screening of anti - hiv therapeutics . in particular , the present invention relates to reporter gene constructs for the detection of the hiv nef and host ask1 protein interaction . furthermore , the invention relates to a functional interaction for nef - ask1 proteins prepared in a recombinant manner , a method for identifying of nef - ask1 interaction which causes activation of pathway to activate apoptosis presumably causing immune evasion for hiv in infected cells . the reporter gene construct according to the present invention , after it had been introduced into cells , in the presence of hiv nef and host ask1 proteins result in the expression of reporter luciferase protein which may be used for quantitative / qualitative interaction of hiv nef and host ask1 protein . the both interacting construct cloned in fluorescence expression vector when transfected in eukaryotic cells inhibits ask1 mediated apoptosis and were reversed by the inhibitors . furthermore , the invention was used to identify the inhibitor for the interaction of nef - ask1 in the cell .

Description:
a “ fusion protein ” refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide . the fusion protein may be formed by the chemical coupling of the constituent polypeptides or it may be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein . a single chain fusion protein is a fusion protein halving a single contiguous polypeptide backbone . the term “ two - hybrid system ” refers to a system comprising two chimeric molecules one of which bears a nucleic acid binding region , the other of which bears an expression control element ( e . g . a transactivation or repressor domain ). the molecules further a cognate binding pair such that one chimeric molecule is capable of specifically binding to the other chimeric molecule . the two - hybrid system further comprises a nucleic acid encoding a protein binding site that is specifically bound by the protein binding domain on the chimeric molecule thereby anchoring the chimeric molecule to the nucleic acid . the domain of the chimeric molecule recognizes and binds to its cognate binding partner on the second chimeric molecule thereby recruiting that molecule to the nucleic acid whereby the expression control element alters ( e . g . activates ) expression of a gene or cdna comprising the underlying nucleic acid . “ transfection ” is used herein to mean the delivery of expressible nucleic acid to a target cell , such that the target cell is rendered capable of expressing said nucleic acid . it will be understood that the term “ nucleic acid ” includes both dna and rna without regard to molecular weight , and the term “ expression ” means any manifestation of the functional presence of the nucleic acid within the cell , including without limitation , both transient expression and stable expression . the term “ transactivator ” refers to a molecule that induces transcription and / or upregulates transcription of a gene or cdna . the transactivator may be a complete “ native ” molecule or a domain of a molecule that is capable of inducing and / or upregulating transcription of a gene or cdna . “ reporter genes ” are genes or cdnas that express an easily assayable ( detectable and / or quantifiable ) product . detection of the assayable product indicates the expression and / or level of expression of the reporter gene . reporter genes are well known to those of skill in the art . they include , but are not limited to , genes expressing bacterial chloramphenicol acetyl transferase ( cat ), beta - galactosidase (. beta .- gal ), green fluorescent protein ( gfp ) and other fluorescent protein , various bacterial luciferase genes , e . g ., the luciferase genes encoded by vibrio harveyi , vibrio fischeri , and xenorhabdus luminescens , the firefly luciferase gene flux , and the like . the term “ transcriptional activators ” refers to proteins , which activate transcription in yeast , plant , insect and mammalian cells . these proteins contain two parts : one directs dna binding and the other , called the activating region , presumably interacts with some component of the transcriptional machinery . activating regions are typically acidic and require some poorly - understood aspect of structure , probably at least in part an alpha - helix . here in the present invention , the “ transcriptional activator system ” utilized is the one , which is formed by fusing a dna - binding fragment of the yeast activator gal4 to a highly acidic portion of the herpes simplex virus protein vp16 . vp16 activates transcription of immediate early viral genes by using its amino - terminal sequences to attach to one or more host - encoded proteins that recognize dna sequences in their promoters . the full length ask1 cdna cloned in pcmvsport6 vector ( bc054503 ) was purchased from saf labs . we designed the ask1 ( a - f ) overlapping fragments of approximately one 1 kb covering the entire ask1 gene ( fig1 ). the forward and reverse primers are designed for amplifying ask1 fragments as shown in table 1 . nef gene was cloned in vp16pcdna plasmid constructed in lab using pcdna 3 ( invitrogen ) as vector backbone , while the fragment containing the cmv promoter , intron , t7 promoter , nuclear localization signal and multiple cloning region was taken from the mammalian two - hybrid assay vector pact . the resulting construct thus containing the cloned nef gene was called vp16pcdna + pact ( seq id no . 1 ). two sets of constructs were generated from the two vectors , pbind and vp16pcdna + pact : ask1 fragments ask1a ( seq id no . 4 ), ask1b ( seq id no . 6 ), ask1c ( seq id no . 8 ), ask1d ( seq id no . 10 ), ask1e ( seq id no . 12 ), ask1f ( seq id no . 14 ), ask1g ( seq id no . 16 ), ask1h ( seq id no . 18 ), ask1i ( seq id no . 20 ) and ask1j ( seq id no . 22 ) were cloned in pbind vector . hiv - 1 nef gene was cloned in vp16pcdna + pact pcr was performed for each gene using the appropriate 5 ′ and 3 ′ primers ( as given in table 1 ). for cloning of ask1 fragment in pbind vector of mammalian two vectors all primer contains bamh1 restriction site in forward primer and xbal restriction site in reverse primer . all constructs were verified by sanger sequencing in an abi automatic sequencing system ( perkinelmer applied biosystems inc , foster city , calif .). the primers were custom - synthesized by sigma , operon and idt . ask1and hiv - 1 nef ( seq id no . 25 ) clones were cloned in peyfp - n1 ( seq id no . 24 ) ( clontech # 6006 - 1 ) vector for detection of apoptosis and c - jun , p 38 phosphorylation analysis . plasmid was constructed by using primer containing saii and bamhi restriction site in forward and reverse primer respectively for ask1 clone and hindiii , saii in forward and reverse primer respectively for hiv - 1 nef clone . primer sequence are given below in table - 2 hek 293 cells were grown in minimal essential medium ( low glucose , sigma ) supplemented with 10 % fetal bovine serum , penicillin ( 100 units / ml ), streptomycin ( 100 μg / ml ) and gentamycin ( 50 μg / ml ) at 37 ° c . with 5 % co 2 . 1 × 10 5 cells were seeded per well in twenty four well plates , one day before transfection . using commercially available exgen 500 ( fermentas ) transfection reagent , all the required endotoxin free plasmid constructs were co - transfected into hek 293 cells . 0 . 75 microgram of each plasmid and 5 . 0 micro liters per well exgen 500 reagent were used . 48 h post transfection the cells were harvested and luciferase activity was monitored using the dual - luciferase ® reporter assay kit ( promega ). the data for basal control were used for the conversion of luciferase activity to fold activation . as a positive control , the protein - protein interaction vectors pactmyod and pbind - id encoding the myod and id control proteins , provided with the kit were used . the transfected cells were lysed in 1 × passive lysis buffer ( plb ) provided with the kit , and the cell extracts ( containing luciferase enzyme ) were added to the luminometer tubes and the luciferase and renilla rlu were measured on berthold luminometer using the respective substrates for 10s along with 2s delay time . the following examples are given by way of illustration and therefore should not be construed to limit the scope of the invention . to identify the region ( s ) in ask1 interacting with nef using mammalian two - hybrid model the ask1 interacts with nef and inhibits apoptosis ( geleziunas et al 2001 ), however , the region of ask1 that is interacting with nef is not known . to identify which region it is interacting , the ask1 gene approximately 4 . 1 kb , was divided in overlapping fragments of approximately 1 kb from n - terminal to cover entire ask1 gene . these fragments were cloned in pbind mammalian two - hybrid vector , which has gal4 dna binding domain . the ask1 fragment cloned in pbind were ; ask1a ( 1 - 1035 ), ask1b ( 959 - 2010 ), ask1c ( 1820 - 2712 ), ask1d ( 2581 - 3152 ), ask1e ( 3153 - 3552 ) and ask1 f ( 3478 - 4100 ), as shown in fig . ( 1 a ). the full length nef gene was cloned in pact mammalian two hybrid vector , which has all the interacting domains ( accession no . gq184335 ). the mammalian two - hybrid vectors cloned with or without nef and ask1 fragments were co - transfected along with g5luc ( luciferase ) vector in atcc crl - 1573 ™ cells . the reporter luciferase expression was measured after 48 hours of transfection . among the ask1 fragments , ask1a , ask1b and ask1d showed 2 . 28 , 1 . 10 and 2 . 89 fold , respectively , luciferase activity compared to negative control pact - pbind . ask1c , ask1d and ask1f showed no luciferase activity compared to control , as shown in fig1 ( b ) . the myod - id protein was taken as positive control for showing interaction of proteins in mammalian - teo hybrid model , as shown in fig1 c ). in this model the set of vectors which is expressing luciferase gene in cells are considered to be interacting with each other . our results show that ask1a , ask1b and ask1d fragment in presence of nef express luciferase gene which shows that they interact with nef where as other fragments of ask1 does not show any tendency for interaction with nef . in mammalian two - hybrid model , ask1a , ask1b and ask1d fragments showed interaction with nef which indicate that these fragments may have tendency in ask1 for its interaction ability . we characterized the ask1a downstream sequence and ask1d upstream sequence in context to nef interaction . constructs were made based by adding upstream and downstream sequences of ask1a and ask1d respectively . larger constructs which were made are ask1g ( contain both ask1a , ask1d and in between sequence ), ask1h ( contain ask1d and upstream sequence ), ask1i ( contain ask1a and downstream sequence ) and ask1j ( contain in between ask1a and ask1d fragment ), as shown in fig2 a . all the ask1 constructs were cloned in pbind mammalian two - hybrid vectors . the ask1 fragments and nef were co - transfected along with g5luc vector , in atcc crl - 1573 ™ cells and luciferase activity was measured 48 hrs post transfection . the ask1g fragment showed maximum luciferase activity 154 fold times compared to negative control pact - pbind , as shown in fig2 c . the ask1i and ask1h showed luciferase activity 2 . 72 and 8 . 41 fold respectively as compared to negative control , as shown in fig2 b . in ask1h having both ask1a and ask1b fragments showed synergistic effect . the ask1j showed no luciferase activity 0 . 5 fold same as negative control was found ( fig2 b ). these results showed that ask1g is the minimal region of ask1 required for interaction with nef , deletion of either ask1a or ask1d reduced substantially interaction with nef and loss of both fragments from ask1g leads to complete loss of interaction . these results indicate that ask1g fragment interacts with nef through ask1a and ask1d regions . to study the effect of ask1g , h , l , j fragments in inducing apoptosis and its inhibition by nef ask1 expression either by external stress stimuli or over expression of ask1 gene activates apoptosis in cells . ask1 is active in oligomeric state . the n and c region have oligomerization sequence . ask1 is oligomerized with c region coiled coil region and at n - region after release of thioredoxin protein . ask1 catalytic domain ( 670 - 940 ) forms a tight dimmer interacting in a head - to - tail fashion ( bunkoczi . et al 2007 , structure 15 , 1215 - 1226 ). the oligomerization activates self phosphorylation of 838 threonine residue in the kinase domain . nef inhibits apoptosis by interacting with ask1 . the ask1g , h , i , j fragments showing interactions in mammalian two - hybrid model were studied for its function to induce apoptosis in atcc crl - 1573 ™ cells and its inhibition by nef . the ask1g , h , i , j fragments alone and with nef were transfected in atcc crl - 1573 ™ cells . the transfected cells were cultured for 48 hours and were labeled with annexinv and pi staining for analyzing aspoptosis . the ask1g , h , l , fragments induces apoptosis in atcc crl - 1573 ™ cells and percentage positive cells for annexinv staining were 42 , 42 and 45 % respectively compared to control cells . the nef co - transfected with ask1g , ask1h and ask1i showed 20 %, 38 % and 26 % respectively annexin v stained cells that show inhibition of apoptosis by 52 %, 10 % and 42 % respectively . the ask1j has 28 % annexin v stained cells compared to nef control 20 % and after nef co - transfection there is no reduction in apoptosis compared to ask1j , as shown in ( fig3 ). these results showed that all fragments of ask1g , h , i , j induces apoptosis in atcc crl - 1573 ™ cells but the apoptosis is inhibited substantially by nef with the ask1g and ask1i fragment . inhibitors were designed from the crystal structure of nef solved in our laboratory ( pankaj et al ., 2011 ) the inhibitors were cultured with cells transfected will nef , ask1g and co - transfected with nef - ask1g . the cells after 48 hours were analysed for apoptosis using annexin v and pi staining . the results show that the nef - ask1 expression in atcc crl - 1573 ™ cells inhibited apoptosis by 50 % compared to ask1 alone . the inhibitors reversed the effect of nef - ask1 interaction that is inhibition of apoptosis by increasing annexin v stained cells same to ask1 cells fig4 . this result suggests that nef - ask1 co - transfected in cells show inhibition of apoptosis and is reversed by inhibitors . effect of inhibitors in restoring apoptosis by reversing jnk phosphorylation inhibited by nef - ask1g interaction in atcc crl - 1573 ™ cells ask1 mapkkk induces apoptosis by jnk and p38 pathway . to identify that the minimal ask1g fragment can activate jnk and p38 pathway to induce apoptosis and the presence of nef can affect this pathway . the ask1 g and nef were transfected alone and co - transfected in atcc crl - 1573 ™ cells . the jnk and p38 pathways were detected by analyzing phosphorylation of jnk or p38 kinases by using phosphorylated antibodies . the results showed that ask1g fragment activated jnk pathway by phosphorylated jnk kinase where as late effect was seen in p38 kinase ( data not shown ). further in co - transfected cell with nef , inhibits the phosphorylation of jnk kinase whereas in presence of peptide the jnk phosphorylation is reversed , same as ask - 1 , inhibited by nef - ask - 1 interaction ( fig5 ). densitometry analysis showed that nef decrease phosphorylation of jnk kinase 2 fold compare to ask1g transfected cells ( fig5 ). nef alone transfected cell show no phosphorylation of jnk kinase compared to control . these results suggest that ask1g activates apoptosis by jnk pathway and this pathway is inhibited with the presence of nef in atcc crl - 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