fluorine has been recognized as a valuable 
 element in medicinal 
 chemistry , and about 2025% known drugs contain fluorine atoms . fluorine is the most electronegative element and has a strong effect 
 on physicochemical and conformational properties of organic compounds . as a consequence , introduction of fluorine atoms 
 into ligands is a promising strategy in lead optimization to strengthen 
 protein  ligand interactions . furthermore , introduction of fluorine 
 into ligand molecules affects physicochemical properties and modulates 
 absorption , distribution , metabolism , and excretion in drug - like molecules . fluorine can enhance ligand affinity through interaction with 
 both 
 polar and hydrophobic groups in proteins . while organic fluorine is a very poor hydrogen bond acceptor , interaction of c  f with polar hydrogen 
 atoms has been observed in protein  inhibitor complexes . an interesting mode of fluorine 
 interactions has been observed for thrombin inhibitors where substitution 
 of hydrogen with fluorine resulted in 5-fold increase in potency . the crystal structure revealed that fluorine 
 is in remarkably close ( 3.1  ) contact to the carbonyl moiety 
 of asn98 . further analysis of the cambridge structural database ( csd ) 
 and protein data bank ( pdb ) showed that short fc = o 
 contacts ( 3.03.7  ) are abundant in both organic compounds 
 and protein  ligand complexes , and the fluorine atom frequently 
 approaches the electrophilic carbonyl carbon atom in an orthogonal 
 arrangement . for example , in the trifluoroacetyl dipeptide 
 anilide inhibitor bound to elastase ( pdb code 2est ) , all three fluorines 
 are involved in close contacts with backbone carbonyl groups . fc = o interactions have 
 been observed with both backbone as well side chain carbonyls , and 
 several studies have recognized these interactions as an attractive 
 approach to increase ligand binding affinity . previous studies have demonstrated 
 that very potent inhibitors 
 can be developed through the use of fluorine substitutions . for example , 
 a low nanomolar inhibitor of dipeptidyl peptidase iv has been developed 
 by the introduction of several fluorine atoms . introduction of trifluoromethyl groups during the optimization of 
 fragment - derived ligands resulted in the development of picomolar 
 inhibitors of cytochrome bc1 complex . systematic incorporation of fluorine 
 at different positions in a series of thrombin inhibitors revealed 
 that introduction of fluorine into the benzyl ring enhanced the binding 
 affinity by 6-fold . as a step toward the 
 identification of fluorophilic hot - spots in proteins , it has been 
 proposed to use f nmr ligand - based screening of fluorinated 
 fragments and a combination of screening 
 and computational analysis . however , 
 a rational approach for designing fluorinated ligands is missing . we previously identified the thienopyrimidine class of compounds 
 which directly bind to menin and inhibit the protein substitution of a propyl group on the thienopyrimidine 
 scaffold with trifluoroethyl , which resulted in the mi-2 - 2 compound , 
 leads to a significant 10-fold increase in the binding affinity . the crystal structure of mi-2 - 2 bound to menin 
 revealed that the cf3 group is involved in close contacts 
 with the protein backbone . backbone 
 interactions offer excellent opportunities to enhance the activity 
 of inhibitors targeting protein  protein interactions . however , 
 introduction of fluorine atoms into ligand molecules might be synthetically 
 challenging or may require multistep synthesis . therefore , a method 
 for rational design of favorable fluorine interactions in protein  ligand 
 complexes would significantly facilitate inhibitor development in 
 drug discovery projects . in order to understand the effect of fluorine 
 substitutions , we synthesized series of mi-2 - 2 analogues systematically 
 changing the number of fluorine atoms in two different groups and 
 determined high - resolution crystal structures of the inhibitors bound 
 to menin . we found that when fluorine atoms in menin inhibitors are 
 involved in the orthogonal multipolar c fc = o 
 interactions , it significantly enhances ligand binding affinity . on 
 the basis of these findings , we developed a computational algorithm 
 named fmap to support structure - based design of favorable c fc = o 
 interactions in protein  ligand complexes , and we demonstrated 
 its applicability to known fluorine - containing small molecule inhibitors . we identified 2559 crystal structures 
 containing a fluorinated ligand and performed an analysis using a 
 python script to select structures in which a fluorine atom is located 
 within 3.5  of either the peptide backbone carbonyl carbon or 
 amide nitrogen . geometrical criteria are selected 
 to cover 80% of the fluorine positions identified in the small 
 molecule ligands observed in our pdb search . calculation of the fluorine 
 sites is initiated by defining an arbitrary number of 29 hypothetical 
 fluorine sites within 3  distance from either the backbone carbon 
 or nitrogen ( table s1 ) . subsequently , fmap uses a series of filters 
 to remove fluorine positions using following criteria : ( 1 ) steric 
 clash ; fluorines within 1.8  of any protein atom are removed . ( 2 ) entirely buried or overly exposed fluorines ; this filter removes 
 positions that are not accessible to small molecules or are entirely 
 exposed , such as protein termini , loops ; this is accomplished through 
 a summation the number of cs closer than 10  and the 
 number of total atoms closer than 5  ; any position with a c 
 count between 15 and 28 and a total atom count less than 30 is retained . ( 3 ) fluorines too close to carbonyl oxygens ; positions are eliminated 
 when closer than 2.7  from a carbonyl oxygen and less than 60 
 degrees off the c = o bond vector , eliminating positions that 
 are too close to the electron lone pairs of carbonyl oxygens . ( 4 ) isolated sites ; this procedure eliminates isolated fluorines , removing 
 positions that are not clustered with other nearby fluorines ; fluorines 
 with less than 5 adjacent fluorines within 3.2  are removed . fmap is written in python , and can be run either as a pymol extension 
 or as a standalone command - line program . results of fmap calculations 
 are displayed in pymol using surface representation that encompasses 
 a volume of 2.83.2  distance from the protein backbone . . theoretical evaluation 
 of the fluorine interaction energy within model complexes was performed 
 with a fluoromethane probe positioned against the peptide bond present 
 in a model compound . as a model of peptide bond we used n - acetylglycine - n - methylamide in extended conformation . geometrical parameters to mapping the interaction energy were defined 
 by setting the origin of the spherical coordinate system onto the 
 position of the carbon atom from the peptide bond carbonyl group . radial distances as well as polar and azimuthal angles were then varied 
 by 0.1  and 10 degrees , respectively . within the cf 
 distance range of 2.54.0  , all the combinations of polar 
 and azimuthal angles were considered except for those resulting in 
 steric clashes . fluoromethane orientation was optimized at the hf/6 - 31+g(d ) 
 level of theory with model peptide and fluorine atoms kept frozen . we selected 
 mp2/6 - 31g(d ) level of theory because it provides appropriate estimation 
 of binding energy in biomolecular complexes . full - length human 
 menin was expressed in a pet32a vector ( promega ) containing n - terminal 
 thioredoxin his6-tag in rosetta ( de3 ) cells . menin was 
 purified using nickel - agarose ( ge healthcare ) followed by ion exchange 
 with q - sepharose ( ge healthcare ) . protein was subsequently cleaved 
 with thrombin followed by nickel agarose purification to separate 
 the thioredoxine tag from menin . purified protein was dialyzed to 
 50 mm tris , 50 mm nacl , 1 mm tcep ph 7.5 buffer . details of menin 
 purification have been published previously . the synthesis and characterization of menin  mll 
 inhibitor is presented in ( see supporting information ) . this assay 
 used fitc - mbm1 peptide of mll ( residues 415 ) at 15 nm with 
 150 nm menin in 50 mm tris , 50 mm nacl , 1 mm dtt ph 7.5 buffer . the ic50 values represent mean values and standard 
 deviations from two to three independent experiments . co - crystallization 
 of menin with small molecule inhibitors was 
 performed with 2.5 mg / ml menin incubated 
 with 3-fold molar excess small - molecule inhibitors ( mi-326 , mi-333 , 
 mi-319 , mi-2 - 3 , mi-836 , mi-859 , or mi-273 ) . crystals were obtained 
 using a sitting - drop technique at 10 c in 0.2 m ammonium acetate , 
 0.1 m hepes , ph 7.5 , and 25% w / v peg 3350 . prior to data collection , 
 crystals were transferred to cryosolution containing 20% peg550 mme 
 and flash - frozen in liquid nitrogen as described previously . x - ray diffraction data for cocrystals of menin 
 with small molecule 
 inhibitors were collected at 21-id - d , 21-id - f , and 21-id - g beamlines at the 
 life sciences collaborative access team at the advanced photon source . structures 
 of the complexes were determined by molecular replacement using molrep 
 with the apo structure of human menin ( pdb code : 4gpq ) as a search model . the model was refined using refmac , coot , and the ccp4 package . in the final stages , refinement was performed with addition of the 
 tls groups defined by the tlsmd server . details 
 of data processing and refinement are summarized in table s2 . coordinates 
 and structure factors have been deposited in the protein data bank . we previously performed extensive 
 medicinal chemistry optimization of the thienopyrimidine class of 
 menin  mll inhibitors and found that substitution of propyl 
 in the mi-2 compound by trifluoroethyl group resulted in a substantial , 
 10-fold increase in the activity of mi-2 - 2 ( figure 1a and table s3 ) . due to difficulties for further substitutions and potential metabolic 
 liability of the thiazoline moiety , we modified this class of compounds 
 by replacing thiazoline with an aromatic thiadiazole ring . although 
 the unsubstituted thiadiazole analogue is very weak , we found that introduction of the trifluoromethyl group 
 substantially improved the activity , resulting in mi-2 - 3 with ic50 = 92 nm ( figure 1a ) . both compounds , mi-2 - 2 and mi-2 - 3 , are potent inhibitors 
 of the menin our previous studies revealed that one fluorine atom from the 
 trifluoroethyl group in mi-2 - 2 forms close contacts with the backbone 
 atoms on menin and is located within 3.0  distance to the backbone 
 carbonyl of his181 , suggesting that this 
 interaction might play an important role in increasing the inhibitory 
 activity of mi-2 - 2 over mi-2 . to understand the molecular basis of 
 high binding affinity of mi-2 - 3 , we determined the crystal structure 
 of its complex with menin . the newly developed mi-2 - 3 with an additional 
 trifluoromethyl group binds to menin in a similar binding mode as 
 mi-2 - 2 ( figure 1b ) . interestingly , the new cf3 group within the trifluoromethyl  thiadiazole 
 moiety also forms close contacts with the menin backbone ( figure 1b ) , and one of the 
 fluorine atoms is located 3.4  from the carbonyl group of met322 . therefore , the fluorine atoms in both cf3 groups of mi-2 - 3 
 are involved in orthogonal multipolar c fc = o 
 interactions with the backbone atoms in two different regions on menin . to assess the contribution of the cf3 group in mi-2 - 3 , 
 we synthesized mi-326 by replacing trifluoromethyl with the methyl 
 group and found that it led to 8-fold decrease in the inhibitory 
 activity ( ic50 = 779 nm for mi-326 , table s3 ) . these two 
 examples , mi-2 - 2 and mi-2 - 3 , emphasize that c  fc = o 
 contribute very favorably to the protein  ligand interactions . ( a ) structures and activities of 
 mi-2 - 2 and mi-2 - 3 . multipolar interactions involving fluorine atoms 
 have been recognized for their pronounced effect on protein ligand 
 interactions , and well - placed fluorine may substantially enhance the 
 activity of small molecule inhibitors . introduction of trifluoromethyl groups in menin inhibitors resulted 
 in a significant improvement of inhibitory activity due to formation 
 of short - distance multipolar interactions with the protein backbone . ligand 
 complexes ( see methods ) . out of 2559 structures 
 containing fluorinated ligands , we found 442 complexes with a fluorine 
 atom within 3.5  of either the backbone carbonyl carbon or amide 
 nitrogen . this search demonstrated that fluorine is frequently located 
 within a short distance of the backbone carbonyl group with the c f 
 bond preferrably oriented in the orthogonal arrangement relative to 
 the plane of the peptide bond ( figure 2a ) . we have also performed theoretical calculations of the interaction 
 energy between the model peptide bond and fluoromethane . we found 
 favorable interaction energy for the c  f positioned above the 
 peptide carbonyl group , which is consistent with the analysis of experimental 
 structures ( figure 2b ) . ( a ) combined analysis of protein  ligand 
 structures from pdb , with fmap predictions of the potential fluorine 
 positions and their representative c  f bonds relative to backbone 
 peptide bond . fmap prediction is 
 shown as purple surface with orange vectors shown for representative 
 c  f bonds . ( b ) binding energy calculations ( kcal / mol ) for interaction 
 of fluoromethane with a model peptide using mp2/6 - 31g(d ) theory level 
 as a function of c  f bond orientation . model peptide is presented in balls and sticks representation ( carbon 
 in cyan , oxygen in red , and nitrogen in blue ) . purple surface represents favorable 
 positions for fluorine atoms to interact with protein backbone . on the basis of the analysis of 
 protein  ligand complexes 
 from the protein data bank ( pdb ) , we developed an algorithm ( fmap ) 
 for mapping sites for fluorine atoms on protein structures to form 
 favorable c the geometric criteria used in fmap have been 
 selected to encompass 80% of fluorine sites found in the experimental 
 structures in pdb . fluorine sites are mapped onto a protein structure 
 through a pymol extension and are represented 
 as a surface spanning 2.83.2  range from the peptide 
 bond ( figure 2a ) . fmap 
 also eliminates unlikely fluorine positions through filters based 
 on unfavorable geometry for multipolar interactions as well as steric 
 clashes with protein atoms ( see methods for 
 a detailed description of fmap ) . we employed fmap to analyze 
 the inhibitor binding site on menin 
 and found that there are two potential sites for accessing close contacts 
 between fluorine and protein backbone . importantly , both sites are 
 occupied by the cf3 groups in the complex of menin with 
 mi-2 - 3 ( figure 2c ) , 
 supporting the utility of fmap for the prediction of fluorophilic 
 sites in protein structures . the first site is relatively small and 
 is occupied by the cf3 group connected to the thiadiazole 
 moiety , whereas the second site is much larger and is occupied by 
 the trifluoroethyl group attached to the thienopyrimidine scaffold . close inspection of the menin - mi-2 - 3 crystal structure revealed that 
 only a single fluorine in each cf3 group has favorable 
 geometry for c , we concluded that most likely 
 not all fluorines are needed for high - affinity interactions of menin 
 with the mi-2 - 3 and mi-2 - 2 inhibitors . fmap analysis suggested that only one fluorine atom 
 in the cf3 group within the trifluoromethyl - thiadiazole 
 moiety of mi-2 - 3 is capable of favorable interactions with the backbone 
 carbonyl of met332 . in order to evaluate the contributions of fluorine 
 atoms to the binding affinity of mi-2 - 3 , we synthesized a series of 
 analogues replacing cf3 with ch3 , ch2f , and chf2 groups ( figure 3 ) . first , we assessed the effect of substituting cf3 by ch3 and found that the absence of the three 
 fluorine atoms in mi-326 results in > 8 fold decrease in the inhibitory 
 activity ( figure 3a ) . the crystal structure revealed that mi-326 binds to menin in a very 
 similar manner as mi-2 - 3 ( figure 3b ) , and the difference in the binding affinity predominantly 
 results from the loss of the fluorine atoms . we then synthesized and 
 tested two additional analogues with two ( mi-319 ) and single ( mi-333 ) 
 fluorines . the inhibitory activity of mi-319 is very similar to mi-2 - 3 
 indicating no differences between cf3 and chf2 groups ( figure 3a ) . surprisingly , mi-333 , which harbors the ch2f group , has 
 about 20-fold weaker activity than mi-2 - 3 and is even 2-fold less 
 potent than mi-326 with no fluorines ( figure 3a ) . to explain this effect , we determined 
 the crystal structures of mi-333 and mi-319 bound to menin . the chf2 group in mi-319 binds in a very similar manner as cf3 with one of the fluorine atoms in a short , 3.2  , distance 
 to the backbone carbonyl of met322 ( figure 3b ) . on the contrary , the single fluorine 
 in mi-333 adopts a position that is tilted approximately 38.5 
 from the plane of the thiadiazole ring and points away from the protein 
 backbone ( 3.7  distance to c = o of met322 ) ( figure 3b ) . as a consequence , fc = o 
 interactions , and no gain in the activity is observed for mi-333 ( figure 3a ) . effect of fluorine substitutions 
 in thiadiazole moiety on activity 
 of menin  mll inhibitors . ( b ) crystal structures of inhibitors bound to menin showing 
 the shortest distances between fluorine and menin backbone . the orientation of the ch2f group relative to 
 the thiadiazole 
 ring was unexpected , emphasizing a strong conformational effect of 
 the fluorine atom . as previously observed , substitution of h by f 
 can profoundly change the conformational preferences of a small molecule 
 because of the size and stereoelectronic effects . although we were able to predict the position of fluorine 
 required for favorable interactions with the protein backbone using 
 fmap , we did not anticipate that ch2f can adopt an orientation 
 where the fluorine points away from the backbone . introduction of 
 the second fluorine into the chf2 group was necessary to 
 achieve an orientation of the c  f bond allowing for favorable 
 c quantum mechanical energy calculations of rotational energy 
 barrier for cfh2 group in mi-333 shows two minima at 55 
 and 55 , and the conformation in the crystal structure is disfavored 
 by about 0.5 kcal / mol ( figure s1 ) . on the contrary , in mi-319 , one 
 fluorine is positioned in the energetical minimum ( s  c  c  f 
 dihedral angle equal to 49 ) while the second fluorine 
 which has less favorable geometry ( s  c  c  f dihedral 
 angle equal to 71 ) can interact with backbone . this example 
 demonstrates that while only single fluorine may interact with backbone , 
 other fluorines might be needed to stabilize the appropriate rotameric 
 state . comparison of the activities of mi-2 - 2 and mi-19 
 indicates that the trifluoroethyl group contributes significantly 
 to the high activity of mi-2 - 2 , and replacement of cf3 with 
 ch3 results in over 20-fold loss in inhibitory activity 
 ( figure 4a ) . fmap analysis 
 for the menin binding site suggests that only single fluorine in cf3 group can form c fc = o interactions 
 with the backbone . to test the contributions of individual fluorines 
 in the cf3 group of mi-2 - 2 , we synthesized two compounds 
 with ch2f ( mi-836 ) or chf2 ( mi-859 ) groups . 
 when compared to mi-19 , addition of the first fluorine enhanced the 
 activity nearly 5-fold , whereas addition of the second fluorine increased 
 the activity further by about 4-fold , making it comparable to mi-2 - 2 
 with cf3 group ( figure 4a ) . to rationalize the effect of these modifications , 
 we determined the crystal structures of mi-836 and mi-859 bound to 
 menin ( figure 4b ) . we found that the single fluorine in mi-836 points toward a hydrophobic 
 site formed by the side chains of leu177 , phe238 , ala182 , and ser155 , 
 and therefore , the 5-fold gain in the activity likely results from 
 favorable hydrophobic contacts . introduction of chf2 in 
 mi-859 allows for the second fluorine to be involved in the c  fc = o 
 interactions with the backbone of his181 , accounting for an additional 
 4-fold improvement in activity . effect of fluorine substitutions in thienopyrimidine 
 moiety on 
 activity of menin  mll inhibitors . ( b ) crystal structures of inhibitors bound to menin 
 showing the shortest distances between fluorine and the menin backbone . model of mi-19 has been 
 made on the basis of the structure of mi-2 - 2-menin complex . ( c ) crystal 
 structure of mi-273 bound to menin showing close contacts of fluorines 
 in cf2cf3 group with menin . very similar ic50 values of mi-859 ( with chf2 ) and mi-2 - 2 ( with cf3 ) indicates that the third 
 fluorine 
 is dispensable for binding . furthermore , the clogp value for mi-859 
 is approximately 0.6 unit lower than for mi-2 - 2 ( clogp = 3.89 and 
 3.32 for mi-2 - 2 and mi-859 , respectively ) . therefore , our approach 
 based on the fmap calculations may be used not only to predict fluorine 
 substitutions in ligand molecules but also to design compounds with 
 fewer number of fluorine atoms and reduced lipophilicity without compromising 
 ligand binding affinity . analysis of the mi-2 - 2-menin structure 
 revealed that the methylene 
 group in the ch2cf3 moiety is positioned closely 
 to the backbone carbonyl groups of ser178 and glu179 and may constitute 
 a further site for fluorine substitutions . however , the fmap analysis 
 revealed that introduction of the cf2 group at this site 
 would not be favorable due to poor geometry of the two fluorines with 
 respect to the carbonyl groups of ser178 and glu179 . to test this 
 hypothesis , we synthesized mi-273 with cf2cf3 group and found that such a substitution results in a 15-fold 
 decrease in the activity when compared to mi-2 - 2 ( figure 4a ) . we determined the crystal 
 structure of mi-273 bound to menin and found that it binds in an identical 
 manner as mi-2 - 2 ( figure 4c ) . the two additional fluorines in the cf2 group 
 of mi-273 are in close distances to the carbonyl oxygens of ser178 
 and glu179 leading to repulsive interactions . fc = o interactions 
 require optimal geometry , and the fmap approach can filter - out the 
 sites that are unfavorable for fluorine atoms in ligand molecules . the 4-fluoro substitution of benzyl group in compound 1 resulted in over 5-fold improvement in inhibitory activity for 2 . analysis of the crystal structure of 2 bound 
 to thrombin revealed short distance between fluorine and backbone 
 carbonyl of asn98 . we performed fmap analysis 
 of this complex and found that fluorine in 2 fits well 
 into the fmap predicted site for the c therefore , fmap could be a valuable tool to predict 4-fluoro 
 substitution in 1 to increase its potency . ( f ) macrocyclic menin  mll inhibitor ( model based on pdb 
 structure 4i80 ) . we also found several 
 other examples of ligands for which the activity 
 of unsubstituted and fluorine substituted analogues have been determined 
 and the crystal structures of fluorine analogues bound to the target 
 proteins are available . in a recent example , potent inhibitors of 
 procaspace-6 have been developed using a fragment - based discovery 
 approach . substitution of the phenyl 
 ring in 3 with a fluorine resulting in compound 4 led to a 6-fold improvement in the binding affinity . the 
 crystal structure demonstrated that fluorine in 4 is 
 in a close , 3.1 to 3.2  , distance from the carbonyl groups of 
 ala195 and ser196 and is involved in favorable c  fc = o 
 interactions ( figure 5b ) . again , the position of fluorine in 4 is consistent 
 with the fmap prediction ( figure 5b ) . structure - based design of hiv-1 entry inhibitors 
 resulted in development of tetramethylpiperidine class of compounds , 
 which bind to the viral envelope glycoprotein gp120 . introduction of fluorine into the phenyl ring in 5 yielded 5-fold more potent inhibitor 6 , and 
 structural analysis of a very close analogue of 6 revealed 
 that fluorine is within the 3.5  distance to the carbonyl group 
 of ser256 , which overlaps well with the fmap prediction for favorable 
 positions of fluorine in the binding sites ( figure 5c ) . introduction of the cf3 group into phenyl ring of 7 resulted in a large , 24-fold increase in the affinity for 8 ( figure 5d ) . although substitution of hydrogen 
 by trifluoromethyl group represents a relatively large structural 
 perturbation , the cf3-group in the crystal structure of 8 bound to -lactamase is mostly solvent exposed , and 
 one of the fluorines is located within a short , 3.2  distance 
 to the carbonyl of thr319 , which fits well into the fmap prediction 
 ( figure 5d ) . structure analysis revealed 
 that this class of inhibitors binds to the same pocket on menin as 
 thienopyrimidine compounds and that the phenyl ring overlaps with 
 the position of trifluoroethyl in mi-2 - 2 . we synthesized analogue 10 with the 3-fluorophenyl 
 group to introduce fluorine pointing toward the carbonyl group of 
 his181 and found that this leads to 2-fold improvement in the inhibitory 
 activity ( figure 5e ) . fmap analysis predicted favorable fluorine substitution at this position 
 and the crystal structure of 10 bound to menin validated 
 that fluorine indeed participates in the c introduction of the aromatic fluorine atom into the hydroxymethylpiperidine 
 class of menin  mll inhibitors has a less pronounced effect 
 than in the thienopyrimidine class likely due to the slightly longer 
 distance between fluorine and the carbonyl of his181 ( 3.2 vs 3.0  , 
 respectively ) and less favorable geometry . interestingly , addition 
 of fluorine into this site also resulted in a significant improvement 
 in the affinity of the macrocyclic peptidomimetic inhibitor of the 
 menin  mll interaction . a compound 
 with fluorine at the meta position of the phenyl ring we modeled the meta - fluoro analogue using the 
 crystal structure of menin with the macrocyclic peptidomimetic inhibitor 
 and found that fluorine is expected to occupy the site that remains 
 in a short distance to the carbonyl of his181 and overlaps well with 
 the fmap predictions ( figure 5f ) . we have also found examples where substitution with 
 fluorine did 
 not have beneficial effect on inhibitory activity despite reasonably 
 good agreement with the fmap predictions . in two such examples , introduction 
 of fluorine into inhibitors of neuronal nitric oxide synthase and 
 c - jun n - terminal kinase 1 led to the modest decrease in the inhibitory 
 activity ( figure s2 ) . this indicates that prediction 
 of fluorine substitutions based solely on geometrical criteria might 
 have potential limitations , and other factors such as effect of fluorine 
 on stereoelectronic or conformational properties of the ligand , structural 
 changes upon ligand binding , might need to be considered in order 
 to further improve the design of c targeting protein  protein interactions 
 ( ppis ) using small molecule inhibitors is considered challenging , 
 and the most  druggable  ppis belong to the protein - peptide 
 class of complexes . interfaces at such ppis frequently feature secondary 
 structure elements such as -helical bundle or addition of -sheet . development of potent small molecule inhibitors targeting such ppi 
 interfaces could significantly benefit from optimization of the inhibitor - backbone 
 contacts . interestingly , analysis of the menin mi-2 - 3 structure reveals 
 that the two cf3 groups form similar c the cf3 group at the thiadiazole interacts with -helix , 
 whereas the cf3 attached to the thienopyrimidine core interacts 
 with -sheet on menin ( figure 6a , c ) . ( a ) details of the interaction of cf3 group in mi-2 - 3 with 
 -helical fragment in menin and fmap prediction . orientation 
 of the -helix is similar as in panel a. ( c ) details of the 
 interaction of cf3 group in mi-2 - 3 with -sheet in 
 menin . ( d ) fmap prediction for idealized -sheet structure shown 
 in similar orientation as in panel c. we have used fmap to analyze accessibility of protein backbone 
 in the secondary structure elements to participate in the c we performed the fmap calculations for idealized secondary 
 structures composed of the poly - ala sequences and found that only 
 a small area of the -helix is accessible to interact with fluorine , 
 whereas a much larger surface area could interact with fluorine in 
 -sheet or -hairpin ( figure 6 ) . the access to protein backbone in helical 
 conformation is small and restricted via amino acid side chains ( figure 6b ) . therefore , there 
 is limited access of the fluorine to the peptide bond in order to 
 participate in the orthogonal c the access to peptide bonds in -sheet 
 conformation is significantly larger , and fluorine may be positioned 
 over a relatively large area to favorably interact with the backbone 
 ( figure 6d ) , facilitating 
 the design of fluorinated ligands . this analysis clearly indicates 
 a potential to design favorable interactions involving fluorine in 
 ligands that bind at ppi interfaces . design of such interactions should 
 be particularly feasible for interfaces involving -sheets due 
 to the relatively large accessibility of protein backbone . fluorine scanning strategy has been previously proposed as an effective 
 approach to improve the activity of small molecule inhibitors . such a strategy is synthetically 
 demanding and requires synthesis of multiple analogues . in an attempt to facilitate design of c  fc = o 
 interactions in protein  ligand complexes we developed the fmap 
 algorithm . ligand 
 complex and calculates sites surrounding a ligand which could be favorably 
 occupied by fluorine atoms . we demonstrated that fmap could be used 
 to rationalize improvement in activity upon introduction of fluorine 
 in thienopyrimidine class of menin inhibitors as well as for several 
 known inhibitors . fmap may also represent a valuable tool for the 
 design of new fluorine substitutions in protein ligands . fmap relies 
 solely on geometrical and structural criteria , and other effects , 
 such as conformational or electronic changes resulting from fluorine 
 substitution are not taken into account , which might represent a limitation 
 of this approach . nevertheless , we expect that fmap can be very useful 
 in the drug discovery projects to rationally design positions for 
 flourine atoms in ligand molecules . it may also support development 
 of ligands with an optimal number of fluorine atoms to improve binding 
 affinity while reducing ligand hydrophobicity and molecular weight . introduction of the cf3 group in menin inhibitors as 
 well as in several examples reviewed in this study results in a substantial 
 gain in the affinity providing that optimal geometry of the c fc = o 
 interactions provide unique opportunities to introduce favorable interactions 
 between small molecule ligands and the polar protein backbone . due 
 to unique orthogonal geometry relative to the protein backbone , these 
 interactions may be introduced into the binding sites where hydrogen 
 bonds are not feasible . we found that substitution of ch3 for cf3 may increase ligand binding affinity as much 
 as 10-fold . however , multipolar interactions involving the cf3 group may not be solely responsible for the increase in binding 
 affinity . the effect of desolvatation of more hydrophobic cf3 group is expected to lead to a larger positive entropy of binding 
 when compared with ch3 . the 
 cf3 is roughly twice the size of a methyl group and due to a larger size and different shape , 
 it may form more optimal van der waals contacts within the binding 
 site . furthermore , the two additional fluorine atoms may participate 
 in hydrophobic interactions with neighboring atoms . in the case of 
 menin inhibitors , we found that not all fluorine atoms in the cf3 group are needed for the high affinity interaction . fc = o interactions may 
 impact conformational equilibrium and favor a rotamer which can not 
 favorably interact with protein backbone or might cause high entropic 
 cost of freezing out a desired rotamer . despite that the h to f substitution 
 represents a relatively minor modification our structural data collected for 
 the menin  inhibitor complexes offers a unique set of data which 
 may facilitate better understanding of the c  fc = o 
 interactions . fc = o interactions 
 have been 
 typically reported for enzyme inhibitors . with increasing 
 interest and demand in development of ppi inhibitors , ligand interactions at solvent 
 exposed interfaces . as we demonstrated for the menin  mll inhibitors , fluorine interaction with the protein backbone may offer such opportunities , 
 particularly at the interfaces involving -helical or -sheet 
 structures . in this study fc = o interactions , 
 which adds a new tool for structure - based design of new inhibitors 
 targeting protein  protein interfaces as well as protein ligands 
 in a more general context . the fmap algorithm may facilitate prediction 
 of fluorine substitutions in ligand molecules and support ligand optimization 
 in drug discovery projects .
multipolar interactions involving 
 fluorine and the protein backbone 
 have been frequently observed in protein  ligand complexes . 
 such fluorine  backbone interactions may substantially contribute 
 to the high affinity of small molecule inhibitors . 
 here we found that 
 introduction of trifluoromethyl groups into two different sites in 
 the thienopyrimidine class of menin  mll inhibitors considerably 
 improved their inhibitory activity . in both cases , trifluoromethyl 
 groups 
 are engaged in short interactions with the backbone of menin . 
 in order to understand the effect of fluorine , we synthesized a series 
 of analogues by systematically changing the number of fluorine atoms , 
 and we determined high - resolution crystal structures of the complexes 
 with menin . 
 we found that introduction of fluorine at favorable geometry 
 for interactions with backbone carbonyls may improve the activity 
 of menin  mll inhibitors as much as 5- to 10-fold . in order 
 to facilitate the design of multipolar fluorine 
 backbone interactions 
 in protein  ligand complexes , we developed a computational algorithm 
 named fmap , which calculates fluorophilic sites in proximity to the 
 protein backbone . 
 we demonstrated that fmap could be used to rationalize 
 improvement in the activity of known protein inhibitors upon introduction 
 of fluorine . 
 furthermore , fmap may also represent a valuable tool 
 for designing new fluorine substitutions and support ligand optimization 
 in drug discovery projects . 
 analysis of the menin 
 mll inhibitor 
 complexes revealed that the backbone in secondary structures is particularly 
 accessible to the interactions with fluorine . considering that secondary 
 structure elements are frequently exposed at protein interfaces 
 , we 
 postulate that multipolar fluorine 
 backbone interactions may 
 represent a particularly attractive approach to improve inhibitors 
 of protein  protein interactions .