neuropilin 1 ( nrp1a)(1)aabbreviations : g , glycine ; p , proline ; a , alanine ; v , valine ; l , leucine ; i , isoleucine ; m , methionine ; c , cysteine ; f , phenylalanine ; y , tyrosine ; w , tryptophan ; h , histidine ; k , lysine ; r , arginine ; q , glutamine ; n , asparagine ; e , glutamic acid ; d , aspartic acid ; s , serine ; t , threonine ; bt , biotinylated ; boc , tert - butyloxycarbonyl ; bsa , bovine serum albumin ; dmem , dulbecco s modified eagle s medium ; dmso , dimethyl sulfoxide ; ebm , endothelial cell basal medium ; elisa , enzyme - linked immunosorbent assay ; fbs , fetal bovine serum ; fmoc , 9-fluorenylmethyl)oxy]carbonyl ; 5-fu , 5-fluorouracil ; hepes , n-2-hydroxyethylpiperazine - n-2-ethanesulfonic acid ; hsqc , heteronuclear single quantum coherence ; huvecs , human umbilical vein endothelial cells ; nrp1 , neuropilin 1 ; nrp2 , neuropilin 2 ; pbf , 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl ; pbs , phosphate buffered saline ; pybrop , bromo - tris - pyrrolidinophosphonium hexafluorophosphate ; pae , porcine aortic endothelial ; sds , sodium dodecyl sulfate ; sema3a , semaphorin 3a ; tips , triisopropylsilane ; tfa , trifluoroacetic acid ; tris , tris(hydroxymethyl)aminomethane ; vegf , vascular endothelial growth factor ; vegfr2 , vascular endothelial growth factor receptor 2 . is a receptor for vascular endothelial growth factor a165 ( vegf - a165 ) and the neuronal guidance molecule semaphorin 3a ( sema3a)(2 ) with key roles in vascular and neuronal development ( figure 1 ) . in endothelial cells , nrp1 enhances the biological signals of vegf - a mediated by binding to its receptor vascular endothelial growth factor 2 ( vegfr2 ) .
nrp1 has also been implicated in tumor growth and angiogenesis ; inhibition by a blocking antibody that prevents vegf - a binding to nrp1 enhanced the antitumor effects of the inhibitory anti - vegf - a antibody , bevacizumab , in mouse xenograft models.(3 ) as an alternative to biological therapeutics , small molecule inhibitors of nrp1 function would be desirable , but development of proteinprotein interaction inhibitors is not a trivial task .
we utilized the bicyclic peptide 1 , corresponding to the c - terminal 28 amino acids of vegf - a165 ( figure 2 ) as a starting point for small molecule design . from this peptide we developed eg00229 , 2 ( figure 2 ) , a small molecule designed to interact with the vegf - a165 binding pocket of nrp1 .
mutational analysis , nmr , and x - ray crystallography establish that the interaction with nrp1 of peptide ligands ( and by inference vegf - a ) and the new small molecules described herein is with the same binding site formed by the loops at the end of the b1 domain.(6 ) these molecules act as inhibitors of vegf - a function , reducing vegf - a receptor phosphorylation and endothelial cell migration .
the in vitro cytotoxic effect of paclitaxel and 5-fluorouracil was enhanced in the presence of 2 .
abbreviations : g , glycine ; p , proline ; a , alanine ; v , valine ; l , leucine ; i , isoleucine ; m , methionine ; c , cysteine ; f , phenylalanine ; y , tyrosine ; w , tryptophan ; h , histidine ; k , lysine ; r , arginine ; q , glutamine ; n , asparagine ; e , glutamic acid ; d , aspartic acid ; s , serine ; t , threonine ; bt , biotinylated ; boc , tert - butyloxycarbonyl ; bsa , bovine serum albumin ; dmem , dulbecco s modified eagle s medium ; dmso , dimethyl sulfoxide ; ebm , endothelial cell basal medium ; elisa , enzyme - linked immunosorbent assay ; fbs , fetal bovine serum ; fmoc , 9-fluorenylmethyl)oxy]carbonyl ; 5-fu , 5-fluorouracil ; hepes , n-2-hydroxyethylpiperazine - n-2-ethanesulfonic acid ; hsqc , heteronuclear single quantum coherence ; huvecs , human umbilical vein endothelial cells ; nrp1 , neuropilin 1 ; nrp2 , neuropilin 2 ; pbf , 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl ; pbs , phosphate buffered saline ; pybrop , bromo - tris - pyrrolidinophosphonium hexafluorophosphate ; pae , porcine aortic endothelial ; sds , sodium dodecyl sulfate ; sema3a , semaphorin 3a ; tips , triisopropylsilane ; tfa , trifluoroacetic acid ; tris , tris(hydroxymethyl)aminomethane ; vegf , vascular endothelial growth factor ; vegfr2 , vascular endothelial growth factor receptor 2 .
nrp1 has a large extracellular ( ex ) domain comprising tandem a1/a2 , b1/2 , and a c domain , a single membrane - spanning domain , and a small cytosolic domain ( cyt ) .
the vegf - a165 c - terminal domain encoded by exons 7 and 8 ( yellow and blue oblongs , respectively ) binds to the extracellular nrp1 b1 domain .
concomitant binding of the vegf homology domain of vegf - a165 ( solid red ovals ) to vegfr2 results in formation of a receptor complex of nrp1 with vegf - a165 and vegfr2 and enhanced intracellular signaling , essential for optimal migration and angiogenesis in development and in tumors .
bicyclic peptide 1 ( c - terminus of vegf ) and small molecule neuropilin inhibitor 2 .
the reported crystal structures and our own computational analysis of the nrp b1 domain using sybyl siteid identified the cleft formed by the loops at one end of the -barrel as a potential binding site ( figure 3a ) .
residues clustered in this region(8 ) were conserved in mammalian nrp1 species and in human nrp2 , a closely related receptor for vegf - a ( figure 3b ) , implying an important functional role in vegf - a binding .
mutational analysis of vegf - a binding to nrp1 was therefore performed to confirm the identity of the binding pocket .
alanine substitution of amino acid y297 , w301 , t316 , d320 , s346 , t349 , y353 , or w411 resulted in complete loss of high affinity biotinylated - vegf - a binding to cos-7 cells transfected with mutant nrp1 cdna constructs ( figure 4a ) .
in addition , alanine substitution of k351 resulted in partial loss of vegf - a binding , while mutation of t337 , p398 , and s416 caused modest decreases in binding and mutation of e319 had no effect ( figure 4a and supporting information figure s1a ) .
loss of binding was not due to impaired expression of nrp1 mutants , as western blot analysis of transfected cos-7 cells indicated similar levels of protein expression of all constructs ( supporting information figure s1b ) . a triple mutant b1 protein ( s346a , e348a , t349a ) was previously shown to prevent vegf - a binding to rat nrp1.(7 ) ( a ) vegf / tuftsin binding site of nrp1 b1 domain ( black arrow ) , with the protein surface and the loops l1 ( green ) , l2 ( yellow ) , l3 ( cyan ) , l4 ( pink ) , l5 ( red ) , and l6 ( black ) shown . model constructed from pdb code 2orz .
( b ) protein sequence alignment of human , mouse , and rat nrp1 ( hnrp1 , mnrp1 , rnrp1 ) with human nrp2 ( hnrp2 ) .
highlighted residues were predicted to be in close contact with bound ligand from the model in panel a. ( a ) mutational analysis of the nrp1 pocket .
cos-7 cells were transfected with expression plasmids for wild - type ( wt ) or mutant nrp1 as indicated .
values presented are the means  sd obtained from three to six independent experiments each performed in duplicate .
( b ) an excerpted region of the 2d n , h hsqc nmr spectrum of human nrp1 b1 in the free state ( black contours ) and with saturating concentrations of tuftsin ( blue ) and 1 ( green ) .
the cross - peak assignments for the free - state nrp1 b1 are indicated in bold .
( c ) solvent accessible surface representations of the structure nrp1 b1 with color highlighting showing those residues whose backbone amide nh cross - peak exhibits a significant chemical shift perturbation ( compound  > 0.15 ; see experimental section ) upon addition of a saturating concentration of tuftsin .
the intensity of the color is scaled to the residue - by - residue value of the compound . ( e ) pymol diagram showing positions of mutated residues on the nrp1 b1 protein surface .
mutations giving 100% inhibition of binding are shown as red , and moderate inhibition is shown as salmon ( k351 ) .
the binding of ligands was assessed by chemical shift perturbation mapping with 2d n , h nmr of n - labeled nrp1 b1 ( figure 4b ) . in each case the residues with significant perturbations of the backbone nh cross - peak chemical shifts were identified ( compound  > 0.15 ; see experimental section ) .
we employed tuftsin as a control ligand for these studies because its binding site has been established by x - ray crystallography.(7 ) titration with 1 , tuftsin ( h - tkpr - oh ) , and the linear peptide ac - dkprr - oh ( c - terminal five residues of vegf - a ) yielded highly similar patterns of chemical shift changes that mapped the vegf - a interaction site ( figure 4c , d and supporting information figure s2 ) .
the largest chemical shift changes were observed for the backbone nh groups of w301 ( adjacent to c - terminal end of loop 1 ) ; t316 , g318 , y322 ( loop 5 ) ; a344 , i345 , k347 , t349 , k351 ( loop 3 ) ; and g414 , m417 ( loop 4 ) ( supporting information figure s3 ) . the binding site revealed by nmr
was identical to that predicted(6 ) and previously shown for tuftsin(7 ) and very similar to a map of the key residues essential for binding ( figure 4e ) .
these data clearly indicate that 1 and the peptide ac - dkprr - oh bind in a common mode to the same site as tuftsin and vegf - a , providing a strong rationale for modeling the interaction of small molecule inhibitors with this binding pocket .
these data imply that small molecules blocking this site could interfere with the binding ( and function ) of vegf , and other molecules that bind this site .
simple linear peptides were synthesized by standard 9-fluorenylmethoxycarbonyl ( fmoc ) solid phase methodology as previously reported.(9 ) for the small molecules a highly flexible mixed solution phase / solid phase synthesis was devised that allowed the preparation of multiple analogues in a timely fashion . the lysine analogue and linking scaffold
were assembled in solution phase and then coupled in one unit to the arginine - o - wang resin using standard coupling conditions ( scheme 1 ) .
a representative example is shown in scheme 2 , which illustrates the range of chemical manipulations applied to different building blocks .
thus , suzuki coupling of 3-nitroiodobenzene 3 with 3-methyphenylboronic acid 4 provided the biphenyl derivative 5 which was oxidized to the acid 6 using potassium permanganate .
coupling to preformed wang - arg resin with the reactive coupling agent bromo - tris - pyrrolidinophosphonium hexafluorophosphate ( pybrop ) gave the nitro compound 7 linked to solid phase .
tin(ii ) chloride reduction followed by coupling with the boc ( tert - butyloxycarbonyl ) protected acid gave the intermediate 8 .
the target compound 9 was then produced by deprotection with trifluoroacetic acid / triisopropylsilane ( tfa / tips ) .
for further testing larger amounts of compounds were required , and these were synthesized by solution phase chemistry as shown in scheme 3 for 2 .
reaction of the thiadiazolesulfonyl chloride 10 with methyl 3-aminothiophene-2-carboxylate 11 was achieved in pyridine at room temperature to give the sulfonamide 12 .
hydrolysis to the acid 13 with lithium hydroxide and amide coupling of 13 with the 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl ( pbf ) protected arginine methyl ester 14 gave 15 .
reagents : ( a ) pd2(dba)3 , pd(pph3)4 , kf , dioxane , reflux 24 h ; ( b ) kmno4 , py , h2o , reflux 3 h ; ( c ) hobt , dci , dmf , wang - arg - oh ; ( d)sncl2h2o , dmf ; ( e ) pybrop , rcooh , 2,6-lutidine , ch2cl2 ; ( f ) tfa , tips , h2o .
reagents : ( a ) pyridine , 20 c ; ( b ) liohh2o , 50 c , thf , meoh , h2o ; ( c ) pybrop , dipea , dcm , 20 c ; ( d ) tfa / h2o 9:1 .
our strategy for designing small molecule inhibitors was initially to perform structureactivity relationship studies of short peptides in assays of vegf - a binding to porcine aortic endothelial ( pae ) cells expressing nrp1 in the absence of other vegf receptors ( pae / nrp1 ) and thereby to identify the essential properties of peptides necessary for inhibition of binding . with this information
, we then constructed a design template for the peptidenrp1 interaction ( scheme 1 , box ) and undertook a systematic search for chemistry scaffolds that would retain the high affinity of the peptides in binding assays .
the c - terminal peptide ac - rxdkprr(9 ) of vegf - a retains significant affinity for nrp1 .
substitution of the nonessential penultimate arginine to alanine 16 and the synthesis of increasingly n terminal truncated peptides produced h - kpar - oh , 17 , a minimal length significantly active peptide ( table 1 ) .
substitution with d - amino acids for the lysine or alanine 18 , 19 lowered inhibitory activity .
the penultimate position was tolerant of proline 20 and phenylalanine 21 in addition to alanine .
the terminal arginine was essential for activity , as replacement with d - arginine 22 , lysine 23 , or glutamine 24 removed activity .
the essential c - terminal arginine was retained with different chemical scaffolds being used to connect it to the lysine ( equivalent to k162 in vegf - a165 ) ( table 2 ) .
these scaffolds were selected on the basis of an approximate modeling fit of the full small molecule mimetic with the h - kpar - oh peptide , and a representative subset is shown .
biphenyl scaffolds 9 , 25 , furanylenephenyl 26 , and cycloalkylaryl scaffolds 27 , 28 , 29 gave modest inhibitions .
however a thiophene - based scaffold 30 had an ic50 of 13 m for inhibition of vegf - a165 binding to nrp1 .
follow - up studies showed that other five - membered ring heterocycles were less effective ( data not shown ) .
reversal of the sulfonamide linkage 31 or replacement of the thiophene with a phenyl ring 32 removed activity , as did removal of the 4-aminophenylsulfonamide group 33 .
the best scaffold exemplified by 30 was progressed with an extensive structureactivity study ; salient features are shown ( table 3 ) .
thus , h - bond acceptor groups in the 2 or 3 position of the aromatic or aryl group were beneficial , 34 and 35 , whereas methylation of the sulfonamide nh removed activity , 36 and 40 .
other 4-substitutions were tolerated , 37 and 38 , while a 2-nitro group , 39 , showed the best activity of the simple substituents .
insertion of a benzothiadiazole heteroaryl produced compound 2 , with an ic50 of 8 m in a cell based assay .
the sulfur atom had a positive effect on potency because the o analogue 42 was less active .
most alterations to the arginine , including the unnatural d - enantiomer 45 or 46 ( table 4 ) or substitution by lysine 47 , resulted in a partial or complete loss of activity ( table 4 ) .
the methyl ester 48 had poor activity , while replacement of the alkyl side chain by phenyl , 49 , also reduced activity .
extension of the alkyl chain to four carbons , 50 , again reduced activity ; however , introduction of a carbonyl group into the side chain of the arginine 51 was partially tolerated .
further manipulations to the carboxylic acid group in the thiadiazole series , such as reduction to the alcohol 54 or conversion to an amino acid mimic 55 were deleterious .
thus , 2 was identified as a non - peptide antagonist of vegf - a binding to nrp1 .
pae / nrp1 cells in 24-well plates were incubated with different concentrations ( 0.1100 m ) of compounds followed by addition of 0.1 nm i - vegf - a165 .
results were obtained from three independent experiments , each performed using triplicate determinations at each concentration of compound , and are presented as the mean  sem .
pae / nrp1 cells in 24-well plates were incubated with different concentrations ( 0.1100 m ) of compounds followed by addition of 0.1 nm i - vegf - a165 .
results were obtained from three independent experiments , each performed using triplicate determinations at each concentration of compound , and are presented as the mean  sem .
pae / nrp1 cells in 24-well plates were incubated with different concentrations ( 0.1100 m ) of compounds followed by addition of 0.1 nm i - vegf - a165 .
results were obtained from three independent experiments , each performed using triplicate determinations at each concentration of compound , and are presented as the mean  sem .
pae / nrp1 cells in 24-well plates were incubated with different concentrations ( 0.1100 m ) of compounds followed by addition of 0.1 nm i - vegf - a165 .
results were obtained from three independent experiments , each performed using triplicate determinations at each concentration of compound , and are presented as the mean  sem .
definitive evidence for the binding site of 2 was provided by monitoring the nrp1 b1 domain 2d n , h - heteronuclear single quantum coherence ( hsqc ) nmr spectrum in the presence of increasing concentrations of the ligand .
n , h hsqc spectra are universally used to monitor the local  chemical environment  of nh groups , and in a ligand titration , the exchange regime exhibited by the perturbed cross - peaks can be related to the ligand binding kinetics such that slowintermediate exchange behavior is typically observed for ligands with a slow off - rate consistent with tight binding .
consistent with the measured ic50 for nrp1 b1 binding , the nmr titration yielded intermediate - to - slow exchange characteristics for the majority of perturbed resonances .
both the distribution of residues that exhibited ligand - dependent chemical shift perturbations ( supporting information figures s2 and s3 ) and the directional pattern of those chemical shift changes ( supporting information figure s4 ) are highly similar to those obtained with 1 , ac - dkprr - oh , and tuftsin .
these data point to the conservation of interactions between the backbone and side chain elements of the terminal arginyl moiety of 2 with the binding pocket on nrp1 b1 . the precise binding mode and ligand conformation of 2
the crystal structure , containing a dimer in the asymmetric unit ( interchain c root - mean - square deviation of < 0.5  ) , was determined at 2.9  resolution , with electron density readily observable for 2 in the binding pockets of both noncrystallographic symmetry related molecules ( figure 5a ) .
the carboxylate group of 2 superimposes almost perfectly with the carboxylate group of tuftsin in the rat nrp1 b1b2 crystal structure ( supporting information figure s5a).(7 ) the guanidine groups of the two ligands also overlay .
the structure also reveals an intramolecular shared hydrogen bond ( figure 5b ) between the carbonyl amide nh and the sulfonamide nitrogen , providing a stable central conformation of the molecule .
a the benzothiadiazole ring adopts a conformation on the same side to the guanidine chain with respect to the central planar portion of the molecule containing the thiophene ring . in chain
the benzothiadiazole ring is involved in stacking interactions with a side chain residue . in chain a this interaction is provided by the m276b side chain of a symmetry related molecule ( not shown ) . in chain b the benzthiadiazole ring stacks against the n300b side chain ( supporting information figure s5b ) .
the part of the pocket binding the arginine seems to be an almost perfect  arginine receptor  , and few changes to the l - arginine were tolerated .
( a ) fom weighted difference map of 2-bound nrp1 b1 crystal structure calculated from the model refined without atomic coordinates for 2 . the calculated electron density ( green mesh )
clearly shows the presence of the ligand in two different conformations in the binding sites a and b. final stick models for 2 in chain a ( left ) and chain b ( right ) are overlaid over the density .
colors are green , red , blue , and yellow for carbon , oxygen , nitrogen , and sulfur , respectively .
the position of the ( putative ) shared hydrogen is indicated by a magenta sphere and arrow .
compound 2 selectively inhibited radiolabeled i - vegf - a binding to pae / nrp1 , but not vegfr2-expressing cells , with an ic50 of 8 m ( table 3 ) and inhibited bt - vegf - a binding to purified nrp1 b1 domain in a cell - free assay with an ic50 of 3 m ( figure 6a ) .
compound 2 also inhibited vegf - a binding to lung carcinoma a549 and prostate carcinoma du145 cells , which express nrp1 , but not vegfr1 and vegfr2 , with similar potency ( supporting information figure s6 ) .
binding of vegf - a to human umbilical vein endothelial cells ( huvecs ) , which express vegfr2 , vegfr1 , and nrp1 , was also inhibited by 2 with an ic50 of 23 m , the reduced potency reflecting the specificity of 2 for nrp1 and its inability to compete with vegf - a binding to other receptors ( figure s6 ) .
the potency of compound 2 in inhibiting vegf - a binding to nrp1 was thus very similar to that of the bicyclic peptide compound 1 , which inhibits i - vegf - a binding to pae / nrp1 and tumor cells with ic50 values of 23 m and inhibits i - vegf - a binding to huvecs with an ic50 of 20 m.(9 ) binding and functional activity of 2 .
( a ) 2 displaces biotinylated vegf from nrp1 b1 in a cell - free assay .
plates precoated with purified recombinant nrp1 b1 domain were incubated for 2 h at room temperature with 0.25 nm bt - vegf - a165 in the presence of the indicated concentrations of 2 , and specific vegf binding was determined .
values presented are the means  sem obtained from three independent experiments each performed in triplicate .
confluent huvecs were pretreated for 30 min with the indicated concentrations of 2 or with an equivalent volume of solvent ( dmso ) and were then treated with or without 25 ng / ml vegf - a165 for 5 min at 37 c .
total vegfr2 and vegfr2 phosphorylated at tyr1175 were then determined in treated cell lysates using a specific elisa .
values presented are the means  sem obtained from three independent experiments each performed in duplicate : (  ) p < 0.05 versus no 2 ; (  ) p < 0.01 versus no 2 ; (  ) p < 0.001 versus no 2 .
huvecs were pretreated for 30 min with the indicated concentrations of 2 or with an equivalent volume of solvent ( dmso ) and placed into top inserts .
the chemotaxis of cells toward either vegf at 25 ng / ml or medium control ( c ) in bottom wells was determined after 4 h of incubation .
values presented are the means  sem obtained from three independent experiments each performed in duplicate : (  ) p < 0.05 for 100 m versus 0 m 2 pretreated cells toward vegf .
a549 cells were incubated in serum - free medium containing paclitaxel at the indicated concentrations in the absence or presence of 100 m 2 .
values presented are the means  sem obtained from three independent experiments each performed in triplicate : (  ) p < 0.01 for the chemotherapeutic drug alone versus drug plus 2 .
the effects of 2 on vegf - a signaling and function were examined in human endothelial cells .
compound 2 reduced vegf - a - induced vegfr2 tyrosine phosphorylation in huvecs in a dose - dependent fashion , with a maximum inhibition of 34% at 100 m ( figure 6b and supporting information figure s7 ) . compound 2 also significantly reduced vegf - a induced migration of huvecs ( figure 6c ) .
these results agree well with our previous findings showing partial inhibition of vegf - a - induced vegfr2 tyrosine phosphorylation by the bicyclic peptide nrp1 antagonist 1 and partial inhibition of both vegfr2 activation and vegf - induced migration by an inhibitory nrp1 antibody .
overall , compound 2 caused a partial inhibition of vegf receptor activity and biological function , consistent with the current model for the role of nrp1 in vegf function , in which nrp1 is required for optimal signaling and certain biological functions downstream of vegfr2 , particularly migration.(1 ) the functional effect of 2 was also examined in a549 lung carcinoma cells , which express nrp1 but no detectable vegfr2 .
treatment of a549 cells with 2 caused a significant reduction in cell viability over a 48 h incubation ( supporting information figure s7 ) .
pretreatment of a549 cells with 2 significantly enhanced the cytotoxic effects of two widely used chemotherapeutic drugs , paclitaxel ( figure 6d ) and 5-fluorouracil ( supporting information figure s8 ) by approximately 4- and 10-fold , respectively . nrp1 down - regulation by sirna has previously been shown to increase the chemosensitivity of pancreatic cancer cells,(10 ) while cytotoxic synergy has been noted for vegf pathway inhibition with a combination of the multikinase inhibitor sorafenib and the proteasome inhibitor bortezomib.(11 )
compound 2 exhibited a biological profile consistent with inhibition of vegf - a binding to nrp1 demonstrating an impairment , but not complete inhibition , of cell migration and vegfr2 phosphorylation .
the viability of carcinoma cells was also decreased by 2 , and it enhanced the potency of cytotoxics .
nrp1 blockers have the potential to target both angiogenesis and tumor growth and metastasis and may provide a valuable alternative targeted cancer therapy , particularly relevant in the light of concern over recent findings showing that anti - vegf agents may stimulate metastasis.(12 ) compound 2 and its analogues make excellent tools for the study of nrp1 biology and provide a point of departure for development of more potent anti - nrp1 drugs in the future .
all commercially available solvents and reagents were used without further treatment as received unless otherwise noted .
nmr spectra were measured with a bruker drx 400 or 600 mhz spectrometer ; chemical shifts are expressed in ppm relative to tms as an internal standard and coupling constants ( j ) in hz .
mass spectra were obtained using a waters zq2000 single quadrupole mass spectrometer with electrospray ionization ( esi ) .
high resolution mass spectra were acquired on a waters lct time - of - flight mass spectrometer with electrospray ionization ( esi ) .
analysis of compounds by reverse - phase lcms was carried out on an analytical c18 column ( phenomenex gemini , 50 mm  3.0 mm , 5 m ) and an ab gradient of 595% for b at a flow rate of 1 ml / min , where eluent a was 0.1% formic acid / water and eluent b was 0.1% formic acid / acetonitrile
. silica gel ( 60  , 4063 m , fisher ) was used for flash column chromatography .
routine analytical thin layer chromatography was performed on precoated plates ( 60f254 , machery - nagel ) .
linear peptides were synthesized by fmoc solid - phase synthesis using wang or 2-chlorotrityl linkers in accordance with our previously reported methods.(9 ) the resins and the amino acid derivatives , fmoc - abu - oh , fmoc(me)-ala - oh , fmoc - ala - oh , fmoc - arg(pbf)-oh , fmoc - asp(otbu)-oh , fmoc - lys(boc)-oh , fmoc - phe - oh , and fmoc - pro - oh were purchased from calbiochem novabiochem ( nottingham , u.k . ) and bachem ag ( bubendorf , switzerland ) .
the biopolymer tools in sybyl were used to build some of the peptides , to check structures for errors , to correct atom types , and to add / remove hydrogens as necessary .
structures were minimized using the tripos force field with a steepest descent gradient of 100 iterations followed by a conjugate gradient of 0.01 kcal / mol or a maximum of 10000k iterations as termination criteria . during minimizations , a
12  nonbonded cutoff was applied and when solvent was not present and ionic strength was set to 4.00 .
the collected structural data were analyzed with the graphic tools of sybyl , version 7.0 .
all calculations were performed on the dual hewlett - packard workstation xw6000 , two 2.8 ghz cpus running redhat enterprise linux ws4 .
the possible binding sites of nrp1 b1 domain were searched using the sybyl siteid module ( sybyl , version 7.0 ; tripos inc . , st .
louis , mo ) . to a stirred solution of the arylsulfonyl chloride ( 10 ) ( 7.68 g , 32.7 mmol , 1.2 equiv ) in pyridine ( 30 ml ) under nitrogen ( balloon ) at 45 c was added dropwise over 1 h a solution of methyl 3-aminothiophene-2-carboxylate ( 11 ) ( 4.28 g , 27.3 mmol , 1 equiv ) in pyridine ( 10 ml ) . the resulting suspension was stirred for 20 h at 20 c .
the reaction was monitored using tlc and was deemed complete after approximately 2 h. water ( 25 ml ) was added and the resulting mixture stirred for 15 min .
the resulting pale - yellow precipitate was isolated by suction filtration and was washed with water ( 3  10 ml ) followed by isohexane ( 3  10 ml ) , then dried ( in vacuo ) to afford the desired product .
the ester 12 ( 9.28 g , 26.1 mmol , 1 equiv ) was stirred with lithium hydroxide monohydrate ( 5.48 g , 130.5 mmol , 5 equiv ) in a tetrahydrofuran / methanol / water mixture ( 5:3:2 ; 20 ml ) at 50 c for 4 h. the reaction mixture was monitored using tlc ; when deemed complete , the solvents were removed in vacuo .
the product was then directly isolated by precipitation using 2 m hydrochloric acid ( 100 ml ) followed by suction filtration .
the precipitate was washed with water ( 3  50 ml ) and then with isohexane ( 3  50 ml ) and dried in vacuo at 40 c for 14 h to afford the desired product .
h nmr ( 400 mhz , dmso - d6 ) :  10.43 ( 1h , br , s ) , 8.448.38 ( 2h , m ) , 7.86 ( 1h , d , j = 8.9 , 7.1 hz ) , 7.75 ( 1h , d , j = 5.5 hz ) , 7.25 ( 1h , d , j = 5.5 hz ) . the acid ( 13 ) ( 5.0 g ,
14.7 mmol , 1 equiv ) and bromo - tris - pyrrolidinophosphonium hexafluorophosphate ( pybrop , 8.25 g , 17.7 mmol , 1.2 equiv ) were suspended in dichloromethane ( 50 ml ) , under nitrogen ( balloon ) , and stirred for 2 min at 20 c .
n , n - diisopropylethylamine ( 23 ml , 132.3 mmol , 9 equiv ) was added in one portion and the orange solution stirred at room temperature for 10 min .
the protected amino acid ( h - arg(pbf)-ome ; 8.40 g , 17.7 mmol , 1.2 equiv ) was added as a single portion , and the reaction mixture was stirred at 20 c for 4 h. the solvent was removed in vacuo and the residue taken up in ethyl acetate ( approximately 200 ml ) .
the solution was washed with 1 m hydrochloric acid ( 10  50 ml ) and then evaporated directly to give a viscous brown gum that was maintained under vacuum for 17 h. the resulting brown glassy solid was carried forward without further purification .
the crude ester 15 ( 11.2 g , 14.7 mmol ) and lithium hydroxide hydrate ( 3.08 g , 73.5 mmol , 5 equiv ) were stirred in tetrahydrofuran / water ( 2:1 , 300 ml ) for 30 min at 20 c .
after this time , lcms indicated complete reaction , the organic solvent was removed in vacuo and hydrochloric acid ( 1 m , aqueous solution , 20 ml ) added to precipitate the product .
the resulting solid was collected by filtration and washed with 1 m hydrochloric acid ( 100 ml ) , water ( 2  100 ml ) , and isohexane ( 3  50 ml ) and then carried forward directly .
h nmr ( 400 mhz , dmso - d6 ) :  11.36 ( 1h , br
s ) , 8.428.34 ( 3h , m ) , 7.84 ( 1h , dd , j = 8.8 and 7.0 hz ) , 7.66 ( 1h , d , j = 5.3 hz ) , 7.19 ( 1h , d , j = 5.3 hz ) , 4.234.20 ( 1h , m ) , 3.063.01 ( 2h , m ) , 2.92 ( 2h , s ) , 2.46 ( 3h , s ) , 2.41 ( 3h , s ) , 1.95 ( 3h , s ) , 1.821.73 ( 1h , m ) , 1.691.60 ( 1h , m ) , 1.401.35 ( 8h , m ) . the acid ( 11.0 g , 14.7 mmol , 1 equiv ) was stirred with trifluoroacetic acid / water ( 9:1 , 150 ml ) for 65 h at 25 c . after this time
the acidic solution was added to an ice / water mixture , causing the immediate precipitation of a pale - green solid that was collected by filtration and washed copiously with ether before being suspended in further ether and subjected to ultrasonic irradiation for 5 min .
h nmr ( 400 mhz , ( cd3)2so / d2o ) :  8.268.23 ( 2h , m ) , 7.78 ( 1h , dd , j = 8.6 and 7.0 hz ) , 7.45 ( 1h , d , j = 5.3 hz ) , 7.17 ( 1h , d , j = 5.3 hz ) , 4.26 ( 1h , dd , j = 9.6 and 4.5 hz ) , 3.163.13 ( 2h , m ) , 1.881.81 ( 1h , m ) , 1.771.70 ( 1h , m ) , 1.601.54 ( 2h , m ) .
c nmr ( 400 mhz , ( cd3)2so ) :  173.0 , 163.2 , 156.8 , 154.7 , 148.1 , 131.9 , 130.1 , 128.7 , 127.0 , 119.8 , 113.7 , 52.0 , 40.3 , 27.3 , 25.4 .
ms m / z 498 [ m + 1 ] . hrms ( m / z ) : [ m ] calcd for c17h20n7o5s3 , 498.0688 ; found , 498.0697 .
nmr spectroscopy was performed at either 500 or 600 mhz h frequency at 25 c .
n or c , n - isotope labeled nrp1 b1 samples were prepared in 50 mm phosphate buffered saline ( 100 mm sodium chloride ) , 1.0 mm dithiothreitol , 1.0 mm ethylenediaminetetraacetic acid , ph 6.0 .
backbone resonance assignments were obtained by analysis of 3d hnca , hn(co)ca , hncacb , hnco , hn(ca)co spectra .
ligand titrations were monitored by 2d n , h hsqc spectroscopy of 0.3 mm nrp1 b1 to which aliquots of buffered ligand ( 200 mm ) were added until saturation was achieved;(13 ) the maximal protein concentration dilution was < 10% .
control experiments were recorded to assess the sensitivity of the nrp1 b1 cross - peak positions to small changes in protein concentration and sample ph .
nmr spectra were processed with nmrpipe(14 ) and analyzed in ccpn analysis.(15 ) cross - peak chemical shift perturbations were declared significant when the compound change in h and n resonance position  = { [ ( n/6.5 ) + h ] } > 0.15 where n and h are measured in ppm . essentially complete backbone nmr assignments were obtained by standard triple resonance nmr spectroscopy of a uniformly double c , n - isotope labeled sample of nrp1 b1 .
the pattern of secondary c chemical shifts obtained is consistent with the known globular structure of nrp1 b1 from x - ray diffraction studies , indicating that the overall structure in solution is unperturbed ( data not shown ) .
recombinant human nrp1-b1 domain was expressed in e. coli strain rosetta ( de3 ) ( novagen ) as a fusion protein containing a tobacco etch virus protease cleavable n - terminal hexahistidine tag .
the protein was purified by combination of immobilized metal affinity chromatography , size exclusion chromatography ( superdex75 ) , and ion exchange chromatography on a sp ff sepharose column .
purified nrp1-b1 protein in 20 mm tris - chloride ( ph 7.9 ) and 50 mm sodium chloride buffer was incubated with a 10-fold molar excess of 2 for 1 h at 4 c .
the protein was concentrated to 10 mg / ml and crystallized in a 1:1 volume mixture with 100 mm mes ( ph 6.0 ) , 10% peg 8k , and 200 mm zinc acetate at 20 c employing hanging drop vapor - diffusion method .
crystals appeared within 3 days , with thick needle morphology and a yellow hue , from compound 2 .
crystals belonged to space group p21 with cell dimensions of a = 40.96  , b = 89.60  , c = 41.60  ,  = 90.00 ,  = 97.72 ,  = 90.00 and contained two nrp1-b1 molecules in the asymmetric unit .
a data set at 2.9  resolution was obtained at the london school of pharmacy by using a r - axisiv image plate detector system and osmic mirrors .
data were merged and indexed using the program d*trek(16 ) in the crystalclear(17 ) software suite .
molecular replacement was performed using phaser(18 ) with apo nrp1-b1 domain ( pdb code 1kex ) .
iterative rounds of building and refinement were carried out in coot(19 ) and refmac5.(20 ) tls groups were generated using the tlsmd web server.(21 ) the dictionary file for 2 was generated using the prodrg web server.(22 ) the structure was refined to final r / rfree values of 21.6/28.2% with reasonable geometry ( table 5 ) .
highest - resolution shell is shown in parentheses . the full - length open reading frame of human nrp1 ( origene )
subsequently plasmid expression constructs ( pcdna3.2/v5-dest ) and adenoviral vectors encoding nrp1 constructs ( pad / cmv / v5-dest ) were generated by lr recombination using the gateway system ( invitrogen ) .
site - directed mutagenesis was performed with the quikchange xl site - directed mutagenesis kit ( stratagene ) .
recombinant human vegf ( vegf - a165 ) was obtained from r & d systems ( abingdon , u.k . ) .
collagen type i , dulbecco s modified eagle s medium ( dmem)/25 mm hepes , 5-fluorouracil , and paclitaxel were purchased from sigma - aldrich ( dorset , u.k . ) .
human umbilical vein endothelial cells ( huvecs ) were purchased from tcs cellworks ( buckingham , u.k . ) and cultured in endothelial cell basal medium ( ebm ) supplemented with 10% fetal bovine serum ( fbs ) , 10 ng / ml human epidermal growth factor , 12 g / ml bovine brain extract , 50 g / ml gentamicin sulfate , and 50 ng / ml amphotericin-8 .
porcine aortic endothelial cells expressing nrp1 ( pae / nrp1 ) were provided by dr .
the cells were grown in ham s f12 medium containing 10% fbs and 25 g / ml hygromycin b. human tumor cell lines including carcinoma lung a549 and prostate du145 cells were provided by quintiles limited ( edinburgh , u.k . ) and grown in rpmi 1640 medium ( invitrogen , paisley , u.k . ) containing 10% and l - glutamine .
huvecs or du145 cells were seeded at the density of 2  10 cells per well of 96-well plates in 0.1 ml growth medium and transfected with adenovirus vectors containing the full - length open - reading frame of human nrp1 .
cos7 cells were obtained from ecacc and maintained in dmem supplemented with 10% fbs ( invitrogen ) .
cos7 cells were grown in 24-well plates to 80% confluency and transfected with nrp1 plasmid constructs using lipofectamine 2000 ( invitrogen ) according to the manufacturer s instructions .
cells were extracted by lysis buffer ( 64 mm tris - hcl , ph 6.8 , 0.2 mm na3vo4 , 2% sodium dodecyl sulfate ( sds ) , 10% glycerol , protease inhibitors for serine , cysteine , and metalloproteases ) .
the whole cell lysate samples were separated by sdspage and transferred to immobilon membranes ( millipore ) .
immunoreactive bands were visualized by chemiluminescence using horseradish peroxidase - conjugated secondary antibodies and enhanced chemiluminescence reagent ( amersham biosciences , bucks , u.k . ) . radiolabeled binding displacement experiments
were performed in cells grown to confluence in 24-well plates and using the indicated concentrations of compounds followed by addition of 0.1 nm i - vegf - a165 ( 12001800 ci / mmol , ge healthcare , u.k . ) as described previously ( jia et al . , 2006).(9 )
( bt)-vegf - a165 binding , confluent adenovirus nrp1-transfected huvecs or du145 cells in 96-well plates were washed twice with phosphate - buffered saline ( pbs ) .
the various concentrations of compounds diluted in binding medium ( dulbecco s modified eagle s medium , 25 mm n-2-hydroxyethylpiperazine - n-2-ethanesulfonic acid ( hepes ) , ph 7.3 , containing 0.1% bovine serum albumin ( bsa ) ) were added , followed by addition of 2 nm bt - vegf - a165 .
after 2 h of incubation at room temperature , the medium was aspirated and washed three times with pbs .
the bound bt - vegf - a165 to nrp1 was detected by streptavidinhorseradish peroxidase conjugates and the enzyme substrate and measured using a tecan genios plate reader at 450 nm absorbance with a reference wavelength at 595 nm .
half maximal inhibitory concentrations ( ic50 ) for selected compounds were obtained from competition curves obtained from i - vegf - a165 and bt - vegf - a165 binding assays in which different concentrations ( 0.1100 m ) of compound were tested , using prism , version 4.0 , software and the equation where top and bottom are the responses , respectively , at the top and bottom of the curve , y is the inhibitory response at a given compound concentration x , and the hillslope describes the steepness of the curve .
r values measuring goodness of fit of the competition curves were also determined using the prism software .
the 96-well plates were precoated with nrp1-b1 protein at 3 g / ml overnight at 4 c . on the following day ,
the plates were treated with blocking buffer ( pbs containing 1% bsa ) and washed three times with wash buffer ( pbs containing 0.1% tween-20 ) .
the various concentrations of compounds diluted in pbs containing 1% dmso were added , followed by addition of 0.25 nm bt - vegf - a165 .
after 2 h of incubation at room temperature , the plates were washed three times with wash buffer .
the bt - vegf - a165 bound to nrp1-b1 was detected by streptavidinhorseradish peroxidase conjugates and the enzyme substrate and measured using a tecan genios plate reader at 450 nm absorbance with a reference wavelength at 595 nm .
nonspecific binding was determined in the absence of nrp1-b1 coated wells of the plates . following treatment of huvecs , phosphorylation at tyr1175 of vegfr2 and total vegfr2 in cell lysates
were detected by pathscan phopho - vegfr2 and total vegfr2 enzyme linked immunosorbant assay ( elisa ) kits ( cell signalling ) according to the manufacturer s instructions .
cell migration was measured in chemotaxis 24-transwell plates with collagen i - coated inserts incorporating polyethylene terephthalate track - etched membranes with 8 m pores ( becton dickinson biosciences ) .
cells were trypsinized , washed , and resuspended in ebm/0.1% bsa to give a final cell concentration of 3  10/ml . then
1.5  10 cells with or without 2 treatment as indicated were loaded into each top inserts .
the chemotaxis transwell plates were incubated at 37 c for 4 h. after the incubation , nonmigrated cells on the top side of the transwell membranes were removed and migrated cells on the under side of the transwell membranes were stained with the reastain quick - diff kit ( reagena , ltd . , toivala , finland ) .
the stained cells from each well were counted in four fields at 100 magnification using an eyepiece indexed graticule ( 100 grids ) .
cell viability was determined by measurement of conversion of the tetrazolium salt xtt to form formazan dye .
carcinoma cells were seeded at a density of 4  10 cells per well of 96-well plates in 100 l of serum - free medium containing various concentrations of 5-fu or paclitaxel as indicated in the absence or presence of 2 at 100 m .
after 44 h of incubation , xtt labeling reagent mixture ( roche diagnostics , east sussex , u.k . )
was added to the cultures and they were incubated for a further 4 h. the formazan product was then measured at 490 nm with a reference wavelength at 595 nm .
 we report the molecular design and synthesis of eg00229 , 2 , the first small molecule ligand for the vegf - a receptor neuropilin 1 ( nrp1 ) and the structural characterization of nrp1ligand complexes by nmr spectroscopy and x - ray crystallography . 
 mutagenesis studies localized vegf - a binding in the nrp1 b1 domain and a peptide fragment of vegf - a was shown to bind at the same site by nmr , providing the basis for small molecule design . 
 compound 2 demonstrated inhibition of vegf - a binding to nrp1 and attenuated vegfr2 phosphorylation in endothelial cells . 
 inhibition of migration of endothelial cells was also observed . 
 the viability of a549 lung carcinoma cells was reduced by 2 , and it increased the potency of the cytotoxic agents paclitaxel and 5-fluorouracil when given in combination . 
 these studies provide the basis for design of specific small molecule inhibitors of ligand binding to nrp1 . 
Introduction
Results and Discussion
Conclusion
Experimental Section