monomeric human topoisomerase 
 1b ( top1 ) regulates dna topology 
 throughout key cellular events such as dna replication and gene transcription .
eukaryotic top1 relaxes both positively and negatively supercoiled 
 dna and is an established anticancer drug 
 target ( its inhibition initiates apoptosis and hence tumor regression ) .
recent single - molecule nanomanipulation and molecular dynamics studies of top1 in the presence and absence of inhibitors and the 
 originally proposed catalytic cycle of 
 top1 may be used to construct a current view of the enzyme s 
 four - step cycle ( figure 1 ) .
drugs that 
 block top1 fall into two distinct classes : ( 1 ) well - characterized 
 interfacial poisons ( ifps ) and ( 2 ) less common catalytic inhibitor 
 compounds ( cics ) .
currently , all dna - intercalating 
 ifps arrest dna strand religation by non - covalent binding at the nick 
 site of the top1dna cleavage complex , poisoning the enzyme midcycle .
known ifps include camptothecin ( cpt ) 
 and its analogues and synthetic compounds , 
 e.g. , indolocarbazoles , indenoisoquinolines , dibenzonaphthyridones , and aromathecins .
some minor - groove binders that engage top1 s dna substrate 
 below the nick site , e.g. , hoechst 33258 and 33342 , prevent strand religation and are non - interfacial top1 
 poisons . the rational design of new ifps 
 and conceptual understanding of how established ifps work is underpinned by x - ray data for the dna 
cics may operate by blocking two key steps in the 
 enzyme s 
 catalytic cycle : substrate binding or covalent cleavage complex formation . 

compounds inhibiting the first step ( cic1 ) are either conventional 
 competitive inhibitors ( binding to top1 ) or unconventional competitive 
 inhibitors ( binding to dna )
. examples of the former are unknown , but 
 unconventional competitive inhibitors exist and are either dna intercalators or minor groove binders or both .
step 2 catalytic inhibitors ( cic2 ) are currently rather obscure ; 
 one recently described indolizinoquinoline-5,12-dione derivative , 
 cy13i , possibly fits this descriptor .
of relevance to this study , dna - binding au porphyrins were classified as top1 catalytic inhibitors , while other au complexes were initially 
 misassigned as top1 ifps and hence reclassified 
 as catalytic inhibitors .
the correct 
 assignment of a compound s mechanism of action ( moa ) with top1 
 is not straightforward .
the inherent problem is that inhibition of 
 supercoiled dna relaxation by top1 alone does not distinguish between 
 the actions of cics and ifps nor does it distinguish between conventional 
 and unconventional competitive inhibition .
since cic s 
 do not trap top1-nicked dna , dna damage by this 
 class of compounds is likely to be lower than that caused by ifps .
the paucity of top1 cics coupled with their 
 anticipated reduced genotoxicity relative to 
 ifps creates significant opportunities for drug discovery .
here we 
 report on a new class of cytotoxic macrocyclic au top1 
 cics ( figure 1 ) and precise delineation of 
 the moa of the lead compound , 3 .
( a ) illustration of key 
 events in the catalytic cycle of human 
 top1 .
step 1 : top1 binds supercoiled dna ( scdna ; 5-ta-3 
 dinucleotide pair as target ) .
step 2 : nucleophilic attack of the 3-phosphate 
 linking the ta pair ( scissile strand ) by y723 ( catalytic tyrosine 
 residue ) affords a covalent dna 
step 3 : the intrinsic torque stored in scdna drives ratchet - like 
 rotation about the non - scissile strand until strand religation occurs 
 with concomitant release of y723 .
the relaxed 
 dna ( rdna ) is then released by the enzyme 
 in step 4 . interfacial poisons ( ifps ) such as camptothecin ( cpt ) bind 
 the nick site via 5-ta-3 intercalation and h - bonding 
 to top1 to form a ternary drugscdna 
cics operate differently by either blocking 
 substrate recognition by top1 ( type 1 competitive inhibitor , cic1 ) 
 or , in principle , preventing the formation of the covalent cleavage 
 complex by blocking the nucleophilic attack of the scissile strand 
 by y723 ( type 2 competitive inhibitor , cic2 ) .
( b ) structures of new 
 cytotoxic pyrrole - based au macrocycles 15 , free base macrocycle 6 , and 
 the ni analogue of 1 , compound 7 .
compounds 15 reflect a design 
 evolution over our recently patented class of cytotoxic bis(pyrrolide - imine ) 
 au chelates .
specifically , 
 we have employed macrocycles to enhance the redox and chemical stability 
 of the metal ion in conjunction with a quinoxaline ring to augment 
 dna intercalation .
direct metalation of the macrocycle ( route a , figure s2 ) was only successful for 1 and 5 ( i.e. , those macrocycles bridged by a propyl 
 chain ) . for compounds 24 with slightly 
 more elaborate alkyl groups linking the imine nitrogen atoms , a metal - templated 
 cyclization had to be employed ( route b ) .
more specifically , the diamine bridge required to effect cyclization 
 of the macrocycle was added after au - binding by the bis(pyrrole - aldehyde ) 
 precursor .
( although uncommon , au ions reportedly template 
 aldehyde and amine condensations . ) we were able to elucidate the structures 
 of compounds 1 , 2b , and 3 by 
 single crystal x - ray diffraction ( table s1 ) despite the ordinarily challenging morphology of the crystals ( fine , 
 brick - red needles ) . because of the high degree of similitude in the 
 structures of 13 , particularly the 
 au ion coordination geometry and macrocycle conformation 
 and the appreciable cytotoxicity of 3 ( vide infra ) , the following discussion is illustrated with selected , typical 
 ion pairs from the asymmetric unit of 3 ( figure 2 ) .
the au ions in salts 1 , 2b , and 3 are nominally square planar 
 with au 
the au  npyrrole distances average 1.98(3 ) 
  for all nine cations and are 1.5% shorter than the au  nimine bonds , which average 2.01(6 )  ( table s2 ) .
this reflects the fact that the pyrrole groups 
 are anionic -donors with more acute c  n  c angles 
 than the corresponding c = n  c angles of the imine donors . 

the coordination group distances are comparable to those of other 
 au complexes ( au  n : 1.9282.216  ) .
the average npyrrole  au  npyrrole and nimine  au  nimine bond angles for the propyl - bridged systems 1 and 2b are 99.6(8) and 96.5(5) , respectively . for 
 the six independent cations of 3 , these angles average 
 97.1(9) and 99.2(6). the larger seven - membered chelate 
 ring of 3 accounts for the somewhat more obtuse mean 
 nimine  au  nimine bond angle .
the cation conformations of 13 deviate mildly from planarity mainly in response to crystal 
 packing constraints .
in each structure , the cations form -stacked 
 dimers with head - to - tail ( figure 2 ) or oblique 
 geometries ( figures s8s14 ) characterized 
 by interplanar spacings of 3.4  , as found in other -stacked 
 polyaromatic compounds .
there are no aurophilic 
 auau contacts between cations ( auau 
 distances > 5  ) .
the au macrocycle pairs within 
 dimers of 13 exhibit overlaps ranging 
 from 74% in 3 to 89% in 1 .
importantly , 
 the -stacking proclivity of the cations highlights their potential 
 as dna intercalators .
view of the x - ray structure ( 100 k ) of two of six independent 
 ion 
 pairs from salt 3 ( 50% thermal ellipsoids for non - h atoms ; 
 h atoms are shown as capped cylinders ) .
atom color code : 
 gray , c ; lilac , n ; orange , p ; green , f ; gold , au ; pale blue , h. crystallographic 
 details for 1 , 2b , and 3 are 
 given in the supporting information .
selected 
 mean bond distances (  ) and angles (  ) for all six independent 
 cations of 3 : au  npyrrole , 1.98(1 ) ; 
 au 
nimine , 2.02(2 ) ; c = nimine , 
 1.30(3 ) ; npyrrole  au 
the mean plane separation ( 3.4  ) reflects notable 
 -stacking for both the head - to - tail and oblique dimers of 3 .
the affinity constants , ka , for non - covalent 
 binding of 15 to calf - thymus dna 
 ( ctdna ) were determined 
 by fluorometric titrations involving the displacement of intercalated 
 ethidium bromide ( eb , figure 3a ) . from the 
 loss of eb fluorescence ( 614 nm ) with increasing concentration of 15 , ka values 
 ranging from 2  10 to 4 
10 m bp were obtained and are similar in magnitude to 
 that reported for a gold(iii ) porphyrin ( 3  10 m bp ) .
the ka values for 15 showed 
 no independent linear correlation with either the steric bulk of the 
 macrocycle s alkyl bridge or the lipophilicity of the cation . 

however , analysis of ka as a function 
 of both variables concurrently ( figure 3b ) 
 gives a significant three - dimensional bivariate linear correlation 
 and confirms that cations with a small alkyl bridge and high lipophilicity , 
 e.g. 5 , exhibit the highest ka values .
the correlation itself suggests that 15 are dna intercalators since not only 
 are the ka values determined by competitive 
 displacement of a known dna intercalator ( eb ) but also the monotonic increase in ka with increasing lipophilicity of the compounds clearly reflects 
 binding principally within the relatively non - polar intrahelical space 
 of the duplex dna target ( i.e. , between -stacked bases ) . to confirm the intercalation data and examine possible roles for 
 the metal ion , electrophoretic mobility shift assays ( emsas ) , thermal 
 denaturation , and dna unwinding experiments were carried out .
regarding 
 the first of these tests , figure 3c shows that 1 induces a greater mobility shift for a plasmid substrate 
 ( phot1 , lanes 59 ) than the archetypal reference compound for 
 dna intercalation ( eb , lanes 24 ) , despite the comparable ctdna 
 affinity constants for the two compounds ( eb , ka = 5(1 )  10 m bp ; compound 1 , ka = 4.0(4 )  10 m bp ) . the large mobility shift induced by 1
note that supercoiled plasmid dna is affected 
 most upon binding of 1 ( figure 3d ) , as evidenced by the 11% mobility shift relative to nicked - open 
 circular dna ( noc dna , 5% shift ) and very marked band broadening apparent 
 at a concentration of only 5 m ( lane 7 ) .
in the case of a negatively 
 supercoiled plasmid with intact double strands , the linking number 
 describing the dna topology , lk , must remain constant 
 irrespective of the extent of local unwinding ( i.e. , change in twist , tw ) induced by the intercalator .
to keep lk constant , wr , which reflects supercoiling of the 
 plasmid , must adjust according to eq 1:1for au macrocycle 1 , intercalation of negatively supercoiled 
 phot1 is expected to reduce 
 the number of supercoils and thus the macromolecule s supercoil 
 density , thereby significantly impeding its mobility in the gel matrix 
 ( as observed ) .
the three - dimensional plot displayed in figure 3d further highlights the dna distribution with increasing 
  in lanes 58 and specifically emphasizes the 
 appearance of a third , non - migratory form of dna ( form iii , lanes 
 7 and 8) .
two possible explanations for the appearance of this immobile 
 dna species at higher concentrations of 1 are : ( i ) aggregation 
 of the dna1 adduct(s ) occurs , trapping the dna 
 in the well , or ( ii ) full charge - neutralization of the dna takes place . 

however , the latter explanation seems unlikely since each adjacent 
 dinucleotide pair in dsdna is bridged by two phosphate groups , each 
 with a formal charge of 1 .
intercalation of a monocationic 
 intercalator can thus only neutralize 50% of the total charge on the 
 dna duplex even if every adjacent dinucleotide pair non - covalently 
 binds to one au macrocycle , which is improbable .
finally , 
 the apparent  disappearance  of the dna in lane 9 ( 
 = 50 m ) signals either reverse migration of all dna1 adducts or complete saturation of the eb binding sites by 1 so that staining of the gel for dna visualization is ineffective . 

the latter explanation is the more likely given that formation of 
 a cationic dna1 adduct ( as would be required for 
 reverse migration of the dna ) is physically highly improbable .
( a ) displacement 
 of intercalated eb from ctdna by 3 studied by emission 
 spectroscopy ( 298 k , ph 7.0 , 15% dmso - tris / hcl 
 buffer , 15 m ctdna , 15 m eb ) .
inset : determination of 
  at 50% loss of eb fluorescence ( c50 ) ; c50 is used to determine 
 the ctdna affinity constant , ka , of 3 .
( b ) graph of ka for 1 , 2a , and 35 as a 
 function of the steric bulk of the macrocycle s alkyl bridge 
 and the hydrophobicity of the au complex , log(po / w ) ( table s3 ) . 

the surface is the best - fit bivariate linear regression function to 
 the data .
( c ) emsa of selected compounds with supercoiled ( sc , form 
 i ) phot1 plasmid dna ( dna , 31.3 ng / well ; tbe buffer , ph 7.8 ) ; some 
 nicked - open circular ( noc , form ii ) dna is present .
the lanes contain 
 phot1 plasmid dna ( lane 1 ) , increasing concentrations of eb ( lanes 
 24 ) , 1 ( lanes 59 ) , 6 ( lanes 
 1012 ) , and 7 ( lanes 1315 ) .
( d ) two- and three - dimensional 
 deconvolution of the dna bands in lanes 58 of the emsa gel 
 shown in part ( c ) . collectively , the emsa 
 data for 1 reflect intercalative 
 binding of dsdna by the au macrocycle .
this conclusion 
 was further tested by concurrent analysis of the metal - free macrocycle 
 ( compound 6 ) and the ni analogue of 1 ( compound 7 ) ; neither induces a dna mobility 
 shift nor in fact displaces eb from dna in solution .
the au ion is thus obligatory for dna intercalation by 15 . that association depends on the presence of au reflects electrostatic binding of the intercalator to the dna ( as 
 neither 6 nor 7 are charged ) and a pivotal 
 iondipole interaction formed between the au ion and a carbonyl oxygen atom of thymine ( vide 
 infra ) . because the mode of interaction of the au macrocycles 
 with dna has to be established unambiguously to determine the mechanism 
 of action underpinning the cytotoxicity of 2a and 3 ( vide infra ) , we performed two additional 
 tests that unequivocally support an intercalative dna binding mode 
 for the compounds ( figure 4 ) . in the first 
 experiment , thermal denaturation ( melting ) of a linear 291-bp dna 
 duplex ( an amplicon from exon 15 of the human actn3 gene )
was studied as a function of the identity and concentration of the 
 added compound ( table s12 ) . from figure 4a , b , the melting point , tm , increased in sigmoidal fashion with increasing concentration of 13 , consistent with the enhanced thermal 
 stability that is expected to accompany uptake of an intercalator .
significantly , no change in tm was observed 
 for 6 over the full concentration range .
the values of 
 tm ( the change in melting point 
 relative to the control ) were + 4.65 , + 3.90 , and + 4.40 c for 
 compounds 13 , respectively , when 
 measured at their highest concentrations ( 50 m ) . from 
 figure 4b , the dose
 response function 
 for 1 gives the saturating value of the melting point 
 increase , tm = + 5.1(4 ) 
 c .
the tm values determined 
 here are in good agreement with those reported for the metallointercalators 
 [ co(pic)2(dppz ) ] ( + 4.4 c ) , where pic = picolinate 
 and dppz = dipyrido[3,2-a:2,3-c]phenazine , and [ ru(nh3)4(dppz ) ] ( + 5.2 c ) as well as organic protoberberine derivatives 
 ( + 2.9 to + 6.4 c ) .
other dppz - based 
 metallointercalators , however , exhibit tm values as large as + 14 c . ( a ) 
 hrm curves for a linear 291-bp dsdna fragment ( ph 8.4 ) derived 
 from the human actn3 gene as a function of the concentration of 1 .
the plot reflects the negative first derivative of the 
 fluorescence intensity ( df / dt , 510 nm ) from the dna - intercalating reporter dye ( cybr green ) used 
 to monitor strand separation .
( b ) plot 
 of the melting temperatures , tm , against 
 the concentrations of three au macrocycles and the metal - free 
 macrocycle 6 .
the zero - slope fit for 6 has 
 an intercept , tm , of 83.0(1 ) c and 
 reflects the mean value of tm for the 
 amplicon ( since 6 does not interact with the dna ) .
the 
 dose  response function for 1 is : tm = tm + ( tm  tm)/(1 + ( c / ec50 ) ) , where c is the molar 
 concentration of 1 .
the fit parameters were : tm = 82.95(9 ) c ; tm = 88.1(4 ) c ; ec50 = 9(2 ) 
  10 m ; p = 0.9(1 ) ;  = 0.013 ; r = 0.996 .
lane 1 , supercoiled 
 phot1 plasmid dna ; lane 2 , relaxed phot1 plasmid ( effected by incubation 
 with 10 units of top1 for 30 min at 37 c ) ; lanes 35 , 
 increasing concentrations of the intercalator control m - amsa added to relaxed phot1 ; lanes 614 , increasing concentrations 
 of compounds 1 , 7 , and 6 added 
 to relaxed phot1 as indicated .
abbreviations : noc , nicked - open circular 
 dna ; sc , supercoiled dna ; rx , relaxed ; ti , dna topoisomer bands ; m - amsa , 4-(9-acridinylamino)methanesulfon - m - anisidide . several factors will determine the measured value of tm ( beyond the identity of the primary intercalating 
 group ) .
these include the nature of the ancillary ligands , the base 
 sequence of the dsdna substrate ( since this governs the density of 
 binding sites for base pair - specific intercalators ) , the solution 
 conditions ( ph , buffers , salts ) , and 
 the experimental method . the method used here evidently affords reliable 
 data over a wide concentration range , permitting measurement of tm
. however , as shown in figure 4a , some caution in evaluating the data is required 
 at higher compound concentrations when the signal is weak ( df / dt < 0.1 ) .
the loss of signal intensity in the experiment reflects two main processes : 
 ( 1 ) the release of cybr green ( an intercalating dye that binds at 
 base pairs in dsdna via minor groove entry ) upon strand separation and , consequently , quenching of its emission 
 by torsional motion in its non - intercalated state .
this is the physical basis underpinning melt analysis with 
 the high - resolution melt ( hrm ) method available in commercial real - time 
 pcr machines .
( 2 ) competition between the dye and intercalator for 
 binding sites along the length of the dsdna substrate .
this is evident 
 from the dose - dependent decrease in the emission signal from the dye 
 at a fixed temperature in figure 4a and accounts 
 for the vanishing signal at high concentrations of 13 .
further , from 
 the data for 6 , the irremissibility of the au ion for dna intercalation is again evident . in the second 
 experiment
, we used a standard dna unwinding assay designed to identify intercalators from their 
 interaction with dna in the presence of top1 ( figure 4c ) .
the method was first described by hsiang et al . in 1985 and elaborated on by pommier et al . in 1987 . in our experiment ,
phot1 plasmid dna was first 
 relaxed by incubation with 10 units of top1 for 30 min at 37 c 
 ( lane 2 ) .
increasing concentrations of the test compounds were then 
 added , and the solutions incubated for a further 30 min interval at 
 37 c prior to workup and analysis .
the dna intercalator control m - amsa gave the expected gaussian distribution of dna topoisomers 
 ( characterized by a linking number change , lk , of 1 ) at a concentration of 50 m 
 after workup .
the same topoisomer band distribution was observed at 
 a concentration of only 5 m in the reaction with 1 ( lane 7 ) , consistent with 1 being a more potent dna 
 intercalator than m - amsa . at a higher concentration 
 ( 50 m ) ,
the reactions with the ni analogue of 1 ( compound 7 ) and the metal - free macrocycle 6 confirm that neither intercalate dna since the signature 
 dose - dependent gaussian distribution of topoisomer bands is noticeably 
 absent in lanes 914 .
similar results were obtained running 
 the reaction the other way ( i.e. , starting 
 with supercoiled phot1 ; figure s54 ) .
the 
 observations for 1 in lane 8 merit further reflection . 
 as discussed in the literature and supporting information
, the method used is incapable of determining 
 whether the scdna product obtained at high concentrations of the intercalator 
 is : ( 1 ) positively or negatively supercoiled or ( 2 ) the result solely 
 of intercalator - induced torque or , alternatively , inhibition of top1 . 
 from lane 8 in figure 4c
, it is therefore possible that 1 may be an inhibitor of top1 , 
 but this can not be concluded with certainty in the absence of definitive 
 mechanistic tests ( e.g. , tests that can discriminate between an ifp 
 and a catalytic inhibitor of the enzyme ) .
compounds 15 were prescreened by the national cancer 
 institute ( nci , 
 bethesda , md ) against their panel of 60 human cancer cell lines ; 2a and 3 were sufficiently cytotoxic to warrant 
 full five - dose screens and 3 proceeded to in 
 vivo hollow - fiber studies ( figures s35s43 , 
 table s6 ) .
the mean ic50 value across all cell lines for the au macrocycle 
 is 16(7 ) m ; the large standard deviation reflects considerable 
 variation in the cytotoxicity of the compound across each group of 
 cell lines ( as highlighted by the radar plot of figure 5 ) .
leukemia , central nervous system ( cns ) , and colon cancer 
 were the most sensitive cancer types to 3 .
importantly , 
 a total of 19 ( out of 57 ) cancer cell lines had ic50 values 
 for compound 3 that were < 5 m , accounting for 
 selection of 3 by the nci for in vivo hollow - fiber assays ( table s7 , figures s42 and 
 s43 ) .
the five most sensitive cell lines were nci - h522 ( non - small 
 lung cancer , ic50 = 280 nm ) , rxf-393 ( renal cancer , ic50 = 1.3 m ) , sf-268 ( cns cancer , ic50 = 1.4 
 m ) , sw-620 ( colon cancer , ic50 = 1.5 m ) , 
 and lox - imvi ( melanoma , ic50 = 1.7 m ) . in the nci s 
 non - tumored animal toxicity assay with female athymic nude mice ( figure s42 ) ,
compound 3 was remarkably 
 well tolerated as reflected by a 100% survival rate after 17 days 
 for doses ranging from 100 to 400 mg kg dose ( intraperetoneal injection ) . despite these encouraging 
 data , 3 was insufficiently cytotoxic in the majority 
 of the cell lines tested in the nci s hollow fiber cytotoxicity 
 assay to warrant progression to the next investigational phase ( tumor 
 xenograft studies in live mice ) . notwithstanding the lower than desired 
 activity of 3 in the hollow fiber assays , we believe 
 that 3 and its structural congeners represent an important 
 group of compounds that have the potential to be developed into novel 
 metallodrug lead candidates with relatively low side - effects because 
 of the in vivo stability of the macrocyclic au complex . going forward , absorption , distribution , metabolism , 
 and excretion studies are clearly warranted in the case of 3 and may well delineate the factors that reduce the efficacy of the 
 compound in live animals .
abbreviations : n , number of cell lines within 
 each cancer category ; gi50 , compound concentration effecting 
 50% growth inhibition ; ic50 , compound concentration effecting 
 100% growth inhibition ; 
 lc50 , compound concentration that induces 50% cell death .
estimated standard deviations are given in parentheses ; large values 
 indicate variable susceptibility of a specific group of cell lines 
 to the test compound ( see figure s41 ) .
( a ) radar plot of log(gi50 ) values vs cancer 
 cell line from the nci-60 cytotoxicity screen for 3 against 
 their panel of 60 human cancer cell lines . comparative data for cisplatin , 
 camptothecin ( cpt ) , and 5-fluorouricil ( 5fu ) are shown to illustrate 
 the cell - line dependent response to the drugs as well as their effective 
 cytotoxic range [ mean log(gi50 ) values
response 
 function for 3 with the cns cancer cell line u251 ( the 
 relevant gi50 value is indicated by the orange arrow on 
 the radar plot ) .
( b ) hierarchical cluster analysis ( group average 
 method , minkowski distances ) of the gi50 data for 3 with analogous data for 26 anticancer drugs with known mechanisms 
 of action ( data taken from the nci database ) . compound 3 clusters with camptothecin ( a top1 ifp ) . since release of an exogenous metal ion by a ligand system 
 in a 
 live
animal is often the cause of acute toxicity , an important question to answer prior to determining the 
 moa of a metallodrug candidate is how stable the compound is in aqueous 
 solution , especially in the presence of cellular reducing agents such 
 as glutathione .
we found that the au macrocycles of this 
 study were both redox stable and essentially non - aggregating under 
 biologically pertinent conditions ( figures s15 
 and s16 ) , which might at least partly explain the fact that 3 was particularly well - tolerated in mice and essentially 
 non - toxic even at the highest test concentration of 400 mg kg . from the graph of log(gi50 )
values 
 for 3 against all 60 tumor cell lines ( figure 5a ) , compound 3 is comparable to cisplatin 
 and midway 
 between the limits of highly and mildly cytotoxic compounds such as 
 topotecan ( a top1 ifp ) and 5-fluorouricil ( a clinically deployed antimetabolite ) , 
 respectively . in order to identify the moa and hence the cellular 
 target(s ) of 3 ,
the gi50 data for 3 were compared with equivalent data for 26 compounds of known moa 
 from the nci s database using hierarchical cluster analysis 
 ( figure 5b ) .
the cytotoxicity profile of 3 is clearly akin to that of cpt , the archetypal top1 ifp . 

although the statistical data strongly suggest that 3 targets top1 in tumor cell cultures , they do not prove that 3 is an ifp of top1 .
unambiguous determination of the moa 
 is essential to definitively assign 3 as a top1 ifp or 
 top1 cic .
cyril and muller s recent solid - phase 
 enzyme inhibition assay capable of distinguishing 
 between top1 ifps and cics was used to elucidate the moa of 3 ( figure 6a ) .
. added ifps trap dna covalently 
 bound to top1 during enzyme turnover , which is subsequently detected 
 by an enhanced fluorescence signal from the dna - sensing dye picogreen . 

cics of top1 do not result in covalently trapped dna , favoring diminished 
 emission from the reporter dye with increasing compound concentration . 
 at concentrations of 100 m ,
14 all give a negative readout ( picogreen relative fluorescence ) akin 
 to the signal obtained from the cic control ( mitoxantrone , mtx ) . from 
 this assay , 14 are clearly catalytic 
 inhibitors of top1 at higher concentrations .
this result was 
 confirmed for 3 using a novel assay designed to trap 
 top1dna complexes using ultra - high enzyme : dna ratios ( figure 6b ) . in lanes 
 15 , increasing [ top1 ] at a fixed [ dna ] in the absence of nacl 
 ( ensuring a high dna affinity for top1 ) increases the yield of noc 
 dna in the reaction .
this is the expected result as we previously 
 reported that endogenous top1 may  cluster  
 at catalytic sites on genomic dna in situ .
importantly , 
 at high [ top1 ] and in the presence of an ifp like cpt , multiple single - strand 
 cleavages afford double - strand breaks and thus linear dna after workup . 

an ifp of top1 is therefore identified by its ability to generate linear dna in the assay .
lanes 1116 confirm that 3 is not an ifp of top1 ( no linear dna ) ; rather , the dose - dependent 
 drop in [ noc dna ] reflects catalytic inhibition of top1 by 3 . from figure 6a , both 1 and 
 mtx give a similar ( positive ) readout to cpt at doses of 0.1 and 1 
 m , suggesting that they are top1 ifps at low concentrations . 

the switch to catalytic inhibition of top1 occurs at higher doses 
 ( 110 m ) .
this was confirmed for 1 by integrating the dose - dependent linear dna band intensities in 
 a gel equivalent to that shown in figure 6b . 
 from figure s56 ,
the concentration of linear 
 dna increases with increasing   up to 500 nm , thereafter 
 exhibiting a sigmoidal decrease consistent with catalytic inhibition 
 of top1 at higher doses .
dual - mode inhibition of topoisomerases is 
 well - known for top2 and is equally feasible , though rarely observed , for top1 .
compounds 
 such as doxorubicin target the dna nick - sites in the enzyme  dna 
 covalent cleavage complex at low doses , poisoning the enzyme , but 
 intercalate the dna substrate at higher concentrations , engendering 
 catalytic inhibition .
mechanistically , 
 a large association constant , ka1 , exists 
 for formation of the ternary drugdna  enzyme covalent 
 cleavage complex , while a smaller association constant , ka2 , exists for intercalation of the enzyme s dna 
 substrate . because the two assays used 
 here each permit a distinction to be made between top1 ifps and catalytic 
 inhibitors in a single experiment , they are ideal for detecting dual - mode 
 inhibitors . consequently , 1 can be firmly assigned as 
 a dual - mode ( ifp - cic ) top1 inhibitor .
compounds 2a4 , in contrast , are simple cics . in the conventional 
 top1 dna relaxation assays ( topogen , inc . ; 

figure 6c ) , 3 showed marked inhibition 
 of the enzyme at 500 nm and complete inhibition of the enzyme at a 
 dose of 5 m ( lanes 9 and 10 ) .
when compared with the activity 
 of the dna intercalator control m - amsa ( lanes 1315 ) , 
 compound 3 engenders equivalent inhibition of the enzyme 
 at one - tenth of the concentration .
collectively , the data in figure 6 demonstrate that the au macrocycles 
 are top1 cics .
furthermore , we estimate that the method - dependent 
 ic50 values for 3 are 49(2 ) nm and 9.2(5 ) 
 m from the assays in figure 6c , b , respectively 
 ( figures s44 and s45 ) .
notably , these assays 
 neither distinguish between conventional ( drug binds enzyme ) and unconventional 
 ( drug binds substrate ) catalytic inhibition of top1 nor between type 
 1 and 2 cics of the enzyme .
however , since the dna affinity of 15 has been established with certainty , 
 the compounds may be tentatively assigned as unconventional 
 cics of top1 .
( it is important to note that concrete assignment of 
 the above mechanism requires an independent experiment to prove that 
 the compounds do not bind to the enzyme itself ; vide infra . ) enzyme target and mechanism assignment .
( a ) relative fluorescence 
 intensity from dna - bound picogreen ( 525 nm ) vs the concentration of 
 two drug standards ( cpt and mitoxantrone , mtx ) and 14 .
the assay measures covalent top1dna trapped by 
 the test compound , distinguishing between ifps and cics .
the data 
 are corrected for background emission ( range indicated by the dashed 
 lines ) .
( b ) proof of catalytic inhibition of top1 by compound 3 using a gel - based product - trapping assay ( high enzyme : dna 
 ratio , 0 mm nacl , [ phot1 ] = 31.3 ng / well in all reactions ) .
lanes 
 15 : enhanced yield of nicked - open circular dna ( noc dna ) with 
 increasing [ top1 ] .
lanes 610 : effect of increasing [ cpt ] from 
 0.5 to 50 m ( 100 units of top1 ) .
lanes 1116 : catalytic 
 inhibition of top1 ( 100 units ) with increasing   attenuates 
 the yield of noc dna ( linear dna , l , is essentially absent above a 
 dose of 1 m ) .
( c ) standard scdna relaxation assay for 3 and the dna - intercalator m - amsa .
lanes 
 13 : phot1 dna alone , dmso control , and top1-mediated relaxation 
 of the substrate ( no inhibitor present ) , respectively .
lanes 412 : 
 inhibition of top1 with increasing   ( complete inhibition 
 occurs at 5 m ) .
m - amsa only completely inhibits 
 top1 at 50 m ( lanes 1315 ) . finally , since hierarchical cluster analysis of the nci-60 
 data 
 for 3 indicated a moderately close link between the in vitro cellular target(s ) of the au macrocycle 
 and zorubicin ( a top2 poison ) , we evaluated the compound s 
 ability to inhibit top2. the results ( figure 
 s55 ) indicate that 3 is a weak catalytic inhibitor 
 of the enzyme , as evidenced by an inhibitory effect commencing only 
 at a relatively high compound dose of 50 m . since no linear 
 dna was detected in the assay , compound 3
evidently , 3 does not target type ii topoisomerases 
 to the same extent as top1 . from this
if top1 s 
 dna substrate 
 is targeted by 3 , then where does intercalative binding 
 of 3 occur ?
one can not naively assume that because 3 is a type 1 cic of top1 ( figure 1 ) , it binds to precisely the same nucleotide sequence as the enzyme . 

we have used a conformational search strategy with an empirical force 
 field parametrized for macromolecular simulations to accurately locate 
 the binding site of 3 . because most molecular mechanics 
 ( mm ) simulation programs lack parameters for metal complexes , the 
 x - ray data for 13 ( nine independent 
 experimental structures ) were first used to develop a force field 
 for the au macrocycles .
we augmented the sp4 force field 
 ( within ammp ) with parameters to model 15 and related au chelates . 
 the new force field rivals density functional theory ( dft ) in structural 
 accuracy but at a fraction of the computational cost ( table s2 ; figures s5 and s6 ) .
we then devised 
 a conformational search strategy ( figures 7a and s7 ) to determine the energetically 
 preferred intercalation point for 3 along the length 
 of the 22-bp dna duplex typically employed for x - ray structure determinations 
 of top1 with its dna substrate ( figure 7d ) .
( in this respect it is noteworthy that classical 
 force fields perform surprisingly well when compared with computationally 
 expensive high - level quantum chemical simulations for calculations 
 of both the structure of dna and , importantly , base - pair stacking 
 energies . )
( a ) conformational 
 search strategy with a 22-bp dna duplex sequence [ part ( d ) ] favored 
 by top1 and a modified sp4 force field for macromolecular simulations
. 
 a macrocycle such as 3 may intercalate between an adjacent 
 base pair in one of four energetically distinct ways .
the search proceeds 
 in a manner akin to stepping down the rungs of a ladder .
the 
 compound intercalates at the 5-ta-3 site ( t10-a11 ) 
 via major groove entry .
( c ) structure of the next - lowest energy conformation 
 ( minor groove t10-a11 intercalation adduct ) of dna3 .
the higher energy of this adduct reflects , in part , the non - planar 
 conformation for the dna - bound au macrocycle relative 
 to the global minimum .
( d ) graph of thermodynamic stability vs base 
 pair ( left ) for the 22-bp dna duplex .
ghelix is the empirical free energy penalty for strand separation 
 at the specified base pair .
right : graph of the relative energies 
 of all simulated dna3 intercalation adducts vs 
 ghelix .
the best - fit straight line 
 for the major groove adducts is given by : erel = 154(6 ) ( ghelix ) 
the lowest - energy conformation 
 ( figure 7b , table s5 ) has 3 intercalated 
 at a 5-ta-3 site ( t10-a11 ) via the major groove .
the 
 butyl chain of the macrocycle juts out into the major groove , oriented 
 downstream ( 5 to 3 ) .
the next lowest - energy conformation 
 ( erel = 45.6 kj mol , figure 7c ) has 3 intercalated 
 via the minor groove ( butyl chain oriented downstream ) at the same 
 dinucleotide pair ( t10-a11 ) .
minor groove intercalation yields a less 
 stable non - covalent adduct than major groove intercalation at a ta 
 step for several reasons , as depicted in figure 8 ( and figures s46 and s47 ) and enumerated 
 at this juncture .
( 1 ) in contrast to the major groove intercalation 
 adduct where both pyrrole rings and parts of the quinoxaline ring 
 of 3 are involved in  stacking 
 with a and t , only the edges of the quinoxaline ring of 3 interact with these bases in the minor groove intercalation adduct . 

( 2 ) the extent of insertion of the macrocycle into the intrahelical 
 space is markedly less in the minor groove intercalation adduct .
( 3 ) 
 the au macrocycle is forced to adopt a somewhat more 
 distorted ( non - planar ) conformation in the minor groove intercalation 
 adduct , which leads to a 5.58 kj mol increase 
 in the torsional strain energy relative to the conformer that best - fits 
 the binding pocket by major groove entry .
( the total energy difference 
 between the two au macrocycle conformations is 4.94 kj 
 mol , with the conformer intercalated via major 
 groove entry lowest in energy . ) ( 4 ) electrostatic interactions between 
 the au ion and heteroatoms ( o , n ) of either a or t in 
 the minor groove adduct are absent ( figure 8b ) .
significantly , when embedded within the t10-a11 binding pocket 
 the lowest - energy major groove intercalation adduct of 3 exhibits a short auo contact ( 3.13  ) involving 
 the nearest carbonyl oxygen of t10 and the au ion .
this 
 electrostatic interaction ( primarily an ion  dipole attraction ) 
 evidently makes a vital contribution to the overall stability of the 
 dna3 non - covalent complex .
perhaps most significantly , 
 the conformational search gives the base pair specificity of 3 when the unique intercalation adducts are ordered from lowest 
 energy ( highest affinity ) to highest energy ( lowest affinity):where superscripts 
 ( j ) and ( i ) are major and 
 minor groove insertions , respectively .
independent verification 
 of the above prediction that the intercalation 
 site for 3 ( t10-a11 ) precisely matches top1 s 
 target site for strand scission is mandatory .
the experimental thermodynamic 
 stabilities of the 10 possible nearest - neighbor interactions ( e.g. , 
 aa / tt , at / ta , etc . ) present in a dna duplex are known and underpin the empirical algorithm used to calculate 
 the free energy penalty for helix unwinding ( ghelix ) by the program web - thermodyn .
we used this method with a sliding window of two adjacent 
 nucleotides to analyze the 22-bp dna duplex of interest ( figure 7d ) .
as shown by the bar graph , the stability of 
 the adjacent nucleotide pairs follows the order : ta < at < ac 
 < ag  ct < aa = tt < ga . the free energy penalty 
 for separating the nucleotide pair 5-ta-3 is thus 
 the smallest ( < 1 kj mol ) . the fact that top1 
 has evolved to effect single - strand scission of duplex dna at a 5-ta-3 
 site , a thermodynamic weak point in the double helix , is noteworthy . 

it is also clear that a metallointercalator such as 3 similarly targets a 5-ta-3 site in duplex dna and 
 that because 3 and top1 share the same dna target ( substrate ) , 
 compound 3 will necessarily function as a type 1 unconventional 
 catalytic inhibitor of the enzyme .
the scatter graph ( figure 7d ) compares the 
 relative energies of the dna3 adducts with ghelix for dinucleotide pair separation .
a good 
 linear relationship exists for adducts formed by binding / entry of 
 the intercalator via the major groove ( but not the minor groove ) . 
 clearly , the stability order ( base pair specificity ) for the major 
 groove dna intercalation adducts of 3 parallels the thermodynamic 
 stability of the dinucleotide pairs along the 22-bp sequence because 
 the intercalator binds via the major groove and has to part the bases , 
 thereby locally unwinding the helix at 
 the intercalation point .
both major and 
 minor groove intercalation 
 of 3 at the energetically favored t10-a11 step in the 
 22-bp duplex lead to a number of characteristic conformational perturbations 
 of the dna .
we used the program w3dna to quantify 
 these effects at the level of individual steps ( i.e. , complementary 
 base pairs ) using the ammp - calculated structure of the intercalator - free 
 22-bp dna duplex ( shown in figure 7a ) as a 
 conformational reference .
figure 9 graphically 
 depicts the most significant of the metallointercalator - induced conformational 
 perturbations .
relative to the reference b - form 22-bp duplex , which 
 has a mean propeller twist ,  , of 11.9(2) , the 
 binding of 3 by either major or minor groove intercalation 
 at the t10-a11 step shifts the propeller twist from the range 30 
 to 15 to between 5 and 0 immediately after 
 the intercalation point , after which  returns to ca .
the reduction in propeller twist 
 reflects the more regular , coplanar arrangement of the dna bases which 
 come into direct contact with the aromatic intercalator through -stacking ; 
 the local ordering of the bases evidently extends from a11 to g12 , 
 but no further .
figure 9b highlights the marked 
 buckling of the base pairs induced by intercalation of 3 at the t10-a11 step through either the major or the minor groove . 

the mean buckling parameter ,  , for the reference b - form 22-bp 
 duplex measures 0(3) , as expected for an unperturbed b - form 
 dna conformation .
the intercalation of 3 , in contrast , leads to substantial buckling of the base 
 pairs up- and downstream of the intercalation point with  averaging 
 3(8) over the full 22-bp duplex for both intercalation 
 adducts .
+ 12 immediately 
 downstream of the t10-a11 step is pronounced ; such a conformational 
 perturbation has been previously noted for the structure of adriamycin - intercalated 
 dna .
the intercalation of 3 at the t10-a11 step is further characterized by reverse opening 
 (  ) of the 10-ta and 11-at base pairs ( max = + 10.1 for the 10-ta pair of the minor groove adduct ) relative 
 to the reference b - form 22-bp duplex for which  = 10(1) 
 ( figure 9c ) .
finally , as expected the rise 
 between adjacent bases in the stack , z , increases 
 at the intercalation point because the intercalator assumes the position 
 of a hydrogen - bonded base pair in the stack .
this is depicted graphically in figure 9c 
 where the 11-at pair clearly exhibits z values 
 of 7.2 and 7.0  for the major and minor groove intercalation 
 adducts , respectively .
the calculated z values 
 are consistent with the experimental values for base rise that accompany 
 the presence of a dna - bound intercalator ( ca .
( a ) view ( roughly perpendicular to the aun4 plane ) of 
 the ammp / sp4-simulated 
 structure of the binding pocket ( t10-a11 ) of the lowest - energy intercalation 
 adduct of 3 with the 22-bp dna duplex given in figure 7d .
the -stacking between the bases and the 
 pyrrole and quinoxaline rings of 3 is highlighted along 
 with the juxtaposition of a t10 carbonyl oxygen atom ( red sphere ) 
 and the au ion ( yellow sphere ) of the intercalator .
( b ) top view of the 
 minor groove intercalation adduct highlighting partial intercalation 
 of the au macrocycle and the practically negligible  
 overlap of the pyrrole rings with the bases of the binding pocket . 

this view explains why the minor groove t10-a11 intercalation adduct 
 is 45.6 kj mol higher in energy than the lowest - energy 
 conformation . in both structures ,
the dna is rendered as a stick model 
 in shades of gray and lilac , h atoms are omitted for clarity , and 3 is shown as a ball and stick model ( c , green ; n , blue ; au , 
 gold ) .
the two inset illustrations show edge - on projections viewed 
 from the major to minor groove of the upper structure in both cases 
 ( left image , van der waals radii including h atoms ; right image , ball 
 and cylinder model with h atoms omitted for clarity ) .
analysis of key conformational parameters for the two 
 lowest - energy 
 dna3 adducts formed by major and minor groove intercalation 
 of the au macrocycle at the t10-a11 step of the 22-bp 
 duplex depicted in figure 7 .
( a ) graph of propeller 
 twist (  ) as a function of base pair index for bases 419 . 

( b ) graph of buckling angle (  ) as a function of base pair index 
 for bases 419 .
( c ) bar graphs of base - pair opening (  , 
 left ) and rise ( z , right ) for selected base 
 pairs close to the intercalation point .
although a range 
 of base pair specificities seem to exist for metallointercalators 
 and are probably ligand and metal ion dependent , the recently determined 
 x - ray structure of dna - bound -[ru(phen)2(dppz ) ] , where phen is 1,10-phananthroline , 
 showed that the most symmetric ( of many ) intercalative binding modes 
 for this cationic minor groove metallointercalator involved a 5-ta-3 
 site in a palindromic duplex dna sequence that was characterized by 
 deep intromission of the dppz ligand into the dna intrahelical space 
 in a manner akin to the major groove intercalation adduct calculated 
 for 3 ( figure 8a ) .
( note that 
 the quinoxaline ring of 3 is essentially two - thirds of 
 the phenazine ring system in dppz , so the intercalative components 
 of the two ligand systems are to a substantial extent comparable . ) 
 interestingly , -[ru(phen)2(dppz ) ] did 
 not intercalate at a 5-at-3 site in an analogous palindromic 
 duplex dna sequence . despite the 
 different trajectories preferred for dna intercalation by 3 and -[ru(phen)2(dppz ) ] , the foregoing 
 experimental observation of ta over at specificity parallels the thermodynamic 
 dna base pair specificity determined here for 3 using 
 macromolecular simulations .
note that the coordinatively saturated 
 metal ion in -[ru(phen)2(dppz ) ] does 
 not interact with any dna bases and that the phen ligands seemingly 
 direct the trajectory of intercalation ( minor groove to major groove ) 
 through non - covalent interactions with the minor groove nucleotides 
 at the binding site .
interestingly , the x - ray structure of dna - bound 
 -[ru(bpy)2(dppz ) ] , where bpy is 2,2-bipyridine , 
 exhibits similar minor groove intercalation , but with a rather different 
 dinucleotide pair specificity to the former ru complex 
 ( binding at central and terminal 5-at-3 and 5-cg-3 
 steps , respectively , being favored ) . since 
 the two ru complexes under discussion differ only in 
 their ancillary ligand pairs ( phen vs bpy ) , the non - innocence of the 
 co - ligands in directing ( at least partly ) the site of dppz intercalation 
 is highlighted .
the intercalation mode calculated here for 3 is therefore distinct , more closely parallels that observed for 
 major groove ta intercalation by -[rh(bpy)2(chrysi ) ] ( where chrysi = chrysene-5,6-quinone diimine ) , and evidently reflects the fact that the au ion is square planar and housed within a macrocycle with 
 practically no steric bulk orthogonal to the mean plane of the quinoxaline 
 and pyrrole ring systems .
figure 8a gives a detailed view of the dinucleotide 
 binding 
 site of the lowest - energy structure of dna3 .
as 
 noted above , in addition to  stacking interactions 
 of adenine and thymine with the pyrrole rings of 3 , a 
 significant 3.13- oau contact involving a 
 carbonyl oxygen atom of t10 and the au ion of 3 indicates that the binding pocket interacts electrostatically with 
 the metal ion .
the simulations therefore explain why au is required for dna intercalation by the compounds . that 3 explicitly targets a ta rather than an at site is also apparent . 

specifically , if the order of the bases in the duplex is mutated to 
 a10-t11 in the simulation , formation of the key thymine oau contact is obviated ( figure s48 ) .
this a10-t11 intercalation adduct is , furthermore , 82.0 kj mol higher in energy than the global minimum energy structure 
 ( t10-a11 intercalation via the major groove ) .
if 3 binds at top1 s t10-a11 target site , can the enzyme still 
 recognize its substrate and form a covalent cleavage complex ?
we answered 
 these questions by simulating the structure of wttop1 non - covalently 
 bound to dna3 ( t10-a11 intercalation ) . from figure 10a , b , the quinoxaline ring of 3 protrudes 
 into the minor groove and sterically blocks the enzyme s probe 
 residue r364 from hydrogen bonding to n3 of g12 .
furthermore , 
 as shown by the overlay of the simulated structure of top1dna3 with the x - ray structure of 
 the top1 y723f mutant ( mtop1 ) bound to the 22-bp dna duplex ( figure 10b ) , intercalation 
 of 3 engenders partial unwinding of the dna helix downstream 
 of the intercalation point . the strand shift measured by
the displacement 
 of g12 is 3.53  , consistent with -stacked 3 assuming the position normally occupied by a nucleobase ( a11 ) and 
 the magnitude of the base rise ( z = 7.2  ; 
 figure 9c ) .
importantly , the scissile strand s 
 t10-a11 phosphodiester link shifts 1.52  downstream , potentially 
 thwarting attainment of the transition state for covalent tyr  o  p 
 bond formation .
our macromolecular simulations clearly predict that 
 top1 will neither bind its dna substrate nor form a cleavage complex 
 in the presence of 3 .
dna intercalation by 3 , 
 the ensuing structural perturbations , 
 and their impact on dna binding by top1 .
( a ) lowest - energy simulated 
 structure of the wild type top1dna3 ternary 
 complex illustrating steric displacement of arg-364 and asp-533 by 
 the quinoxaline ring of 3 protruding out the minor groove . 

these  probe residues  of the enzyme are key to dna 
 substrate recognition .
( b ) overlay of the simulated structure displayed 
 in part ( a ) with the x - ray structure of the y723f top1 mutant ( mtop1 ) 
 bound to dna ( pdb code : pdb1a36 ) .
( c ) dna binding by 
 mtop1 in the presence and absence of 3 determined by 
 spr ( surface plasmon resonance ; 37 c , 41 l min flow rate , ph 7.4 , 10% dmso ) .
the on - chip duplex dna sequence is 
 illustrated beneath the graph ; the enzyme s ta dinucleotide 
 target is highlighted .
the biotinylated 20-bp duplex was anchored 
 to avidin bound to the sensor chip surface .
a conceptual illustration 
 of the relevant equilibrium is given above the graph of spr response 
 functions .
the foregoing 
 predictions were experimentally verified by determining whether or 
 not mtop1 , a catalytically inactive mutant analogue of top1 , binds 
 to an oligonucleotide target fixed to a surface plasmon resonance 
 ( spr ) chip in the presence and absence of 3 ( figure 10c ) . the mutant enzyme ( mtop1 )
contains a point 
 mutation at the active site tyrosine ( y723f ) which does not appreciably 
 alter overall protein structure or dna binding affinity but destroys 
 the ability of the enzyme to engage dna in a cycle of cleavage and 
 religation ( which would complicate our analysis of dna binding ) . in 
 this experiment ,
the surface of the spr chip was derivatized with 
 a biotinylated 20-bp dna duplex before passing a solution of mtop1 
 over the chip .
an spr response function commensurate with protein 
 uptake ( 40100 s ) followed by saturation ( 100160 s ) 
 and desorption ( > 160 s ) was obtained .
after flushing the chip with 
 buffer , a solution of mtop1 and 3 ( 50 nm ) was passed 
 over the chip as before .
no spr response was detected ( baseline signal 
 in figure 10c ) , consistent with fast uptake 
 of 3 by the dna and inhibition of enzyme binding by the 
 immobilized dna3 intercalation adduct .
finally , we garnered experimental proof that 3 binds 
 to the ta sites of the 20-bp oligonucleotide .
specifically , a solution 
 of 3 passed over the dna - embellished spr chip afforded 
 three stepwise association response functions prior to discrete desorption 
 of 3 ( figure s50 ) .
the data 
 reflect the presence of the three 5-ta-3 sites along 
 the synthetic 20-bp dna duplex and the fact that each has a unique 
 microscopic affinity constant for 3 .
the macroscopic kd values for 1 and 3 ( table s4 and figure s53 ) were 2.8 and 
 3.4 m , respectively , broadly in accord with their ctdna affinity 
 constants ( figure 3a ) .
importantly , 3 did not bind to mtop1 ( figure s52 ) , indicating 
 that the enzyme is neither a primary nor a secondary target for the 
 compound . based on the above spr data , dna binding experiments , 
 and enzyme 
 targeting assays
, the lead compound of this study may be confidently 
 assigned as an unconventional type 1 catalytic inhibitor of top1 . 

going forward , it will be interesting to ascertain whether the in vitro mechanism of action for 3 applies in vivo ( i.e. , in a chromatin setting ) .
although such assays 
 exist for ifps of top1 , non - emissive 
 top1 cics such as 15 , which are 
 unsuitable for confocal microscopy , currently present several unmet 
 challenges regarding detection of endogenous top1 inhibition within 
 cell nuclei .
in summary , we have 
 synthesized and characterized a new class of 
 nominally planar cationic au macrocycles that incorporate 
 two pyrrole - imine units linked to a quinoxaline moiety on one side 
 and an alkyl chain bridge on the opposite side . from inception ,
physical 
 measurements of dna binding by the compounds indicate that they are 
 intercalators with high affinity constants ( ka > 10 m bp ) for ctdna that 
 correlate interdependently with the lipophilicity of the salt and 
 the steric bulk of the alkyl chain bridge within the macrocycle .
hierarchical 
 cluster analysis of nci-60 cytotoxicity data for the most active compound 
 ( salt 3 ) indicated that 3 correlates most 
 closely with the topoisomerase ib ( top1 ) poison camptothecin .
several 
 topical enzyme inhibition assays were used to prove that 3 is a catalytic inhibitor ( and not a poison ) of top1 .
since catalytic 
 inhibition of human topoisomerase ii ( top2 ) by 3 was 2 orders of magnitude weaker than its inhibition of 
 top1 , compound 3 is a type i - specific agent .
new 
 mm parameters were developed for the sp4 force field for macromolecular 
 simulations from the nine independent x - ray structures of the au macrocycles determined herein for parametrization .
a conformational 
 search strategy was devised to locate the lowest energy intercalation 
 site ( adjacent nucleobase pair ) within a 22-bp dna duplex commonly 
 used as a top1 substrate .
the simulations showed that 3 intercalates dna at the enzyme s 5-ta-3 dinucleotide 
 target sequence via major groove entry ( the minor groove adduct being 
 > 45 kj mol higher in energy ) and that a crucial 
 auo electrostatic interaction accounts for the observed 
 base pair specificity .
macromolecular simulations of a ternary non - covalent 3dnatop1 complex suggested that the molecular 
 mechanism of action of dna - bound 3 is to block substrate 
 recognition by the enzyme through steric repulsion .
surface plasmon 
 resonance studies confirmed ( 1 ) that top1 fails to bind its dna substrate 
 in the presence of 3 , ( 2 ) that 3 does not 
 bind to top1 itself , and ( 3 ) that the base specificity of 3 deduced by the macromolecular simulations ( ta ) is correct .
the overarching conclusion of this multifaceted study is that the 
 most cytotoxic au macrocycle , lead compound 3 , is an unconventional type 1 catalytic inhibitor of human top1 .
 topoisomerase 
 ib ( top1 ) is a key eukaryotic nuclear enzyme that 
 regulates the topology of dna during replication and gene transcription . 
 
 anticancer drugs that block top1 are either well - characterized interfacial 
 poisons or lesser - known catalytic inhibitor compounds . here 
 we describe 
 a new class of cytotoxic redox - stable cationic au3 + macrocycles 
 which , through hierarchical cluster analysis of cytotoxicity data 
 for the lead compound , 3 , were identified as either poisons 
 or inhibitors of top1 . 
 two pivotal enzyme inhibition assays prove 
 that the compounds are true catalytic inhibitors of top1 . 
 inhibition 
 of human topoisomerase ii ( top2 ) by 3 was 
 2 orders of magnitude weaker than its inhibition of top1 , confirming 
 that 3 is a type i - specific catalytic inhibitor . 
 importantly , 
 au3 + is essential for both dna intercalation and enzyme 
 inhibition . 
 macromolecular simulations show that 3 intercalates 
 directly at the 5-ta-3 dinucleotide sequence targeted 
 by top1 via crucial electrostatic interactions , which include  
 stacking and an auo contact involving a thymine 
 carbonyl group , resolving the ambiguity of conventional ( drug binds 
 protein ) vs unconventional ( drug binds substrate ) catalytic inhibition 
 of the enzyme . 
 surface plasmon resonance studies confirm the molecular 
 mechanism of action elucidated by the simulations . 
Introduction
Results and Discussion
Conclusions