human immunodeficiency 
 virus type 1 ( hiv-1 ) , the causative agent 
 of acquired immune deficiency syndrome ( aids ) , remains a significant 
 challenge to global health . since its initial identification 
 in 1983
, hiv-1 infection has reached the status of a pandemic . in 
 2009 alone , there were approximately 2.7 million new infections and 
 about 2.0 million deaths from aids related causes .
currently there is no cure for hiv-1 infection . while progression 
 of the disease can be controlled by highly active antiretroviral therapy 
 ( haart ) , a combination of drugs designed to inhibit different stages 
 in the virus life cycle , the complexity 
 of the haart regimen , and the ability of the virus to evolve resistance 
 suggest that alternative drug targets for hiv-1 treatment and prophylaxis 
 are needed .
one potentially attractive 
 target for pharmacological interference 
 in the hiv-1 life cycle is the virus requirement for a programmed 
 1 ribosomal frameshift ( 1 prf ) in order to express 
 its enzymes .
ribosomal frameshifting is 
 a recoding mechanism common among viruses with polycistronic ( multiple 
 open reading frames , or orfs , in a single gene ) genomes .
it allows 
 viruses to translate polypeptides in different orfs by avoiding the 
 stop codon(s ) present in the single mrna transcript . in hiv-1 ,
gag , the precusor of the viral structural 
 proteins , is produced via normal translational rules , while pol , the 
 precursor of the viral enzymes , is synthesized as a fused gag - pol polyprotein via 1 prf .
this occurs with a frequency 
 of 510% of ribosomes translating the full - length viral mrna . 

a critical molar ratio of gag - pol to gag protein is required for hiv-1 
 replication and infectivity ; alterations to this ratio have been shown 
 to be detrimental .
1 prf in hiv-1 
 is controlled by two cis acting mrna elements : a 
 heptameric slippery sequence ( u uuu uua ) , with the 0 frame indicated 
 by spaces and where the frameshift actually occurs , and a downstream 
 two - stem helix immediately following the slippery sequence , also known 
 as the frameshift stimulatory signal ( hiv-1 fss , figure 1 ) . while several mechanisms have 
 been proposed to account for the frameshift , it is currently hypothesized that this event results from an incomplete 
 translocation for a limited number of ribosomes , due to resistance 
 of the fss to unwinding .
modification 
 of the slippery site or stimulatory sequence ( either via natural variation 
 or laboratory mutations ) in ways affecting frameshifting efficiency 
 translates to a decrease in viral replication .
these and other results have led several groups to propose 1 
 prf as a potential target for developing antiretroviral agents for 
 hiv-1 .
( left ) proposed secondary structure 
 of the hiv-1 fss , supported by nmr structural studies .
( right ) htlv-2 
 fss stem - loop sequence used as a specificity control in this work . 

note that when the slippery sequence occupies the decoding site of 
 the ribosome , the lower stem is unwound and it is the upper stem - loop 
 that acts as the effective frameshift stimulatory signal .
nmr structural analyses indicate that the hiv-1 
 fss rna consists 
 of a g - c rich upper stem - loop structure , separated from a flexible lower stem by a gga trinucleotide bulge 
 ( figure 1 ) .
the bulge 
 produces a roughly 60 bend between the upper and lower stems . 

this stability is believed 
 to play a vital role in the stimulation of the frameshift , since the 
 ribosome must unwind the stem during translation .
the highly structured acaa tetraloop 
 is uncommon among tetraloops but is conserved 
 among all hiv-1 group m subtypes except the uncommon h and j subtypes . 

likewise , the heptameric slippery sequence is conserved across all 
 hiv-1 group m subtypes .
shape analysis of the intact hiv genome suggests 
 a more complex structure for the fss rna , although the upper stem - loop 
 is retained . since
other viruses also 
 rely on frameshifting , targeting frameshift - regulating 
 structures may have general 
 utility beyond the context of hiv .
for example , human t - cell leukemia 
 virus type 2 ( htlv-2 ) uses two 1 prf events similar to hiv-1 
 in order to synthesize fused gag - pro and gag - pro - pol precursor proteins .
the rna responsible for the 1 prf essential 
 for expression of gag - pro in htlv-2 also consists of two cis - acting rna elements , a heptanucleotide ( aaaaaac ) slippery site and 
 a stem - loop shown in figure 1 .
this htlv-2 fss served as a sequence specificity control 
 in the experiments we describe herein .
the first attempted use 
 of a synthetic molecule to alter hiv-1 
 frameshifting and thereby influence viral replication was reported 
 by green and co - workers in 1998 .
the 
 authors showed that 1,4-bis[n-(3-n , n - dimethylpropyl)amidino]benzene tetrahydrochloride , 
 a bis guanidinium - containing compound termed 
, was able to stimulate 1 frameshifting , 
 alter the 
 gag - pol : gag ratio , inhibit hiv-1 replication in cem cells ( a lymphocytic 
 cell line ) , and interfere with the formation of viral particles in 
 chronically infected ch-1 cells ( a cos cell line stably transfected 
 with hiv - gpt , an hiv derivative in which the e. coli gpt gene replaces hiv env(28 ) )
. increases in reverse transcriptase ( rt ) following treatment with 
 1.5 mm rg501 were also observed , as would be 
 expected for an increase in gag - pol production .
recently , the butcher 
 group confirmed that this compound indeed binds the hiv-1 fss rna 
 with weak affinity ( kd  360 m ) , 
 and they carried out nmr structure analysis , indicating that rg501 binds in the major groove of the upper stem - loop .
unfortunately , as noted by the authors , rg501 is a relatively nonselective binder and interacts 
 with other rnas .
it stimulates frameshifting in viruses with different 
 fss , and likely that also interacts with 
 the ribosome.rg501 is also toxic , a likely result of its 
 lack of selectivity .
moreover , its interference with hiv replication 
 begins at concentrations below those observed to affect frameshifting .
other compounds such as guanidinoneomycin , idarubicin , and doxorubicin have been 
 shown to bind the hiv-1 fss .
doxorubicin was found to bind with a kd of 2.8 m , decreased frameshifting in 
 a rabbit reticulocyte assay , and also significantly reduced overall 
 translation .
a screen of an arg - rich peptide library revealed a sequence 
 able to significantly reduce frameshifting , but this displayed no 
 selectivity for the hiv-1 fss relative to other frameshift - stimulating 
 constructs and likely also interacts with the ribosome .
thus , as far as we are aware , there are no reported 
 examples of synthetic molecules able to alter hiv-1 frameshifting 
 and interfere with viral infectivity via selective , high - affinity 
 binding to the fss rna .
building on our laboratory s 
 longstanding interest in understanding 
 the factors that drive affinity and sequence selectivity in small 
 molecule recognition of rna , we previously 
 reported the use of an 11,325-member resin - bound dynamic combinatorial 
 library ( designed based on the structure 
 of dna - binding , bisintercalating peptide antibiotics ) to identify 
 a compound ( 1 ) able to bind the hiv-1 fss upper stem - loop with moderate 
 affinity ( kd = 4.1  2.4 m 
 immobilized on an surface plasmon resonance ( spr ) chip via one of 
 its amine groups ; kd = 350  110 
 nm in solution as measured by fluorescence ) and good selectivity .
subsequent efforts 
 revealed that replacement of the disulfide bridge with an olefin ( to 
 produce compounds 2 and 3 ) could be accomplished without any 
 reduction in affinity .
these studies also indicated that affinity 
 was essentially abolished if the -surface area of the molecule 
 was reduced , while the peptidic portion of 1 was required for sequence - selective binding .
while the binding ability of 2 and 3 was intriguing , 
 subsequent 
 preliminary experiments indicated the compounds were unable to inhibit 
 virus in a pseudotyped hiv-1 assay ( data not shown ) .
given these results , 
 we hypothesized that further increases in affinity were essential . 

we anticipated that increasing the  surface area of 1 could represent a viable strategy 
 for enhancing the affinity for the hiv-1 fss without significant reductions 
 in selectivity .
in particular , incorporation of a benzo[g]quinoline moiety to produce 4 and 5 was viewed as attractive .
this hypothesis was supported in part by parallel efforts 
 in our laboratory on the design and synthesis of compounds targeting 
 cug repeat rna in which incorporation of a benzo[g]quinoline was found to enhance affinity and to provide compounds 
 with activity in vivo in a mouse model of type 1 myotonic dystrophy .
synthesis of 4 and 5 proceeded via cross - metathesis 
 of half - structure 6 , by analogy 
 to our previous work .
we also synthesized 7 and 8 (  one - armed  
 benzo[g]quinoline - bearing structures ) , as well as 
 compounds 9 and 10 , to test the effect of sequential removal 
 of the putative intercalators .
compound 11 was synthesized in order to ascertain the effectiveness of 
 the olefin bioisostere in improving cellular availability and bioactivity 
 relative to an easily reduced disulfide .
synthesized compounds were first analyzed for binding to the hiv-1 
 fss by surface plasmon resonance ( spr ) .
this technique allows the 
 equilibrium constant ( kd ) and kinetic 
 rate constants ( kon , koff ) to be determined in a label - free format .
a 5-biotin labeled hiv-1 fss rna upper 
 stem - loop ( sequence as in figure 1 ) was immobilized 
 on a streptavidin - functionalized sensor chip .
compound solutions in 
 hbs buffer ( 0.01 m hepes , 0.150 m nacl , ph = 7.4 ) were flowed over 
 the rna , and the reference - subtracted sensorgrams were recorded .
the 
 associative and dissociative phases of the experimental sensorgrams 
 for at least five different concentrations were globally fit to a 
 1:1 langmuir equation to obtain the binding constants . under these conditions , benzo[g]quinoline - containing 
 compounds
4 and 5 bound the hiv-1 fss with affinities ( kd ) of 89 nm and 102 nm , respectively , in each 
 case representing an approximately 50-fold increased affinity over 2 and 3 ( table 1 ) .
binding by benzo[g]quinoline containing monomer 6 was 200-fold weaker relative to that of the dimers 4 and 5 .
 also resulted 
 in an approximately 200-fold decrease in affinity for 7 and a 30-fold decrease for 8 , while removal of both benzo[g]quinoline groups completely abrogated binding between 10 and the hiv-1 fss rna , consistent with our 
 prior results , indicating the importance of the heterocyclic group 
 for affinity .
disulfide linked compound 11 bound the target rna with a kd of 0.741 m , which is 7-fold weaker relative to the 
 olefin analogs 4 and 5 .
no binding was observed between the 2-ethyl 
 benzo[g]quinoline carboxylic acid and the hiv-1fss 
 by spr .
confirming their anticipated selectivity for the hiv-1 fss , 
 compounds 410 showed no binding to the htlv-2 fss by spr 
 at concentrations up to 3 m ( supporting 
 information p s12 ) .
conditions : ( a ) hbs buffer 
 ( 0.01 m hepes , 0.150 m nacl , ph = 7.4 ) ; ( b ) 20 mm hepes , 150 mm nacl , 
 5 mm mgcl2 , and 0.005% tween-20 . the dissociation phase for spr traces obtained at 
 the highest concentrations 
 for compounds 4 and 5 were not well fit by a single - exponential function . 

to control for the possibility of compound aggregation interfering 
 with the measurement , we therefore reexamined 
 these compounds using a running buffer containing detergent ( 20 mm 
 hepes , 150 mm nacl , 5 mm mgcl2 , and 0.005% tween-20 ) .
this 
 had only a marginal impact on the fitted kinetic parameters ( table 1 ) , suggesting aggregation is not a significant complicating 
 factor in the assay .
an spr chip with a very low density of rna provided 
 similar dissociation constants ( supporting information ) . to confirm thermodynamic dissociation constants measured 
 by spr 
 with a fully solution - phase method , and to further assess sequence 
 selectivity ,
saturable quenching of benzo[g]quinoline fluorescence 
 was observed as a function of added unlabeled rna .
dissociation constants 
 obtained in this manner ( table 2 ) for 4 and 5 binding the hiv-1 fss rna are in general agreement with those 
 obtained via spr . by fluorescence
we observe a 5-fold selectivity 
 for 4 binding the fss rna vs total 
 yeast trna , while selectivity for fss rna vs fss dna was 4-fold .
the 
 selectivity of 5 for fss rna vs 
 trna was somewhat lower ( 2.14-fold ) .
curiously , unlike 4 , 5 had 
 no selectivity for fss rna vs fss dna .
thus , as 2 and 3 were previously 
 found to have no measurable affinity for trna , we conclude that the 
 increase in affinity obtained on replacement of the quinoline moieties 
 of 2 and 3 with benzo[g]quinoline does come 
 with a cost of some decrease in selectivity .
this result is in contrast 
 to what was observed in a related series of compounds optimized for 
 binding triplet repeat rna sequences relevant to type 1 myotonic dystrophy .
it is likely that these differences have a structural 
 foundation , and further experiments will be needed to explore this 
 hypothesis .
reported error is the standard 
 deviation on each measurement taken in triplicate with 1 min spacing 
 between measurements . before analyzing the effect 
 of compounds on hiv-1 frameshifting in cells
, we verified that they 
 were capable of crossing cell membranes , and not toxic at reasonable 
 concentrations .
human embryonic kidney cells ( hek 293 ft ) were treated 
 with 4 , 5 , and 11 at concentrations 
 of 50 , 100 , 200 , and 500 m for 12 h. cells were then washed 
 using standard methods to ensure that 
 cell staining was not simply due to surface capture .
the inherent 
 fluorescence of the benzo[g]quinoline chromophore 
 in these compounds allowed direct visualization of cell penetration 
 via fluorescence microscopy ( figure 2 shows 
 results for 50 m treatment ; additional images are provided 
 in the supporting information ) , confirming 
 that cells internalized all three compounds .
compounds did not appear 
 to localize in any specific subcellular structures but were visible 
 throughout the cytoplasm and nucleus .
. 
 bright - field ( gray ) and false - color 
 fluorescence images of compound 4 , 5 , and 11 incubated with hek 293 ft cells at a concentration 
 of 50 m .
compounds were 
 incubated with hek 293 ft cells cultured 
 in dmem for 24 h , after which a 10:1 media to wst-1 cell proliferation 
 reagent mixture replaced the growth media for 2 h. absorbance was 
 then measured at 450 and 690 nm .
disulfide linked compound 11 proved toxic at concentrations above 
 10 m , while no significant toxicity for compounds 4 and 5 occurred 
 at concentrations below 60 m ( figure 3 ) .
the higher toxicity observed for 11 relative to 4 and 5 is not surprising and likely reflects 
 the lability of the disulfide bond in the reducing environment of 
 the cell .
toxicity of hiv-1 fss ligands in hek 293 ft cells , assessed via 
 wst-1 cell proliferation assay .
results are shown for exposure to 4 ( blue triangles ) , 5 ( green triangles ) , or 11 ( red circles ) , in comparison to addition of buffer 
 alone ( black squares ) .
compounds were next evaluated for 
 their ability to alter hiv-1 fss - dependent 
 frameshifting , using a dual - luciferase reporter assay . in this system , the fss sequence has been inserted 
 between renilla ( rluc ) and firefly ( fluc ) luciferase 
 genes .
several constructs were employed . in the pdualhiv(1 ) 
 construct , fluc is in the 1 reading frame relative to rluc , 
 while , in the pdualhiv(0 ) construct , fluc is in the 0 reading frame 
 relative to rluc . in both cases ,
fluc is expressed only as a fused 
 rluc - fluc protein , but in the case of pdualhiv(1 ) , a 1 
 frameshift is required . to ascertain
if compound - dependent effects 
 on frameshifting are specific to hiv-1 , we also carried out an analogous 
 1 prf assay using constructs in which the htlv-2 frameshift 
 site was inserted between rluc and fluc genes , such that fluc is only synthesized 
 by a 1prf event [ pdualhtlv-2 ( 1 ) ] .
hek 293 ft cells were transiently transfected separately 
 with pdualhiv(1 ) and pdualhiv(0 ) plasmids . the frameshift 
 efficiency ( defined as the ratio of firefly to renilla luciferase activities ) in the pdualhiv(1 )
transfected cells 
 was measured in the presence of varying concentrations of compounds 
 ( 050 m ) . a statistically significant ( p < 0.005 ) dose - dependent increase in fluc to rluc ratios ,
> 50% 
 at 50 m , was observed following treatment with 4 or 5 ( figure 4 ) .
control compound 10 , which showed no measurable binding to the hiv-1 fss by spr , 
 had no significant effect on 1 prf in pdualhiv(1 ) 
 transfected cells under similar conditions . in hek
293 ft cells transfected 
 with the pdualhiv(0 ) plasmids , the fused renilla - firefly 
 luciferase protein is synthesized by conventional translation rules , 
 i.e. without ribosomal frameshifting .
therefore , to confirm that the 
 observed effect in pdualhiv(1 ) cells is a direct effect of 
 compound on frameshift - dependent translation , pdualhiv(0 ) transfected 
 cells were treated analogously with compounds 4 and 5 .
no change 
 in fluc to rluc ratio relative to untreated pdualhiv(0 ) transfected 
 hek 293 ft cells was observed , suggesting that 4 and 5 specifically 
 stimulate 1 frameshift translation in cells .
disulfide linked 11 stimulated hiv-1 frameshifting by 
 approximately 25% ( supporting information ) ; this lower effect compared to the cases of 4 and 5 is consistent 
 with its lower affinity to the hiv-1 fss and likely lability in the 
 cell .
treatment of hek 293 ft transfected with the pdualhtlv-2 ( 1 ) 
 construct with compounds 4 , 5 , or 11 resulted in no change in fluc / rluc ratio compared to the 
 case of untreated cells .
this is consistent with in vitro spr results 
 in which no binding was observed between these compounds and the htlv-2 
 fss rna .
compounds 4 and 5 increase frameshifting ( > 50% ) in a dual - luciferase 
 assay incorporating the hiv-1 fss but have no effect on frameshifting 
 in analogous assay incorporating the htlv-2 fss .
relative frameshift 
 efficiency in hek 293 ft cells treated with 4 , 5 or control peptide 10 after transfection with pdualhiv 
 ( 1 ) or pdualhtlv-2 ( 1 ) .
the frameshift efficiency 
 was calculated as the ratio of fluc to rluc in pdualhiv(1 ) 
 or pdualhtlv-2 ( 1 ) transiently transfected cells .
this ratio 
 was arbitrarily set to 100% for plasmid - transfected cells , but not 
 exposed to compounds .
error is sem on three replicates for each concentration . in order for the observed 1 frameshift 
 effect of compounds 
 to be significant , it was important that it correlate to an equivalent 
 reduction in viral replication . to assess this
, we analyzed the effect 
 of compounds in a single - round infection with pseudotyped hiv in hek 
 293 t producer cells and tzm - bl target cells ( a hela cell line ) .
the wild type hiv proviral vector ( pdhiv3-gfp ) 
 codes for all hiv-1nl43 genes except nef ( which is replaced with gfp ) and env , thus preserving gag and pol , and the frameshift required 
 for production of the gag - pol polyprotein .
however , we observed 
 a statistically significant ( p < 0.001 ) and concentration - dependent 
 decrease in infectivity of the pseudotyped hiv-1 virions when producer 
 cells were treated with 4 and 5 ( figure 5 ) . 

the decrease in infectivity was > 90% at a concentration of 20 m 4 , and > 90% at a concentration of 
 40 
 m for 5 .
the core peptide 
 alone ( 10 ) showed a modest decrease 
 in infectivity at a concentration of 40 m , likely via nonspecific 
 effects , given its lack of affinity for the fss .
the protease 
 inhibitor indinavir was tested in parallel to calibrate the assay ; 
 its ec50 was found to be 14.8 nm , in line with previously 
 reported values .
treatment of viral producer 
 cells with compounds 4 and 5 yields 
 a strong inhibition of the infectivity of pseudotyped hiv-1 virions 
 into tzm - bl reporter cells .
infectivity is measured as relative luminescence 
 from the stably expressed firefly luciferase expressed via the hiv 
 ltr promoter in the tzm - bl cell line .
error bars indicate standard 
 deviation on the mean ( n = 3 ) . while virus titer was determined in these experiments using 
 an 
 elisa assay to normalize viral load into target cells , a compound - dependent 
 decrease in viral production was also readily observable via fluorescence 
 microscopy , as the pseudotyped hiv carries gfp as a marker ( figure 6 ) .
phase - contrast images of these cells showed no 
 morphological changes , consistent with wst-1 results ( supporting information ) .
the concentration range 
 required for a decrease in viral infectivity by 4 and 5 was similar 
 to the range at which a significant increase in 1 frameshift 
 was observed in hek 293 ft cells , supporting the hypothesis that these 
 compounds exert their antiviral activity primarily by altering 1 
 prf . in order to provide further support for this hypothesis , viral 
 particles were isolated from supernatants from these experiments by 
 spinning through a 20% sucrose cushion , and probed via western blot 
 for the presence of reverse transcriptase ( rt ) . as rt is produced 
 only as part of the gag - pol fusion , increasing amounts of rt relative 
 to capsid protein p24 ( a structural protein produced as part of gag ) 
 would be required given an increase in frameshifting .
indeed , that 
 is what is observed as a function of added 4 or 5 ( figure 7 ) .
the amount of rt in unprocessed gag - pol ( p160 ) 
 relative to mature rt ( p66 + p51 ) also increased significantly ; this 
 may indicate that treatment with compounds 4 and 5 also inhibits pol 
 processing .
however , the pattern of p160 cleavage intermediates observed 
 on treatment with indinavir is significantly different , indicating 
 that 4 and 5 likely do not directly interact with viral protease 
 ( supporting information p s19 ) .
compound - dependent 
 changes in viral production may be observed 
 directly via fluorescence microscopy .
proviral expression of gfp is 
 reduced in the presence of compounds 4 and 5 compared to 10 and the untreated control ( 40 mm 
 concentration shown ) .
images were acquired 24 h after treatment prior 
 to harvesting viral particles for tzm - bl single - cycle infectivity .
equal 
 loads of viral particles ( measured by p24 elisa ) were isolated from 
 media of viral producer cells by spinning through a 20% sucrose cushion 
 at 100,000 g .
the viral particles were western blotted 
 for rt ( abcam ) and p24 ( nih aids reagent program , cat # 3537 ) .
densitometry 
 of nonsaturated bands was used to calculate ratios for p160/(p66+p51 ) 
 ( listed below the rt blot ) and the ratio of total ( all bands ) rt / p24 
 are ( listed below the p24 blot ) .
designing small molecules 
 that target specific rna sequences and 
 elicit a desired rna mediated biological response constitutes one 
 of the signature challenges in chemical biology . in this paper , we have demonstrated that a moderate - affinity  hit  

compound for the hiv-1 fss rna , obtained from a resin - bound dynamic 
 combinatorial library screen , can be transformed into high - affinity 
 binders .
an intriguing observation from our initial screen was the finding that a symmetrical compound 
 was selected , despite the target rna being nonsymmetrical .
this work 
 confirms that both putative intercalators are required for high - affinity 
 binding , as compounds 7 and 8 had only weak affinity for the fss
. 
 structural analysis will be essential to fully understand the binding 
 mode of 4 and 5 , but the recognition of a nonsymmetrical rna 
 binding site by a symmetrical , dimeric molecule is not unprecedented ; 
 for example , arya and colleagues have described the use of neomycin 
 dimers for recognition of hiv tar rna , and the hergenrother group designed deoxystreptamine dimers that 
 bound nonsymmetrical rna loops .
likewise , 
 structural information should prove useful for understanding the significantly 
 higher selectivity of 4 for fss 
 rna over fss dna and trna relative to 5 .
these enhanced compounds are able to alter frameshifting 
 in a dual 
 luciferase assay in hek 293 ft cells and strongly inhibit viral infectivity 
 in a pseudotyped hiv assay .
relative to previously studied compounds 
 targeting frameshifting in hiv , compounds 4 and 5 produce a roughly 
 equivalent increase in rt at a 37.5-fold lower concentration than rg501 . as such , compounds 4 and 5 represent promising 
 leads for further study , as well as interesting tools to further investigate 
 the mechanism of 1 prf .
since the pseudotyped hiv used in 
 this assay is only capable of one round of replication , we anticipate 
 that stronger effects will be observed with wild - type hiv in a spreading 
 infection assay .
of course , further enhancements in affinity and selectivity 
 would likely improve activity as well . as discussed above , previous 
 high - throughput screening efforts targeting frameshift - modulating 
 compounds in which bicistronic reporters provided the assay readout 
 largely yielded compounds acting on the ribosome . given this observation , 

it has been suggested by others that assays focused on direct binding 
 to the fss rna could be advantageous , as such compounds could potentially 
 increase frameshifting by inhibiting unwinding of the upper stem - loop .
the results presented herein support this hypothesis . 
 in the context of hiv biology ,
our results with compounds 4 and 5 indicate that an increase in frameshifting and a concomitant 
 increase in the gag - pol / gag ratio decreases viral fitness .
this is 
 consistent with the work of mak and colleagues , who increased the 
 gag - pol / gag ratio via cotransfection .
commercially 
 available reagents were obtained 
 from sigma - aldrich chemical co. ( st . louis , mo ) , tci america ( portland , 
 or ) , fisher scientific , emd chemicals ( gibbstown , nj ) , advanced chemtech 
 ( louisville , ky ) , and alfa aesar , and were used without further purification 
 unless otherwise noted .
water used for reactions and aqueous workup 
 was glass - distilled from a deionized water feed .
reaction progress was monitored 
 by analytical thin - layer chromatography ( tlc ) using em silica gel 
 60 f-254 precoated glass plates ( 0.25 mm ) .
compounds were visualized 
 on the tlc plates with a uv lamp ( dual wavelength ;  = 254 nm , 
  = 360 nm ) .
synthesized compounds were purified using flash 
 column chromatography on em silica gel 60 ( 230400 ) mesh or 
 alternatively via preparative reversed phase hplc .
cells were cultured 
 in dulbecco s modified eagle s medium ( dmem ) , supplemented 
 with 10% fbs and 1% penicillin1% streptomycin . premixed wst-1 
 cell proliferation reagent was purchased from clontech , and luminescence 
 assays were carried out using a promega dual - luciferase assay kit 
 following manufacture s instructions .
h nmr spectra were recorded at 
 25 c on either a bruker avance 400 ( 400 mhz ) or bruker avance 
 500 ( 500 mhz ) instrument and processed using mestrenova nmr processing 
 software .
chemical shifts (  ) are reported in parts per million 
 ( ppm ) downfield from tetramethylsilane and referenced to the residual 
 protium signal in the nmr solvents .
data are reported as follows : 
 chemical shift , multiplicity ( s = singlet , d = doublet , t = triplet , 
 m = multiplet , and q = quartet ) , coupling constant ( j ) in hertz ( hz ) , and integration .
c spectra were recorded 
 at 25 c on a bruker avance 500 instrument operating at 126 mhz . 

chemical shifts (  ) are reported in ppm downfield from tetramethylsilane 
 and referenced ( except in d2o ) to the primary carbon resonance 
 in the nmr solvent .
high - resolution mass spectra ( hrms ) were acquired 
 at the university of buffalo chemistry department mass spectrometry 
 facility , buffalo , ny . compounds 
 were synthesized following 
 procedures analogous to those previously reported .
all compounds were produced in > 95% purity , as determined by 
 analytical 
 hplc .
briefly , 4 and 5 were synthesized 
 from olefin precursor monomer 6 , which was assembled 
 on wang resin by standard fmoc solid phase peptide synthesis ( spps ) 
 methods .
one half of the resin - bound monomer was cleaved using 50% 
 tfa in dcm with 1% tes , and was used as solution phase partner in 
 an olefin metathesis reaction employing grubbs second generation 
 catalyst .
the olefin products ( isomer ratio of z / e = 2:3 ) were isolated using reverse phase hplc on a c18 
 column ( waters , xbridge prep c18 5 m obd , 19 mm  250 
 mm ) using gradient elution from 5 to 100% acetonitrile/0.1% tfa in 
 water/0.1% tfa .
olefin geometries were assigned by comparing the olefin 
 proton chemical shifts of the e - olefin downfield 
 to the z - isomer and also by the infrared spectra of the compounds 
 as described previously .
compounds 7 and 8 were synthesized by a slight modification 
 to the procedures described above .
the tripeptide ( phe - pro - allylgly ) 
 was first assembled using fmoc peptide coupling chemistry on wang 
 resin .
2-ethyl benzo[g]quinoline carboxylic acid 
 was coupled to half the material and cleaved with tfa .
this was then 
 employed as the solution phase component in olefin cross - metathesis 
 with the remaining bead - bound portion of fmoc - protected tripeptide .
surface 
 plasmon resonance ( spr ) experiments were performed on a biacore - x 
 instrument ( biacore , inc . , uppsala , sweden ) on a cm5 sensor chip . 
 approximately 2000 ru of streptavidin ( rockland immunochemicals ) was 
 immobilized in both flow cells using edc / nhs chemistry . 5-biotinylated - rna 
 ( either 500 nm 5-biotin - hiv-1 fss or 500 nm 5-biotin - htlv-2 
 fss rna sequences , obtained commercially from integrated dna technologies 
 inc . )
was captured onto the streptavidin surface in one flow cell 
 to a density of approximately 3001200 ru .
the streptavidin 
 surface in the second flow cell was then blocked with biotin solution 
 and served as a reference cell to correct for any nonspecific binding . 

binding constant measurements were carried out for each compound once 
 on a  low density  ( approximately 300 ru ) chip and once 
 on a  high - density 
( approximately 1200 ru ) chip in 
 order to ensure binding was not influenced by rna density .
kinetic 
 binding experiments were carried out by flowing various concentrations 
 of compound in ( a ) hbs buffer ( 0.01 m hepes , 0.150 m nacl , ph = 7.4 ) 
 at a 60 l per min flow rate or ( b ) 20 mm hepes , 150 mm nacl , 
 5 mm mgcl2 , and 0.005% tween-20 at a 50 l per min 
 flow rate over the captured rna sequence .
where necessary , a 20 s , 
 0.5 or 1 m aqueous nacl injection was sufficient for regeneration 
 ( compounds displaying fast off - rates did not require regeneration 
 of the chip between injections ) .
binding constants were obtained by 
 global fit ( conditions ( a ) ) of association and dissociation phases 
 of at least five referenced - subtracted and blank - corrected sensorgrams 
 to a 1:1 langmuir binding equation , or via individual fits ( conditions 
 ( b ) using biaevaluation software ) .
fluorescence titrations were 
 carried out in 20 mm hepes , 150 mm nacl , using a cary eclipse spectrofluorometer . 

all titrations started at a volume of 500 l with the compound 
 at 1 m .
rna was then titrated in from a high - concentration 
 stock ( 10 m for hiv-1 fss rna ; 40 m for hiv-1 fss dna 
 and trna ) in 110 l increments . after rna
was added , 
 the solution was thoroughly mixed in the cuvette via pipetting and 
 allowed to stand for 10 min to reach equilibrium .
each measurement 
 was taken three times with a 1 min waiting period between scans to 
 confirm equilibrium was reached .
hek 293 ft cells grown to 80% confluence 
 at 37 c and 5% co2 were exposed to compounds at a 
 concentration of 50 m for 12 h in a 96-well tissue culture 
 plate .
after removal of the culture media [ dmem containing 10% fbs , 
 1% penicillin - streptomycin ( gibco ) ] , the cells were washed twice with 
 pbs to remove excess and surface - bound compounds .
cells were then 
 imaged while in buffer under a fluorescence microscope ( olympus ix70 ) 
 in the 96-well plate using 358-nm excitation and 460-nm emission filters .
hek 293 ft cells were plated in a 96-well 
 tissue culture plate in dmem ( 10% fetal bovine serum , 1% penicillin - streptomycin ) 
 and allowed to grow to 80% confluence at 37 c under co2 .
varying compound concentrations ( up to 0.5 mm ) and control ( identical 
 volumes of sterile h2o ) in triplicate were incubated with 
 cells for 24 h at 37 c .
ten microliters of premix wst-1 cell 
 proliferation reagent ( clontech ) was added to each well including 
 blanks ( dmem ) , followed by a 2 h incubation at 37 c .
hek 293 ft cells were plated in 96-well plates at densities of 1.5 
  10 cells / well 6 h before transfection in dmem ( 10% 
 fetal bovine serum , 1% penicillin - streptomycin ) .
cells were transiently 
 transfected in separate wells with 0.2 g of plasmid dna ( pdualhiv(0 ) , 
 pdualhiv(1 ) , pdualhtlv-2(0 ) , or pdualhtlv-2(1 ) ) , using 
 lipofectamine 2000 transfection reagent ( invitrogen ) and following 
 the manufacturer s protocol .
five hours after transfection , 
 various compound concentrations ( 050 m ) were added 
 directly to the cells in triplicate and incubated for 36 h at 37 c . 

the culture media was gently aspirated , washed with 1 pbs , and 
 lysed with 200 l 1 passive lysis buffer .
rluc and fluc 
 activities were measured with 5 l of cell lysate and 25 l 
 of luciferase reagent using the dual - luciferase assay system ( promega ) 
 in the same wells .
fluc and rluc luminescence values were measured 
 on a modulus microplate reader ( turner biosystems ) .
relative frameshift 
 efficiencies were calculated by comparing the fluc / rluc luminescence 
 ratio of cells treated with compounds to untreated cells .
the antiviral activity of 4 , 5 , and 10 was measured by single - round 
 infectivity assay with pseudotyped hiv-1 using hek293 t producer cells . 

the hiv-1 proviral vector ( pdhiv3-gfp ) codes for all hiv-1nl43 genes except nef ( replaced with gfp ) and env , thus preserving gag and pol , and the frameshift required for production of the gag - pol polyprotein . 

a single - round infectivity assay was conducted by transient transfection 
 of the viral vector with vsv - g coat protein vector at a ratio of 1:0.5 
 using fugene hd ( promega ) .
the virus producer cells were dosed with 
 compounds four hours after transfection , and viral particles were 
 harvested from the media 24 h after transfecting by filtering through 
 a 0.45-m syringe filter .
the infections were performed using tzm - bl 
 reporter cells that contain stably integrated firefly luciferase that 
 is driven by the hiv - ltr promoter .
triplicate 
 infections in 96-well plates at 10,000 cells / well with 500 pg p24/well 
 proceeded for 48 h before the addition of steadyglo reagent ( promega ) 
 to each well for 30 min .
luminescence was measured as a quantitative 
 metric for changes in viral infectivity in the presence of compound .
 the 
 life cycle of the human immunodeficiency virus type 1 ( hiv-1 ) 
 has an absolute requirement for ribosomal frameshifting during protein 
 translation in order to produce the polyprotein precursor of the viral 
 enzymes . while an rna stem - loop structure ( the  hiv-1 frameshift 
 stimulating signal  , or hiv-1 fss ) controls the frameshift 
 efficiency and has been hypothesized as an attractive therapeutic 
 target , developing compounds that selectively bind this rna and interfere 
 with hiv-1 replication has proven challenging . building on our prior 
 discovery of a  hit  
 molecule able to bind this stem - loop , 
 we now report the development of compounds displaying high affinity 
 for the hiv-1 fss . 
 these compounds are able to enhance frameshifting 
 more than 50% in a dual - luciferase assay in human embryonic kidney 
 cells , and they strongly inhibit the infectivity of pseudotyped hiv-1 
 virions . 
Introduction
Results and Discussion
Discussion and Conclusions
Experimental Section