owing to its pivotal roles in hiv-1 replication 
 ( structural and 
 regulatory ) , the capsid ( ca ) protein has gained attention as a promising 
 therapeutic target .
moreover , the seminal finding that retroviral 
 species - specific host cell restriction factors target the incoming 
 capsid core highlights the enormous therapeutic potential of targeting 
 the capsid protein .
the hiv-1 ca , which 
 is translated as the central region of the gag polyprotein , has primary 
 functions in viral assembly and in packaging the cellular protein 
 prolyl isomerase , cyclophilin a ( cypa ) . after the capsid protein has been liberated by proteolytic processing , 
 it rearranges into the conical core structure that surrounds the viral 
 genome at the center of the mature virus .
the hiv-1 capsid shell is composed of approximately 250 ca hexamers 
 and 12 ca pentamers , comprising about 1500 monomeric ca proteins in 
 all .
ca itself is composed of two domains : the n - terminal 
 domain ( cantd ) and the c - terminal domain ( cactd ) .
several structures of ca protein constructs have been determined 
 including the ntd hexamer , the single ca protein , and the cantd linked to ma , as well as several structures of a disulfide - linked 
 ca hexamer .
these structures reveal that 
 six ntds form the rigid core of hexameric ca , and six ctds form the 
 hexamer s much more flexible outer ring , with dimeric interactions 
 between ctds of neighboring hexamers holding the capsid together .
the structural arrangement in the disulfide - constrained 
 hexamer has been echoed in a recent cryo - electron microscopic study 
 of the mature capsid , with the ntd interface between the studies being 
 extremely similar .
in contrast , the ctd 
 interfaces displayed much more variation between the two studies indicating 
 an inherent flexibility in this region . the wealth of structural 
 information available on the hiv-1 ca 
 has prompted the search for small molecule inhibitors of this key 
 viral protein . since the initial discovery of cap-1 ( n-(3-chloro-4-methylphenyl)-n-[2-[([5-[(dimethylamino)-methyl]-2-furyl]-methyl)-sulfanyl]ethyl]urea ) , several other small molecule inhibitors of the 
 assembly and functions of hiv-1 have been discovered including multiple 
 cap-1 derivatives , several benzodiazepine- 
 and benzimidazole - based inhibitors , a number of 
 diverse chemical scaffolds that target the hydrophobic cavity of the 
 ctd of hiv-1 capsid , and the potent compound 
 pf-3450074 ( pf74 ) .
interestingly , the 
 binding site of pf74 partially overlaps with that of a capsid host 
 binding protein , cleavage and polyadenylation specific factor 6 ( cpsf6 ) , 
 which functions in pre - mrna processing .
moreover , a very recent study by lamorte et al . has identified an inhibitor class that binds to capsid 
 assemblies and artificially increases its stability .
this mechanism 
 of inhibition is in contrast to other inhibitors of ca that primarily 
 function by destabilization
. taken together , these studies demonstrate 
 that the ca is an attractive therapeutic target and many of its intrinsic 
 properties can be modulated by small molecules to achieve an antiviral 
 effect .
we have previously used a structure - based drug design 
 strategy 
 to utilize structural and biochemical information about the capsid 
 ntd to design inhibitors of the ntd 
ntd interface that would 
 interfere with hiv-1 replication at an early , preintegration stage . 

this effort yielded compound i - xw-053 ( 1 ) , which was 
 found to be specific for hiv-1 , to have a large therapeutic spectrum , 
 and to bind to hiv-1 ca with micromolar affinity . despite its high micromolar activity against hiv-1 , 
 the novel chemotype 
 and binding site on hiv-1 ca , coupled with its broad - spectrum anti - hiv 
 activity , suggests that compound 1 would serve as a good 
 starting point for the development of high - efficacy analogues through 
 chemical optimization .
therefore , in this study we performed a first - stage 
 structure  activity analysis in order to improve the efficacy 
 of this compound while determining groups critical for its anti - hiv 
 activity .
this endeavor yielded the identification of compound 34 ( 5-iodo-2-(3-nitrophenyl)-1h - phenanthro[9,10-d]imidazole ) , 
 which represents an 11-fold improvement in antiviral potency over 
 the parental i - xw-053 compound .
we have previously 
 described compound 1 as a novel 
 antiviral that targets a unique site on the hiv-1 ca protein and inhibits 
 by affecting virus uncoating .
however , compound 1 inhibits 
 the replication of hiv-1 in the high micromolar range .
therefore , 
 we sought to improve upon this potency while still retaining the novel 
 qualities of this compound .
screening for analogues of compound 1 in commercial vendor chemical libraries resulted in the 
 identification of 56 hit molecules with a unique set of functional 
 groups attached to the core imidazole structure ( table 1 ) .
these compounds were purchased for biological testing in 
 a pbmc - based hiv-1 replication assay as previously described .
we chose to evaluate the compounds against hiv-189bz197 as this isolate in our 
 hands is one of the most refractory to inhibition using gag - targeting 
 small molecules .
compound toxicity against the 
 peripheral blood mononuclear cells ( pbmcs ) was assessed in parallel 
 using an mts assay as previously described and was used to determine 
 the therapeutic index ( ti value ; cc50/ic50 ) 
 of the test molecules .
however , they could be further subclassified 
 into four categories : core-1 triphenyl imidazole core ( containing 
 31 molecules ) , core-2 an imidazol-2(3h)-ylidene core 
 with six compounds , core-3 tetrahydro phenanthro imidazole with 16 
 compounds , and core-4 phenanthro[9,10-d]imidazol-2(3h,4h,7h)-ylidene containing 
 three molecules that had a combination of the second and third core 
 groups . in the triphenyl imidazole core containing molecules , chemical 
 modifications at
either the ortho , para , or meta positions on any 
 of the three phenyl rings lead to only a modest increase in potency . 

these compounds also had low ti values indicating that the increase 
 in efficacy could be attributed to a concomitant increase in the toxicity 
 of the compounds . among the imidazol-2(3h)-ylidene 
 core compounds ,
a clear sar was observed with halogen modifications 
 to the basal phenyl groups at the para positions leading to a 2-fold 
 increase in potency and a > 3 ti value ( compound 3 ) . 

similarly , 
 an ester group at the ortho position on one of the basal phenyl group 
 lead to a 3-fold increase in potency and > 3 ti value ( compound 14 ) .
however , addition of large aryl or heteroaryl groups 
 lead to no significant change in efficacy or toxicity ( compound 42 ) and a loss of efficacy ( compound 52 ) .
compound 49 containing a nitro substitution on one of the basal phenyl 
 groups had a minor effect on both efficacy and toxicity . among the 
 tetrahydro phenanthrolines ,
halogen , nitro , or thiomethyl modifications 
 at the para position of the apical phenyl ring lead to an increase 
 in potency with low toxicity values leading to an improvement in the 
 ti value .
in addition , the efficacy of these compounds was further 
 improved with a halogen modification at the para position on the phenanthroline 
 group .
the nitro substitution at the para or meta position lead to 
 a marked increase in efficacy and low toxicity as in compounds 29 , 31 , 33 , 37 , and 34 .
compound 30 with the thiomethyl modification 
 had the best efficacy but with a ti value of 3.4 indicating an increase 
 in toxicity too . however , compound 34 demonstrated an 
 ic50 value of 14.2  1.7 m and no toxicity 
 up to the highest test concentration of 100 m ( figure 1 ) . while compound 29 had similar efficacy 
 as compound 34 , it exhibited 46.3% toxicity in cells 
 at 100 m .
the last group that had a phenanthro[9,10-d]imidazol-2(3h,4h,7h)-ylidene core structure did not show any gain in potency 
 or ti value .
the identification of compound 34 represents 
 an 11-fold improvement in potency over the parental compound 1 ( i - xw-053 ) .
effect of compound 34 on the replication 
 of hiv-189bz167 and the viability of primary human pbmcs . 

black squares 
 show the effect of compound 34 on the infection of primary 
 human pbmcs by the clade b primary isolate hiv-189bz167 .
virus infection is expressed as the percentage of infection ( measured 
 by reverse transcriptase activity ) observed in the presence of compound 
 relative to the level of infection observed in the absence of the 
 compound .
red circles show 
 the effect of the compound on the viability of the pbmcs and demonstrate 
 that compound 34 is not toxic up to the highest concentration 
 tested ( 100 m ) . the ic50 value for compound 34 against hiv-189bz167 = 14.2  1.7 m . given the 
 favorable properties of 
 compound 34 , we next sought to demonstrate its interaction 
 with hiv-1 ca .
hiv-1nl4 - 3 ca protein was purified as 
 previously described and attached to 
 the surface of a high density glh sensor chip ( bio - rad laboratories , 
 hercules , ca ) using standard amine coupling .
compound 
 dilutions were injected simultaneously over both these surfaces and 
 the responses recorded .
the compound 34ca interaction 
 follows a similar kinetic profile as the parental compound 1 with rapid association and rapid dissociation rates .
however , unlike 
 the parental compound , 34 appears to interact with ca 
 with a 1:1 stoichiometry .
the equilibrium dissociation constant for 
 the compound 34ca interaction is approximately 
 5- to 6-fold stronger than the compound 1ca interaction . 

moreover , there is remarkable agreement between the ic50 values and the kd values for compound 34 .
sensorgrams depicting the interaction of compound 34 with sensorchip - immobilized hiv-1nl4 - 3 ca .
colored lines indicate experimental data , whereas black 
 lines indicate fitting to a simple 1:1 binding model .
the individual 
 rate constants were marginally out of the dynamic range of the instrument 
 and are therefore not reported .
the equilibrium dissociation rate 
 constant ( kd ) for this interaction was determined to be 
 11.8  4.7 m .
having demonstrated 
 potency and maintenance of target specificity for compound 34 , we next sought to extract structure  activity relationships 
 ( sar ) from our total data . to derive the sar of these molecules , we 
 calculated two ligand efficiency indices , namely binding efficiency 
 index ( bei ) and surface efficiency index ( sei ) , using the experimentally derived ic50 values 
 and other molecular descriptors such as molecular weight ( mw ) , p(ic50 ) which is computed as log10(ic50 ) and polar surface area ( psa ) ( table 1 ) . 

bei is defined as the ratio of p(ic50 ) to mw per kilodalton 
 and sei as the ratio of p(ic50 ) to psa per 100  .
since non - hydrogen atoms play a major role in both hydrophobic 
 and hydrophilic interactions , their contributions to ligand efficiency 
 can be measured directly in terms of the mw and the psa of the compound . 

hence the parameters bei , which correlates binding efficiency with 
 mw , and sei , which correlates binding efficiency with psa , have been 
 hypothesized to be important measures of indices for deriving sar 
 of compounds .
however , in our study on determining the sar of 
 the compounds , the bei and the sei values by themselves were not very 
 confirmatory .
in contrast , a ratio of bei to sei when plotted against 
 ti values ( figure 3 ) shows a clear demarcation 
 when a cutoff value of 1 and 3 for the bei to sei ratio and ti values 
 respectively are chosen .
these cutoff values were chosen based on 
 a hypothetical molecule that has an ic50 value of 20 m , 
 molecular weight of 500 da , and a psa of 50 , which resulted in a bei 
 to sei ratio of 3.39 . a 3-fold difference between effective versus 

based on these 
 cutoff values , seven molecules were chosen that included two molecules 
 from core-2 ( compounds 3 and 14 ) and five 
 from core-3 ( compounds 29 , 30 , 33 , 34 , 45 ) .
none of the molecules from core-1 
 or 4 were chosen under this stringent criteria suggesting that bei , 
 sei , and ti can be used in this combination to derive lead compounds 
 from a data set that seems to have a bottomless sar .
additionally , 
 combining core-2 and 3 did not improve the druggability of the molecules . 

based on the features described in figure 3 , compounds 29 and 34 were chosen as lead 
 compounds with compound 34 as a representative member 
 of the core .
molecular docking studies suggested that compounds 34 and 29 bind in a similar mode with conserved 
 interactions at the same preferred binding site as the parent compound 1(14 ) but with a slightly modified 
 binding mode ( figure 4 ) .
this binding mode 
 suggests that compounds 29 and 34 have interactions 
 with the binding pocket residues that include strong electrostatic 
 interactions such as arene - cation interactions with gln179 , lys170 , 
 arg173 , asp166 and other residues such as tyr169 , glu35 , ser178 , and 
 ser41 .
in addition , the pocket is lined by hydrophobic residues such 
 as ala174 , leu172 , val165 , pro34 , and pro147 that are predicted to 
 contribute favorably to the binding of these compounds .
these putative 
 binding pocket residues are located in the regions that are important 
 for ca ntd 
extensive mutagenesis of the ntd domain has been performed previous 
 to this study . a recent study by manocheewa et al .
mutations in this area of the capsid fall 
 broadly into two categories : ( 1 ) those that destabilized the capsid 
 assembly and
several of the above - mentioned residues that 
 may constitute the binding site for compound 34 have 
 been previously mutated and their effect on viral replication ascertained .
ntd interface 
 when mutated have adverse effect on hiv-1 and therefore could potentially 
 constitute a novel site of vulnerability for small molecule targeting 
 and disruption of ca protein function .
scatter plot of the numerical 
 values of ratio of bei to sei against 
 ti values for all the compounds is shown with color coding for the 
 various core structures .
compounds with best ti values correlating 
 with higher bei to sei ratio cluster together and are represented 
 inside an orange rectangle .
schematic representation of the binding 
 mode of compounds 34 ( a ) and 29 ( b ) are 
 shown .
the binding site 
 residues are colored by their nature , with hydrophobic residues in 
 green , polar residues in purple , and charged residues highlighted 
 with bold contours .
blue spheres and contours indicate matching regions 
 between ligand and receptors . hydrogen bonded interactions are shown 
 by green arrows , ionic interactions in magenta lines , and arene  h 
 interactions in green lines extending across the aryl rings .
using a combination of alanine mutagenesis and 
 spr , we sought to 
 investigate this proposed binding mode .
eight mutant ca proteins containing 
 ile37ala , lys170ala , glu180ala , asn139ala , gln179ala , ser41ala , pro38ala , 
 and arg173ala , as well as the wild - type hiv-1nl4 - 3 ca protein were immobilized on a sensor chip and exposed to a single 
 concentration of compound 34 .
the results of this analysis 
 are shown in figure 5 and suggest that residues 
 lys170 , ser41 , and gln179 when mutated to alanine lead to loss of 
 favorable interactions with compound 34 .
however , residues 
 arg173 and pro38 when mutated to alanine appeared to increase the 
 interaction of compound 34 with hiv-1 ca . from the docking 
 model
this increase may be due to a strengthening of arene - h interactions 
 with the ligand .
when mutated to 
 alanine did not produce any significant change in binding capacity 
 when compared to wild - type , suggesting these interactions could be 
 compensated by other residues in the binding pocket .
effect of mutation of 
 capsid residues in and around the proposed 
 compound 34 binding site on compound binding .
the interaction 
 of compound 34 at a concentration of 50 m with 
 wild - type and mutant versions of the ca protein was assessed using 
 spr . to allow comparison ,
responses at equilibrium were normalized 
 to the theoretical rmax , assuming a 1:1 
 interaction .
the residues lys170 and 
 ser41 , which altered compound 34 interactions when mutated , 
 have been studied previously .
mutation of lys170ala was shown by chang et al . to have no effect 
 on the production of virus - like particles or on the in vitro assembly 
 of the purified protein .
mutation of 
 ser41ala by cartier et al . also had no effect on hiv-1 viral particle 
 production and release .
similarly , mutation 
 of gln179 to alanine had no effect on viral replication kinetics .
taken together , this indicates that the reduction 
 in binding of compound 34 to these mutations is not due 
 to an overall gross perturbation of the native wild - type structure 
 of the ca protein but most likely reflects disruption of individual 
 contacts .
the mutation data also correlates with the difference 
 in binding 
 mode observed between the parent compound 1 and compounds 29 and 34 .
mutation of residues ile37 , asn139 , 
 and arg173 abrogated the binding of compound 1 but had 
 no effect on compound 34 , while lys170 had a major effect 
 on compound 34 but not compound 1 .
however , 
 unlike both compound 1 and ck026 ( compound 1 s parent ) , whose modeling suggested 
 the possibility of two potential binding sites , both compounds 29 and 34 had a strong preference for one of 
 these binding sites . these  binding 
 site preferences  suggested from the modeling studies are reflected 
 in different stoichiometric binding ratios for compound 1 and compound 34 demonstrated using spr and itc . in conclusion , we have designed a novel hiv-1 inhibitor , compound 34 , which binds to a previously untargeted site on the hiv-1 
 ca protein .
compound 34 offers an 11-fold increase in 
 efficacy to its parent compound 1 and was designed by 
 utilizing insightful medicinal and computational chemistry methods . 

furthermore , using biomolecular docking and spr methods we validated 
 that compound 34 may bind to the same site as compound 1 but with a different orientation .
compound 34 is a polycyclic compound and often there are concerns that such 
 planar polycyclic compounds could intercalate with dna and cause genotoxicity . 

however , compound 34 contains a nitro substitution that 
 may be utilized to overcome the mutagenic potential and also add other 
 substituents to remove the planarity of the fused rings .
similar studies 
 were performed on lead compounds with acridine core by changing the 
 position of the nitro group substitution that reduced the mutagenic 
 potential of those compounds .
future 
 studies will involve focused medicinal chemistry based optimization 
 of compound 34 to improve these metabolic and toxicity 
 liabilities .
in addition , cocrystallization of these compounds with 
 ca protein will confirm the differences in binding mode between compounds 1 and 34 , to aid in structure based optimization 
 to further improve potency .
assays involving hiv-1 infection of human peripheral 
 blood mononuclear cells ( pbmcs ) were performed as described previously .
briefly , fresh pbmcs , seronegative for hiv-1 and hbv , isolated from 
 blood samples of the screened donors ( biological specialty corp . , 

colmar , pa ) by using lymphocyte separation medium ( cellgro ; mediatech , 
 inc . ,
manassas , va ) , were stimulated by incubation in 4 g / ml 
 phytohemagglutinin ( pha ; sigma ) for 48 to 72 h. mitogenic stimulation 
 was maintained with the addition of 20 u / ml recombinant human interleukin-2 
 ( rhil-2 ; r&d systems , minneapolis , mn ) to the culture medium . 

pha - stimulated pbmcs from at least two donors were pooled , diluted 
 in fresh medium , and added to 96-well plates at 5  10 cells / well .
cells were infected with the hiv-1 group m isolate 89bz167 
 ( subtype b , cxcr4-tropic ; obtained courtesy 
 of dr .
nelson michael through the nih aids research and reference 
 reagent program , division of aids , niaid , nih [ catalog no .
7692 ] ) 
 at a final multiplicity of infection [ moi ] of  0.1 in the 
 presence of nine different concentrations of the test compounds ( triplicate 
 wells / concentration ) and incubated for 7 days .
viral replication in 
 the presence and absence of the test compounds was determined by analysis 
 of rt activity in the cell free supernatants .
concomitantly , the cytotoxicity of the test compounds was measured 
 by the addition of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium ( celltiter 96 reagent , mts ; promega ) according 
 to the manufacturer s instructions . the therapeutic index ( ti ) 
 of the test molecules was computed as the ratio of the half maximal 
 cytotoxicity concentration ( cc50 ) to the half maximal inhibitory 
 concentration ( ic50 ) .
all molecular descriptors were computed 
 using moe ( ver 10.1 ) and the ic50 values were obtained 
 from experimental methods described above .
docking studies with the 
 56 test compounds along with compound 1 were performed 
 as previously described and targeting the ntd  ntd binding interface 
 pocket .
briefly , three - dimensional structures 
 of all 56 test compounds were modeled using the ligand builder module 
 of moe .
the compounds were optimized using semiempirical mopac using 
 am1 parameters as adapted in moe .
the crystal structure of hiv-1 capsid 
 was obtained from protein databank ( pdb code 3h4e ) and was prepared 
 for docking by adding hydrogen atoms and energy minimizing the structure 
 as described previously .
molecular dockings 
 were performed using gold ( ver 4.1 ) using 20 independent runs for 
 each ligand and the protein  ligand complexes were scored using 
 goldscore , chemscore , and a customized consensus scoring scheme .
all binding assays 
 were performed on a proteon xpr36 spr interaction array ( bio - rad , 
 hercules , ca ) .
proteon glh sensor chips were preconditioned with two 
 10-s pulses each of 50 mm naoh , 100 mm hcl , and 0.5% sds .
the system 
 was then equilibrated with 0.005% tween-20 in phosphate - buffered saline 
 ( pbs ) .
individual ligand flow channels were activated for 5 min at 
 25 c with a mixture of 1-ethyl-3-[3-(dimethylamino)propyl carbodiimide 
 hydrochloride ) ( 0.2 m ) and sulfo - n - hydroxysuccinimide 
 ( 0.05 m ) . immediately after chip activation , the ca protein , purified 
 as previously described , was prepared 
 at 100 g / ml in sodium acetate ph 4.5 and injected across ligand 
 flow channels for 5 min at a flow rate of 30 l min .
then , after unreacted protein had been washed out , excess active 
 ester groups on the sensor surface were capped by the injection of 
 50 l of 1 m ethanolamine ( ph 8.0 ) at a flow rate of 5 l / min . 

a reference surface was similarly created by immobilizing a nonspecific 
 antibody arc4033 ( antimouse / rat interferon- : biosource ; invitrogen , 
 carlsbad , ca ) and was used as background to correct nonspecific binding 
 and for instrument and buffer artifacts .
the standard errors in the 
 immobilization levels from the six spots within each channel were 
 less than 4% .
a stock solution 
 of compound 34 was prepared by dissolving in 100% dmso 
 to a final concentration of 10 mm . to prepare the sample for analysis
, 
 20 l of dmso was added to 30 l of the compound stock 
 solution , which was then subsequently added to pbs - t buffer ( 20 mm 
 na - phosphate , 150 mm nacl , and 0.005% polysorbate 20 , ph 7.4 ) to a 
 final volume of 1 ml and mixed thoroughly
. preparation of analyte 
 in this manner ensured that the concentration of dmso was matched 
 with that of running buffer with 5% dmso .
lower concentrations of 
 the compound were then prepared by 2-fold serial dilutions into running 
 buffer ( pbs - t buffer ( 20 mm na - phosphate , 150 mm nacl , and 0.005% 
 polysorbate 20 , ph 7.4 .
these compound dilutions were then 
 injected over the control and ca surfaces at a flow rate of 100 l / min , 
 for a 2 min association phase , followed by a 2 min dissociation phase 
 using the  one - shot kinetics 
specific regeneration of the surfaces 
 between injections was not needed due to the nature of the interaction .
data were analyzed using the proteon 
 manager software version 3.0 ( bio - rad ) .
the responses of a buffer 
 injection and responses from the reference flow cell were subtracted 
 to account for injection artifacts and nonspecific binding .
wild - type and mutant 
 hiv-1 ca proteins were attached to the surface of a glh sensor chip 
 by standard amine chemistry as described above .
compound 34 was injected over these surfaces at a concentration of 50 m 
 at a flow rate of 100 l / min , for a 2 min association phase , 
 and the response at equilibrium recorded . for comparison , and to take 
 into account minor differences in the ligand density of the mutant 
 surfaces , responses were normalized to the theoretical maximum response 
 ( rmax ) for a given surface , assuming a 
 1:1 interaction .
 despite the considerable successes 
 of highly active antiretroviral 
 therapy ( haart ) for the treatment of hiv / aids , cumulative drug toxicities 
 and the development of multidrug - resistant virus necessitate the search 
 for new classes of antiretroviral agents with novel modes of action . 
 
 the hiv-1 capsid ( ca ) protein has been structurally and functionally 
 characterized as a druggable target . 
 we have recently designed a novel 
 small molecule inhibitor i - xw-053 using the hybrid structure based 
 method to block the interface between ca n - terminal domains ( ntd  ntd 
 interface ) with micromolar affinity . in an effort to optimize and 
 improve the efficacy of i - xw-053 , 
 we have developed the structure 
 activity relationship of i - xw-053 compound series using ligand efficiency 
 methods . 
 fifty - six analogues of i - xw-053 were designed that could 
 be subclassified into four different core domains based on their ligand 
 efficiency values computed as the ratio of binding efficiency ( bei ) 
 and surface efficiency ( sei ) indices . 
 compound 34 belonging 
 to subcore-3 showed an 11-fold improvement over i - xw-053 in blocking 
 hiv-1 replication in primary human peripheral blood mononuclear cells 
 ( pbmcs ) . 
 surface plasmon resonance experiments confirmed the binding 
 of compound 34 to purified hiv-1 ca protein . molecular 
 docking studies on compound 34 and i - xw-053 to hiv-1 
 ca protein suggested that they both bind to ntd  ntd interface 
 region but with different binding modes , which was further validated 
 using site - directed mutagenesis studies . 
Introduction
Results and Discussion
Experimental Section