Rational Design of Orthogonal Multipolar Interactions with Fluorine in Protein-Ligand Complexes

Article abstract of DOI:10.1021/acs.jmedchem.5b00975

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.

Full text of DOI:10.1021/acs.jmedchem.5b00975

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Article
Rational design of orthogonal multipolar interactions
with fluorine in protein-ligand complexes
Jonathan Wesley Pollock, Dmitry Borkin, George Lund, Trupta Purohit,
Edyta Dyguda-Kazimierowicz, Jolanta Grembecka, and Tomasz Cierpicki
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00975 • Publication Date (Web): 19 Aug 2015
Downloaded from http://pubs.acs.org on August 19, 2015
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Rational design of orthogonal multipolar interactions with fluorine in proteinꢀligand complexes
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Jonathan Pollock
†
, Dmitry Borkin
†, George Lund†, Trupta Purohit†, Edyta DygudaꢀKazimierowicz‡,
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Jolanta Grembecka
†
and Tomasz Cierpicki†*
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†
‡
Department of Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA
Molecular Modeling and Quantum Chemistry Group, Department of Chemistry, Wrocław University of
Technology, Wyb. Wyspiańskiego 27, 50ꢀ370 Wrocław, Poland
*Corresponding author:
Tel: 734ꢀ615ꢀ9324
tomaszc@umich.edu
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Abstract
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Multipolar interactions involving fluorine and protein backbone have been frequently observed in proteinꢀ
ligand complexes. Such fluorineꢀbackbone 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 thienopyrimidine class of meninꢀMLL 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 analogs systematically changing the
number of fluorine atoms and 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 meninꢀMLL inhibitors as much as 5 to 10ꢀfold. In order to facilitate the
design of multipolar fluorineꢀbackbone interactions in proteinꢀligand complexes we developed a
computational algorithm named FMAP, which calculates fluorophilic sites in proximity to 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 meninꢀMLL 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 fluorineꢀbackbone
interactions may represent a particularly attractive approach to improve inhibitors of proteinꢀprotein
interactions.
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Introduction
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Fluorine has been recognized as a valuable element in medicinal chemistry and about 20ꢀ25% known
drugs contain fluorine atoms. 1ꢀ3 Fluorine is the most electronegative element and has a strong effect on
physicochemical and conformational properties of organic compounds.3 As a consequence, introduction
of fluorine atoms into ligands is a promising strategy in lead optimization to strengthen proteinꢀligand
interactions. Furthermore, introduction of fluorine into ligand molecules affects physicochemical
properties and modulates absorption, distribution, metabolism, and excretion in drugꢀlike molecules.2, 3
Fluorine can enhance ligand affinity through interaction with both polar and hydrophobic groups
in proteins.4 While organic fluorine is very poor hydrogen bond acceptor 5, interaction of CꢀF with polar
hydrogen atoms has been observed in proteinꢀinhibitor complexes.1, 6, 7 An interesting mode of fluorine
interactions has been observed for thrombin inhibitors where substitution of hydrogen with fluorine
resulted in 5ꢀfold increase in potency 8. The crystal structure revealed that fluorine is remarkably close
(3.1 Å) contact to the carbonyl moiety of Asn98. Further analysis of Cambridge Structural Database
(CSD) and Protein Data Bank (PDB) showed that short F···C=O contacts (3.0−3.7 Å) are abundant in
both, organic compounds and proteinꢀligand complexes and the fluorine atom frequently approaches the
electrophilic carbonyl carbon atom in an orthogonal arrangement. 2, 4, 8, 9 For example, in trifluoroacetyl
dipeptide anilide inhibitor bound to elastase (PDB code 2EST) all three fluorines are involved in close
contacts with backbone carbonyl groups. Orthogonal multipolar C–F···C=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.2, 9, 10
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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.7 Introduction of trifluoromethyl groups during
the optimization of fragmentꢀderived ligands resulted in the development of picomolar inhibitors of
Cytochrome bc1 Complex.11 Fluorine scanning has been proposed as an effective strategy for ligand
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optimization.8, 10 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 6ꢀ
fold.8 As a step towards the identification of fluorophilic hotꢀspots in proteins, it has been proposed to use
19F NMR ligandꢀbased screening of fluorinated fragments12 and a combination of screening and
computational analysis.13 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 proteinꢀprotein interaction (PPI) between menin and MLL with nanomolar affinity.14
Substitution of a propyl group on the thienopyrimidine scaffold with trifluoroethyl, which resulted in the
MIꢀ2ꢀ2 compound, leads to a significant 10ꢀfold increase in the binding affinity.15 The crystal structure of
MIꢀ2ꢀ2 bound to menin revealed that the CF3 group is involved in close contacts with the protein
backbone. This demonstrates that fluorineꢀbackbone interactions offer excellent opportunities to enhance
the activity of inhibitors targeting proteinꢀprotein interactions. However, introduction of fluorine atoms
into ligand molecules might be synthetically challenging or may require multiꢀstep synthesis. Therefore, a
method for rational design of favorable fluorine interactions in proteinꢀligand complexes would
significantly facilitate inhibitor development in drug discovery projects. In order to understand the effect
of fluorine substitutions we synthesized series of MIꢀ2ꢀ2 analogs 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–F···C=O interactions, it significantly enhances ligand binding affinity. Based on these
findings, we developed a computational algorithm named FMAP to support structureꢀbased design of
favorable C–F···C=O interactions in proteinꢀligand complexes, and demonstrated its applicability to
known fluorineꢀcontaining small molecule inhibitors. This study should facilitate rational development of
fluorinated ligands for drug discovery applications.
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Materials and Methods
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PDB search of fluorine containing proteinꢀligand complexes
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We performed a search of the PDB to identify proteinꢀligand complexes containing fluorine atoms. 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 Å of either the peptide
backbone carbonyl carbon or amide nitrogen. We have accepted structures with 2.2 Å resolution or better
for further analysis. We found total of 442 proteinꢀligand complexes, which fit these criteria for detailed
analysis.
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FMAP fluorine site mapping algorithm and filtering criteria
FMAP calculates favorable positions for fluorine to form C–F···C=O interactions with the protein
backbone. 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 Å 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 Å 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 Cαs closer than 10 Å and the number of total atoms closer than 5 Å; any position with a Cα
count between 15ꢀ28 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 Å 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 Å are
removed.
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FMAP is written in python, and can be run either as a pymol extension or as a standalone commandꢀline
program. Results of FMAP calculations are displayed in Pymol using surface representation that
encompasses a volume of 2.8ꢀ3.2 Å distance from the protein backbone. FMAP is freely available from
the authors upon request and requires the Biopython module.16, 17
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Calculation of interaction energy
Theoretical evaluation of the fluorine interaction energy within model complexes was performed with
fluoromethane probe positioned against the peptide bond present in a model compound. As a model of
peptide bond we used NꢀacetylglycineꢀNꢀmethylamide 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 peptide bond carbonyl group. Radial distances as well as polar
and azimuthal angles were then varied by 0.1 Å and 10 degrees, respectively. Within the C···F distance
range of 2.5ꢀ4.0 Å, all the combinations of polar and azimuthal angles were considered except for those
resulting in steric clashes. Fluoromethane orientation was optimized at the HF/6ꢀ31+G(d) level of theory
with model peptide and fluorine atoms kept frozen. The resulting structures were used for MP2/6ꢀ31G(d)
interaction energy calculation. Counterpoise correction was applied to reduce the basis set superposition
error.18 We selected MP2/6ꢀ31G(d) level of theory because it provides appropriate estimation of binding
energy in biomolecular complexes.19 All the quantum chemical calculations were performed using
Gaussian09 program.20
Expression and purification of menin
Full length human menin was expressed in a pET32a vector (Promega) containing Nꢀterminal thioredoxin
His6ꢀtag in RosettaTM (DE3) cells. Menin was purified using nickelꢀagarose (GE Healthcare) followed by
ion exchange with QꢀSepharose (GE Healthcare). Protein was subsequently cleaved with thrombin
followed by nickel agarose purification to separate the thioredoxine tag from menin. Purified protein was
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dialyzed to 50 mM Tris, 50 mM NaCl, 1 mM TCEP pH 7.5 buffer. Details of menin purification have
been published previously.21
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Chemistry
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The synthesis and characterization of meninꢀMLL inhibitor is presented in (See SI).
Characterization of activity of menin−MLL inhibitors
Activity of small molecules to inhibit the meninꢀMLL interaction was determined by fluorescence
polarization (FP) assay. This assay used FITCꢀMBM1 peptide of MLL (residues 4ꢀ15) at 15 nM with 150
nM menin in 50 mM Tris, 50 mM NaCl, 1 mM DTT pH 7.5 buffer. Detailed protocol has been described
previously.21 The IC50 values represent mean values and standard deviations from two to three
independent experiments.
Crystallization of the menin complexes with small molecule inhibitors
Coꢀcrystallization of menin with small molecule inhibitors was performed with 2.5 mg/mL menin 15
incubated with 3ꢀfold molar excess smallꢀmolecule inhibitors (MIꢀ326, MIꢀ333, MIꢀ319, MIꢀ2ꢀ3, MIꢀ836,
MIꢀ859, or MIꢀ273). Crystals were obtained using a sittingꢀdrop 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 flashꢀfrozen in liquid nitrogen as
described previously.15
Crystallographic data collection and structure determination
Xꢀray diffraction data for coꢀcrystals of menin with small molecule inhibitors were collected at 21ꢀIDꢀD
and 21ꢀIDꢀF beamlines at the Life Sciences Collaborative Access Team at the Advanced Photon Source.
Data was processed with HKLꢀ2000.22 Structures of the complexes were determined by molecular
replacement using MOLREP with the apo structure of human menin (PDB code: 4GPQ) as a search
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model. The model was refined using REFMAC 23, COOT 24, and the CCP4 package.25 In the final stages
of refinement was performed with addition of the TLS groups defined by the TLSMD server.26 Validation
of the structures was performed using MOLPROBITY 27 and ADIT.28 Details of data processing and
refinement are summarized in Table S2. Coordinates and structure factors have been deposited in the
Protein Data Bank.
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Results and discussion
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Trifluoromethyl groups in meninꢀMLL inhibitors form close contacts with protein backbone
We previously performed extensive medicinal chemistry optimization of thienopyrimidine class of
meninꢀMLL inhibitors and found that substitution of propyl in the MIꢀ2 compound by trifluoroethyl
group resulted in a substantial, 10ꢀfold increase in the activity of MIꢀ2ꢀ2 (Fig 1a and Table S3).15 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. While unsubstituted
thiadiazole analog is very weak14, we found that introduction of the trifluoromethyl group substantially
improved the activity, resulting in MIꢀ2ꢀ3 with IC50 = 92 nM (Fig 1a). Both compounds, MIꢀ2ꢀ2 and MIꢀ
2ꢀ3, are potent inhibitors of the meninꢀMLL interaction with the IC50 values below 100 nM (Fig 1a). Our
previous studies revealed that one fluorine atom from the trifluoroethyl group in MIꢀ2ꢀ2 forms close
contacts with the backbone atoms on menin and is located within 3.0 Å distance to the backbone carbonyl
of His181,15 suggesting that this interaction might play an important role in increasing the inhibitory
activity of MIꢀ2ꢀ2 over MIꢀ2. To understand the molecular basis of high binding affinity of MIꢀ2ꢀ3 we
determined the crystal structure of its complex with menin. The newly developed MIꢀ2ꢀ3 with an
additional trifluoromethyl group binds to menin in a similar binding mode as MIꢀ2ꢀ2 (Fig 1b).
Interestingly, the new CF3 group within the trifluoromethylꢀthiadiazole moiety also forms close contacts
with the menin backbone (Fig 1b) and one of the fluorine atoms is located 3.4 Å from the carbonyl group
of Met322. Therefore, the fluorine atoms in both CF3 groups of MIꢀ2ꢀ3 are involved in orthogonal
multipolar C–F···C=O interactions with the backbone atoms in two different regions on menin. To assess
the contribution of the CF3 group in MIꢀ2ꢀ3 we synthesized MIꢀ326 by replacing trifluoromethyl with the
methyl group and found that it led to ~8ꢀfold decrease in the inhibitory activity (IC50 = 779 nM for MIꢀ
326, Table S3). These two examples, MIꢀ2ꢀ2 and MIꢀ2ꢀ3, emphasize that C–F···C=O contribute very
favorably to the proteinꢀligand interactions.2, 8, 10
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Development of FMAP algorithm to predict multipolar C–F···C=O interactions
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Multipolar interactions involving fluorine atoms have been recognized for their pronounced effect on
proteinꢀligand interactions, and wellꢀplaced fluorine may substantially enhance the activity of small
molecule inhibitors.2, 8ꢀ10 Introduction of trifluoromethyl groups in menin inhibitors resulted in a
significant improvement of inhibitory activity due to formation of shortꢀdistance multipolar interactions
with the protein backbone. We therefore sought whether such interactions could be rationally designed.
First, we analyzed geometry of fluorineꢀbackbone interactions in known highꢀresolution crystal structures
of proteinꢀligand complexes (see Methods). Out of 2559 structures containing fluorinated ligands we
found 442 complexes with a fluorine atom within 3.5Å 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 CꢀF bond preferrably oriented in the orthogonal arrangement relative to
the plane of the peptide bond (Fig 2a). This exemplifies a presence of multipolar C–F···C=O interactions
as described in details in the previous studies.2, 9 We have also performed theoretical calculations of the
interaction energy between the model peptide bond and fluoromethane. We found favorable interaction
energy for the CꢀF positioned above the peptide carbonyl group, which is consistent with the analysis of
experimental structures (Fig 2b).
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Based on the analysis of proteinꢀligand 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–F···C=O interactions with the protein backbone. 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 Pymol29 extension and are represented as a surface
spanning 2.8ꢀ3.2 Å range from the peptide bond (Fig 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
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are occupied by the CF3 groups in the complex of menin with MIꢀ2ꢀ3 (Fig. 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 while the second site is much larger and is
occupied by the trifluoroethyl group attached to the thienopyrimidine scaffold. Close inspection of the
meninꢀMIꢀ2ꢀ3 crystal structure revealed that only a single fluorine in each CF3 group has favorable
geometry for C–F···C=O interactions with backbone. Based on this analysis we concluded that most
likely not all fluorines are needed for high affinity interactions of menin with the MIꢀ2ꢀ3 and MIꢀ2ꢀ2
inhibitors.
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Interactions of trifluoromethylꢀthiadiazole moiety with menin
FMAP analysis suggested that only one fluorine atom in the CF3 group within the trifluoromethylꢀ
thiadiazole moiety of MIꢀ2ꢀ3 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 MIꢀ2ꢀ3 we synthesized a
series of analogs replacing CF3 with CH3, CH2F and CHF2 groups (Fig 3). First, we assessed the effect of
substituting CF3 by CH3 and found that the absence of the three fluorine atoms in MIꢀ326 results in >8
fold decrease in the inhibitory activity (Fig 3a). The crystal structure revealed that MIꢀ326 binds to menin
in a very similar manner as MIꢀ2ꢀ3 (Fig 3b), and the difference in the binding affinity predominantly
results from the loss of the fluorine atoms. We then synthesized and tested two additional analogs with
two (MIꢀ319) and single (MIꢀ333) fluorines. The inhibitory activity of MIꢀ319 is very similar to MIꢀ2ꢀ3
indicating no differences between CF3 and CHF2 groups (Fig 3a). Surprisingly, MIꢀ333 which harbors the
CH2F group has about 20ꢀfold weaker activity than MIꢀ2ꢀ3, and is even twoꢀfold less potent than MIꢀ326
with no fluorines (Fig 3a). To explain this effect we determined the crystal structures of MIꢀ333 and MIꢀ
319 bound to menin. The CHF2 group in MIꢀ319 binds in a very similar manner as CF3 with one of the
fluorine atoms in a short, 3.2 Å, distance to the backbone carbonyl of Met322 (Fig 3b). On the contrary,
the single fluorine in MIꢀ333 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 Å distance to C=O of Met322) (Fig 3b).
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As a consequence, the fluorine is too far to be involved in a favorable multipolar C–F···C=O interactions
and no gain in the activity is observed for MIꢀ333 (Fig 3a).
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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.2 Although, we were able to predict the position of fluorine required for favorable interactions with
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 CꢀF bond allowing for favorable C–F···C=O interactions and
substantial improvement in activity. Analysis of the crystal structure of MIꢀ333 shows that SꢀCꢀCꢀF
dihedral adapts 38.5˚ angle. Quantum mechanical energy calculations of rotational energy barrier for
CFH2 group in MIꢀ333 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 MIꢀ319, one fluorine is positioned in
the energetical minimum (SꢀCꢀCꢀF dihedral angle equal to ꢀ49˚) while the second fluorine which has less
favorable geometry (SꢀCꢀCꢀF 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.
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Interactions of trifluoroethyl group in thienopyrimidine core with menin
Comparison of the activities of MIꢀ2ꢀ2 and MIꢀ19 indicates that the trifluoroethyl group contributes
significantly to the high activity of MIꢀ2ꢀ2, and replacement of CF3 with CH3 results in over 20ꢀfold loss
in inhibitory activity (Fig 4a). FMAP analysis for the menin binding site suggests that only single fluorine
in CF3 group can form C–F···C=O interactions with the backbone. To test the contributions of individual
fluorines in the CF3 group of MIꢀ2ꢀ2 we synthezised two compounds with CH2F (MIꢀ836) or CHF2 (MIꢀ
859) groups. When compared to MIꢀ19, addition of the first fluorine enhanced the activity nearly 5ꢀfold
while addition of the second fluorine increased the activity further by about 4ꢀfold, making it comparable
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to MIꢀ2ꢀ2 with CF3 group (Fig 4a). To rationalize the effect of these modifications we determined the
crystal structures of MIꢀ836 and MIꢀ859 bound to menin (Fig 4b). We found that the single fluorine in
MIꢀ836 points towards a hydrophobic site formed by the side chains of Leu177, Phe238, Ala182 and
Ser155, and therefore the 5ꢀfold gain in the activity likely results from favorable hydrophobic contacts.
Introduction of CHF2 in MIꢀ859 allows for the second fluorine to be involved in the C–F···C=O
interactions with the backbone of His181 accounting for additional 4ꢀfold improvement in activity.
Very similar IC50 values of MIꢀ859 (with CHF2) and MIꢀ2ꢀ2 (with CF3) indicates that the third
fluorine is dispensable for binding. Furthermore, the cLogP value for MIꢀ859 is approximately 0.6 unit
lower than for MIꢀ2ꢀ2 (cLogP = 3.89 and 3.32 for MIꢀ2ꢀ2 and MIꢀ859, 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.
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Analysis of the MIꢀ2ꢀ2ꢀmenin 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 MIꢀ273 with CF2CF3 group and
found that such a substitution results in ~15ꢀfold decrease in the activity when compared to MIꢀ2ꢀ2 (Fig
4a). We determined the crystal structure of MIꢀ273 bound to menin and found that it binds in an identical
manner as MIꢀ2ꢀ2 (Fig 4c). The two additional fluorines in the CF2 group of MIꢀ273 are in close distances
to the carbonyl oxygens of Ser178 and Glu179 leading to repulsive interactions. This further emphasizes
that favorable C–F···C=O interactions require optimal geometry and the FMAP approach can filterꢀout
the sites that are unfavorable for fluorine atoms in ligand molecules.
Application of FMAP to rationalize fluorine substitutions in known ligands
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One of the first well documented examples of the C–F···C=O interactions favorably contributing to
proteinꢀligand interaction was described for the tricyclic class of thrombin inhibitors.8 The 4ꢀfluoro
substitution of benzyl group in compound 1 resulted in over 5ꢀfold 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 Asn98.8 We performed FMAP analysis of this complex and found that fluorine in 2
fits well into the FMAP predicted site for the C–F···C=O interactions (Fig 5a). Therefore, FMAP could
be a valuable tool to predict 4ꢀfluoro substitution in 1 to increase its potency.
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We also found several other examples of ligands for which the activity of unsubstituted and
fluorine substituted analogs have been determined and the crystal structures of fluorine analogs bound to
the target proteins are available. In a recent example, potent inhibitors of procaspaceꢀ6 have been
developed using a fragmentꢀbased discovery approach.30 Substitution of the phenyl ring in 3 with a
fluorine resulting in compound 4 led to a 6ꢀfold improvement in the binding affinity. The crystal structure
demonstrated that fluorine in 4 is in a close, 3.1 to 3.2 Å, distance from the carbonyl groups of Ala195
and Ser196 and is involved in favorable C–F···C=O interactions (Fig 5b). Again, the position of fluorine
in 4 is consistent with the FMAP prediction (Fig 5b). Structure based design of HIVꢀ1 entry inhibitors
resulted in development of tetramethylpiperidine class of compounds, which bind to the viral envelope
glycoprotein gp120 31. Introduction of fluorine into the phenyl ring in 5 yielded 5ꢀfold more potent
inhibitor 6 and structural analysis of a very close analog of 6 revealed that fluorine is within the 3.5 Å
distance to the carbonyl group of Ser256, which overlaps well with the FMAP prediction for favorable
positions of fluorine in the binding sites (Fig 5c). Another interesting example of the C–F···C=O
interactions has been observed for the βꢀlactamase inhibitor. Introduction of the CF3 group into phenyl
ring of 7 resulted in a large, 24ꢀfold increase in the affinity for 8 (Fig 5d).32 Although substitution of
hydrogen by trifluoromethyl group represents relatively large structural perturbation, the CF3ꢀgroup in the
crystal structure of 8 bound to βꢀlactamase is mostly solvent exposed and one of the fluorines is located
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within a short, 3.2 Å distance to the carbonyl of Thr319, which fits well into the FMAP prediction (Fig
5d).
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We have recently exploited the idea to introduce C–F···C=O interactions into the
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hydroxymethylpiperidine class of the meninꢀMLL inhibitors.33 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 MIꢀ2ꢀ2.33 We synthesized analog 10 with the 3ꢀfluorophenyl
group to introduce fluorine pointing towards the carbonyl group of His181 and found that this leads to 2ꢀ
fold improvement in the inhibitory activity (Fig 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–F···C=O interactions (Fig 5e). Introduction of the aromatic fluorine atom into the
hydroxymethylpiperidine class of meninꢀMLL 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 Å, 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 meninꢀMLL interaction 34. Compound with fluorine at the meta position of the phenyl ring
has 4ꢀfold better binding affinity when compared with the unsubstituted analog.34 We modeled the metaꢀ
fluoro analog 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 (Fig 5f).
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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 cꢀJun Nꢀterminal kinase 1 led
to the modest decrease in the inhibitory activity (Figure S2).35, 36 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
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upon ligand binding, might need to be considered in order to further improve the design of C–F···C=O
interactions.
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Designing C–F•••C=O interactions at PPI interfaces
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Targeting proteinꢀprotein interactions (PPIs) using small molecule inhibitors is considered challenging
and the most “druggable” PPIs belong to the proteinꢀpeptide class of complexes.37ꢀ39 Interfaces at such
PPIs frequently feature secondary structure elements such as αꢀhelical bundle40 or addition of βꢀsheet.41
Development of potent small molecule inhibitors targeting such PPI interfaces could significantly benefit
from optimization of the inhibitorꢀbackbone contacts. Interestingly, analysis of the menin MIꢀ2ꢀ3
structure reveals that the two CF3 groups form similar C–F···C=O contacts but with the two structurally
different backbone conformations. The CF3 group at the thiadiazole interacts with αꢀhelix while the CF3
attached to the thienopyrimidine core interacts with βꢀsheet on menin (Fig 6a,c).
We have used FMAP to analyze accessibility of protein backbone in the secondary structure
elements to participate in the C–F···C=O interactions. We performed the FMAP calculations for idealized
secondary structures composed of the polyꢀAla sequences and found that only small area of αꢀhelix is
accessible to interact with fluorine while much larger surface area could interact with fluorine in βꢀsheet
or βꢀhairpin (Fig 6). The access to protein backbone in α−helical conformation is small and restricted via
amino acid side chains (Fig 6b). Therefore, there is limited access of the fluorine to the peptide bond in
order to participate in the orthogonal C–F···C=O interactions. On the contrary, the access to peptide
bonds in βꢀsheet conformation is significantly larger and fluorine may be positioned over a relatively
large area to favorably interact with backbone (Fig 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
βꢀsheets due to the relatively large accessibility of protein backbone.
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Fluorine scanning strategy has been previously proposed as an effective approach to improve the
activity of small molecule inhibitors.2, 8, 10 Such a strategy is synthetically demanding and requires
synthesis of multiple analogs.8, 10 In an attempt to facilitate design of C–F···C=O interactions in proteinꢀ
ligand complexes we developed the FMAP algorithm. FMAP uses a crystal structure of a proteinꢀligand
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.
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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 CꢀF bond relative
to the backbone carbonyl is achieved. Such C–F···C=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 protein backbone, these interactions may be introduced into the
inding sites where hydrogen bonds are not feasible. We found that substitution of CH3 for CF3 may
increase ligand binding affinity as much as 10ꢀfold. However, multipolar interactions involving 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 larger positive entropy of binding when compared
with CH3.42 The CF3 is roughly twice the size of a methyl group1 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
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additional fluorine atoms may participate in hydrophobic interactions with neighboring atoms. In case of
menin inhibitors we found that not all fluorine atoms in the CF3 group are needed for the high affinity
interaction. However, introduction of CFH2 or CF2H groups to achieve favorable C–F···C=O interactions
may impact conformational equilibrium and favor a rotamer which cannot 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, it may have a complex impact on ligand binding
affinity. Our structural data collected for the meninꢀinhibitor complexes offers a unique set of data which
may facilitate better understanding of the C–F···C=O interactions.
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The C–F···C=O interactions have been typically reported for enzyme inhibitors.8, 10, 30, 32 With
increasing interest and demand in development of PPI inhibitors, efficient approaches are needed to
optimize proteinꢀligand interactions at solvent exposed interfaces. As we demonstrated for the meninꢀ
MLL inhibitors, fluorine interaction with the protein backbone may offer such opportunities, particularly
at the interfaces involving αꢀhelical or βꢀsheet structures. In this study, we developed the FMAP approach
to streamline the design of C–F···C=O interactions, which adds a new tool for structure based design of
new inhibitors targeting proteinꢀprotein 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.
Acknowledgments
This work was supported by the American Cancer Society grants (RSGꢀ11ꢀ082ꢀ01ꢀDMC to T.C. and
RSGꢀ13ꢀ130ꢀ01ꢀCDD to J.G.), NIH R01 CA181185 to T.C., NIH R01 CA160467 to J.G., Leukemia and
Lymphoma Society (LLS) TRP grant (6116ꢀ12) to J.G., LLS Scholar (1215ꢀ14) to JG. Use of the
Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy
(DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under
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Contract No. DEꢀAC02ꢀ06CH11357. Use of the LSꢀCAT Sector 21 was supported by the Michigan
Economic Development Corporation and the Michigan Technology TriꢀCorridor (Grant 085P1000817).
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Figure 1. Inhibitors of the meninꢀMLL interaction containing CF3 groups. a) Structures and activities of
MIꢀ2ꢀ2 and MIꢀ2ꢀ3. b) Crystal structure of MIꢀ2ꢀ3 bound to menin. Short C–F···C=O distances are shown
using dashed lines.
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Figure 2. Prediction of favorable C–F···C=O interactions using FMAP algorithm. a) Combined analysis
of proteinꢀligand structures from PDB, with FMAP predictions of the potential fluorine positions and
their representative CꢀF bonds relative to backbone peptide bond. Positions of fluorine atoms derived
from the proteinꢀligand complexes found in PDB are shown as cyan points. FMAP prediction is shown as
purple surface with orange vectors shown for representative CꢀF bonds. b) Binding energy calculations
(kcal/mol) for interaction of fluoromethane with a model peptide using MP2/6ꢀ31G(d) theory level as a
function of CꢀF bond orientation. Distance between fluorine and carbonyl carbon is set to 3Å. Fluorines
are represented as small balls and the CꢀF bonds are represented as sticks. Model peptide is presented in
balls and sticks representation (carbon in cyan, oxygen in red and nitrogen in blue). c) FMAP prediction
for the meninꢀMIꢀ2ꢀ3 complex. Purple surface represents favorable positions for fluorine atoms to interact
with protein backbone.
Figure 3. Effect of fluorine substitutions in thiadiazole moiety on activity of meninꢀMLL inhibitors. a)
Structures and activities of inhibitors. ꢀꢀG values are calculated relative to MIꢀ326. b) Crystal structures
of inhibitors bound to menin showing the shortest distances between fluorine and menin backbone.
FMAP prediction is shown as purple surface.
Figure 4. Effect of fluorine substitutions in thienopyrimidine moiety on activity of meninꢀMLL
inhibitors. a) Structures and activities of inhibitors. ꢀꢀG values are calculated relative to MIꢀ19. b)
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Crystal structures of inhibitors bound to menin showing the shortest distances between fluorine and
menin backbone. FMAP prediction is shown as purple surface. Model of MIꢀ19 has been made based on
the structure of MIꢀ2ꢀ2ꢀmenin complex. c) Crystal structure of MIꢀ273 bound to menin showing close
contacts of fluorines in CF2CF3 group with menin. FMAP prediction is shown as purple surface.
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Figure 5. Analysis of FMAP calculations for known inhibitors containing fluorine atoms. Crystal
structure of proteinꢀinhibitor complexes showing close C–F···C=O contacts and FMAP predictions (in
purple). The structures of inhibitors and activities are also reported. a) thrombin inhibitor (PDB code
1OYT). b) procaspaseꢀ6 inhibitor (PDB code 4NBL). c) gp120 inhibitor (PDB code 4DKO). d) βꢀ
lactamase inhibitor (PDB code 4E3N). e) meninꢀMLL inhibitor (PDB code 4OG6). f) macrocyclic meninꢀ
MLL inhibitor (model based on PDB structure 4I80).
Figure 6. FMAP predictions for αꢀhelix and βꢀsheet structures. a) Details of the interaction of CF3 group
in MIꢀ2ꢀ3 with αꢀhelical fragment in menin and FMAP prediction. b) FMAP prediction for idealized αꢀ
helix for a single peptide bond. Orientation of the αꢀhelix is similar as in panel a. c) Details of the
interaction of CF3 group in MIꢀ2ꢀ3 with βꢀsheet in menin. d) FMAP prediction for idealized βꢀsheet
structure shown in similar orientation as in panel c.
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