Identification, Biochemical and Structural Evaluation of Species

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Identification, biochemical and structural evaluation of speciesspecific inhibitors against Type I methionine aminopeptidases Chandan Kishor, Tarun Arya, Ravikumar Reddi, Xiaochun Chen, Venkateshwarlu Saddanapu, Anil Kumar Marapaka, Rajesh Gumpena, Dawei Ma, Jun O Liu, and Anthony Addlagatta J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400395p • Publication Date (Web): 16 Jun 2013 Downloaded from http://pubs.acs.org on June 16, 2013

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Journal of Medicinal Chemistry

IDENTIFICATION, BIOCHEMICAL AND STRUCTURAL EVALUATION OF SPECIES-SPECIFIC INHIBITORS AGAINST TYPE I METHIONINE AMINOPEPTIDASES

Chandan Kishora, Tarun Aryaa, Ravikumar Reddia, Xiaochun Chenb, Venkateshwarlu Saddanapua, Anil Kumar Marapakaa, Rajesh Gumpenaa, Dawei Mac, Jun O. Liud,*, and Anthony Addlagattaa,*

a

Center for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, AP500 007, India b

Department of Pediatrics, University of Maryland School of Medicine, 655 W. Baltimore, Street, Baltimore, MD 21201, USA.

c

State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China

d

Departments of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205, USA

*Address for correspondence: Dr. Anthony Addlagatta, [email protected], Tel.: +91-040-27191860, Fax: +91-40-27160387. Jun O. Liu, [email protected], Tel.: +1 410 955 4619; Fax: +1 410 955 4620.

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Abstract Methionine aminopeptidases (MetAPs) are essential enzymes that make them good drug targets in cancer and microbial infections. MetAPs remove the initiator methionine from newly synthesized peptides in every living cell. MetAPs are broadly divided into Type I and Type II classes. Both prokaryotes and eukaryotes contain Type I MetAPs while eukaryotes have additional Type II MetAP enzyme. Though several inhibitors have been reported against Type I enzymes, subclass specificity is scarce. Here, using the fine differences in the entrance of the active sites of MetAPs from M. tuberculosis, E. faecalis and human, three hotspots have been identified and pyridinylpyrimidine based molecules were selected from a commercial source to target these hotspots. In the biochemical evaluation, many of the 38 compounds displayed differential behavior against these three enzymes. Crystal structures of four selected inhibitors in complex with human MetAP1b and molecular modeling studies provided the basis for the binding specificity.


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Introduction In all living cells, ribosome based protein synthesis begins with the amino acid methionine. Methionine aminopeptidases (MetAPs) are responsible for specifically cleaving the initiator methionine in about 70% of all proteins in living cells.1 MetAPs are typically classified into two types: Type I (MetAP1) and Type II (MetAP2), differentiated by the presence of a 60 amino acid insert in the later.2 MetAP1 enzymes are further classified into Type Ιa, Ιb, Ιc and Ιd. Type Ιb, Ιd and Type II MetAPs are expressed in eukaryotes while Type Ιa and Ic are present in prokaryotes.3-5 While most of the prokaryotes contain a single gene (MetAP1a), actinomycetes have a second gene (MetAP1c). Importance of MetAP function in a living cell is underscored by the demonstration that knockout or specific inhibition of MetAP in bacteria that express only one MetAP gene is lethal.1, 6-12 In yeast, slow growth phenotype was observed when individual genes were disrupted; however double knockout of both Type Ib and Type II MetAP encoding genes is lethal.13 In humans, all three MetAP isoforms are overexpressed in cancers and inhibition of individual enzymes for different cancers was beneficial.4, 14, 15 In addition, inhibition of MetAP2 by fumagillin leads to inhibition of endothelial cell proliferation and hence angiogenesis.16, 17 MetAPs are first-row transition metalloenzymes with five conserved metal ion-binding residues in the active site. The highly hydrophobic binding pocket for methionine side chain is comparatively less conserved. The natural products fumagillin, ovalicin and their synthetic derivatives displayed more than a million fold specificity towards MetAP2 enzyme but failed in the clinical trials due to the neurotoxicity and other side effects.18, 19 In spite of large difference



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in the activities by the natural products, mode of inhibition is similar in both Type I and Type II MetAPs i.e. by covalent modification of a conserved histidine residue in the active site.20 Apart from these natural products, there have been no other compounds that displayed such a high selectivity towards a specific MetAP. This is partly due to the high structural and sequence similarity of the active site among all MetAP isoforms. Given that MetAPs are promising drug targets, several molecular scaffolds including pyridinylpyrimidine, triazole, thiobendazole, bengamide, pyridine-2-carboxylic acid, thiazole-4carboxylic acid have been reported as potential inhibitors.9,

12, 21-24

Pyridinylpyrimidine

derivatives inhibit Type I MetAP better than Type II enzymes by binding to a third metal ion near entrance of the active site.10, 21 Molecular, cellular and structural biology based experiments established that these pyridinylpyrimidine inhibitors could specifically target MetAP1b in human cells and reduce the cancer cell proliferation.21 Though these inhibitors show selective and efficient inhibition against MetAP1 enzymes, the limitation is in the selectivity among sub-types of Type I MetAPs. Recently, using modeling tools, we have reported the possibility of differentiating sub-forms of Type I MetAPs from different species by appropriate substituents on the pyridinylpyrimidine moieties.23 In this manuscript, we report the discovery of fine differences between various Type I MetAP active sites based on the analysis of protein data bank (PDB) structures, identification of pyridinylpyrimidine based inhibitors and their differential inhibition of three Type I enzymes: Type Ia from Enterococcus faecalis (EfMetAP1a); Type Ib from human (HsMetAP1b); and Type Ic from Mycobacterium tuberculosis (MtMetAP1c). Finally, results from the protein X-ray crystallography methods to confirm the structural basis for the mode of inhibition are reported.


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Results and discussion In drug discovery and development process, understanding molecular recognition between macromolecules and their ligands is important. Efficient recognition requires both affinity and specificity.25 There have been technological advancements in drug discovery in terms of computational methods, combinatorial synthetic chemistry, high-throughput screening against target proteins and model systems, structural biology and bioinformatics. Achieving high specificity is not only a challenge but also a bottle neck in drug development process.26 Though MetAPs have been pursued for more than two decades, there has been limited success in identifying specific inhibitors except for the fumagillin family of natural products. Crystal structures are available for all sub-classes of MetAP except for Type Id. Since MetAPs display strict substrate specificity towards methionine, the S1 pocket is hydrophobic.27 The S1’ pocket where the two conserved histidines are present is a constricted entrance to the active site and the S2’ is open to the bulk solvent (Figure 1a). Fumagillin and ovalicin covalently bind to one of the histidine residues (H212 in HsMetAP1b) in the S1’ pocket.19 The same histidine was also demonstrated to bind to the third metal ion for mediating the binding of pyridinylpyrimidine based molecules in Type I MetAPs.21, 28 Pyridinylpyrimidine molecules extend from S1 pocket to S2’ pocket (Figure 1).21 Due to the limitation of the space in the S1 pocket, molecules with substitution on only B-ring display inhibition against MetAPs. A similar pattern was observed for the Pyridine-2-carboxylic acid–amide derivatives where the thiazole ring is buried in the hydrophobic S1 pocket while the substituted pyridine ring points out.14 Both these examples suggest that the A-ring should be unsubstituted while B-ring can have substitution.



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Structural differences in enzymes near the B-ring binding region To further understand the differences between various Type I MetAPs, we have carried out bioinformatic studies using the Hotspot Wizard and homology driven secondary structure alignments (HSSP).29,

30

Hotspot Wizard is a tool to identify positions that are important for

activity and substrate specificity but leaves out the residues indispensable for protein function. Analysis of hotspot wizard output suggests of three different spots in MetAP1 family near the entrance of the active site i.e. hot spot 1 (HS1): E61 (14%) of EfMetAP1a, Y196 (33%) of HsMetAP1b and K98 (21%) of MtMetAP1c; HS2: M179 (14%) of EfMetAP1a, N314 (44%) of HsMetAP1b and V216 (28%) of MtMetAP1c; HS3: V166 (34%) of EfMetAP1a, C301 (51%) of HsMetAP1b and T203 (43%) of MtMetAP1c (Figure 1b). Note that the numbers given in the brackets for different residues is the percentage of identity of that particular residue in the HSSP file of the of respective PDB structure (3TB5 for EfMetAP1a, 2G6P for HsMetAP1b and 1YJ3 for MtMetAP1c). EfMetAP1a belongs to Type Ia enzyme family that is found only in microbes, has minimum sequence and structure required for catalysis (Figure S1b). However, the N-termini of Type Ib and Type Ic enzymes is extended by about 120 and 40 amino acids, respectively. Parts of these extensions form the roof to the entrance of the active site (Figure 1b). Modeling of pyridinylpyrimidine molecules Pyridinylpyrimidine molecules have been demonstrated to bind to one of the histidine (H212 in human) using an extra metal ion in the entrance of the active site.14,

21

Analysis of these

structures suggest the possibility of placing three substituents that can interact with HS1, HS2 and HS3 (Figure 1b). R1 substituent in the B-ring is near the HS1 region and makes direct contact. Based on previous structures, large substituents are permitted at this position. R2


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substituent in the B-ring faces the bulk solvent. With flexibility and hydrophilicity, R2 can be a bulkier substitution. On the other hand, space near the R3 group is limited and only small substituents can be accommodated. R2 and R3 substituents scan the HS2 and HS3 regions of the MetAP proteins. To test the compatibility of any substitution, we also planned a small substituent (R4) in the A-ring. To probe the differences in the enzyme pockets between Type Ia, Type Ib and Type Ic, we have selected pyridinylpyrimidine based molecules from a commercial source with different substitutions at R1, R2, R3 and R4. Choice of target MetAP enzymes To represent one member of each class of Type I enzymes, EfMetAP1a, HsMetAP1b and MtMetAP1c were chosen for this study. In addition, these three proteins are also good targets in infectious disease such as urinary tract infection, tuberculosis and cancer. Protein expression and purification was carried out according to reported procedures.3,

23,

28

A total of 38

pyridinylpyrimidine based molecules were obtained from Maybridge (London) with functional variation that can probe the fine differences between Type I MetAP enzymes (Table 1). The colorimetric assay as described earlier was adopted for the biochemical screening.31 Specific inhibition A group of compounds inhibited all three enzymes (MB01, MB04, MB06, MB12, MB13, MB15, MB18, MB19, MB20, MB29, MB30, MB31, MB33, XC22 and Shoc1) to different extents (Table 1). Note that all these compounds have some common features: large but flexible or small substitutions at R1, smaller ones at R2, R3 and no substitution at R4. MB02, MB03, MB09, MB10, MB11, MB21, MB23, MB35 and MB36 are inactive against any of the enzymes. Rigid substitutions at R1, rigid and large substitution at R2 limits the inhibition ability of these



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molecules to bind to the enzyme competitively. One exception in this group is with MB09 that has a flexible group at R1 but inactive against all three enzymes (explained below). Of the three enzymes, human MetAP1b which has tyrosine (Y196) at HS1 is more sensitive followed by EfMetAP1a that has acidic glutamate residue (E61). MtMetAP1c with lysine (K98) at HS1 is the least sensitive of all. A trifluoromethyl group at R4 in the A-ring abrogated inhibitory effects of MB35 and MB36 against any of the enzymes tested. Note that MB04 (morpholino ring at R1), MB12 (N-methylpiperazino ring at R1) are similar to MB35 (a piperidine ring at R1) except that the later has a trifluromethyl group at R4. While MB04 and MB12 inhibit all three enzymes, the MB35 does not inhibit any of the MetAPs. A similar analogy can be drawn between MB15 (1chloro-4-phenoxy group at R1) that is similar to MB36 (a thioether equivalent at R1). With a trifluromethyl group at R4 MB36 is not active against any of the enzymes while MB15 inhibits all three MetAPs with high efficiency. These two examples confirm that lack of inhibition by MB35 and MB36 is due to the presence of trifluromethyl group at R4, which cannot be accommodated in the S1 pocket of MetAP. Crystal Structures – overall mode of binding of pyridinylpyrimidines To establish the molecular basis of differential inhibition potentials of these compounds, we obtained crystal structures of human MetAP1b in complex with selected compounds (MB01, MB17, XC22 and shoc1). As expected, in line with the previous observations, these pyridinylpyrimidines recruit a third metal ion to bind to the enzyme.14, 21 In all structures, the third metal ion coordinates with NE2 atom of the H212 in the protein, two nitrogen atoms of the inhibitor and three water molecules. Differential behavior of regioisomers


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Compounds MB17 and XC22 are structurally closely related, differing in the positioning of trifluoromethyl group on the pyridine ring and the presence of an extra methyl group at R3 in the pyrimidine ring in XC22. While XC22 inhibits all the three enzymes to different degrees, MB17 inhibits only the human enzyme with about ten-fold reduction in efficiency (Table 1). Crystal structure of XC22 in complex with human enzyme revealed that the entire substituent on the pyridinylpyrimidine is well ordered and extends into the grove under the roof between HS1 and HS2 regions (Figure 2a and Figure 2c). On the other hand, the 6-trifluoromethyl-pyridine moiety in the MB17 is disordered and extends into the solvent region (Figure 2b and 2c). If substituent of the MB17 would also have to bind in similar orientation as XC22, the trifluoromethyl group would experience steric clashes with either protein or the chloro substitution at R2 of the inhibitor itself. This explains a lowered affinity of the MB17 compared to XC22 for the human enzyme. While one of the conformations of the disordered R1 substituent of MB17 is stabilized by Y196 (HS1 residue), the other is stabilized by the inhibitor itself. Lack of activity against any of the microbial enzymes could be due to the charged amino acids present in the HS1, which does not allow the binding of the disordered substituent of the MB17. Additional hydrogen bonds improve the affinity Shoc1 is similar to MB30 except for an additional formamide group on the substituent. Shoc1 is well ordered in complex with human MetAP1b (Figure 3a). The formamide group makes two hydrogen bonds with two water molecules, which in turn interact with the protein (Figure 3b). In our previous study, we have reported the crystal structure of MB30 in complex with human MetAP1b in which the inhibitor does not make any hydrogen bonds with protein or solvent except for the interaction with the third cobalt ion.21 About three-fold improvement in the potency of the shoc1 compared to MB30 against the human enzyme was observed while it is



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marginally improved against the microbial enzymes (Table 1). The additional hydrogen bonds through water molecules seem to play a role in the improvement of the activity. Rigid substitutents at R1 cause inhibition of all three MetAPs Common feature between MB01, MB04 and MB12 is the presence of a six-membered heterocycle that is rigid; they inhibited all the three MetAPs within 10-fold difference. To understand the structural basis for this inhibition, crystal stricture of HsMetAP1b was determined in complex with MB01 (Figure 4). While there is no change in the position of the pyridinylpyrimidine moiety, the R1 substituent, unlike other structures presented above, is rigid and extends straight towards the bulk solvent making contact with protein surface. Most of the contacts are with main chain of the protein, which is common among all the enzymes. Best inhibition is observed against the HsMetAP1b, followed by MtMetAP1c and EfMetAP1a, respectively. This can be explained by probable protection of the inhibitor from solvent by the roof region in the human enzyme and to a less extent in MtMetAP1c. This roof is absent in the EfMetAP1a, which explains a weakest inhibition of this enzyme. Docking studies Compounds with similar substituents such as MB07, MB08 and MB09 display different inhibition profile. While MB09 is completely inactive, MB07 is active against only human enzyme and MB08 is active against human and EfMetAP1a (Table 1). It is not apparent how the differences in their structures account for their distinct activity. To understand the differences further, docking studies were carried out with these three inhibitors against all three enzymes (Figure 5). The fluorophenyl group of MB07 forms π-π interaction with side chain of Y196 in human enzyme. The other two microbial enzymes have polar residues that are generally charged


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at pH 7.5, resulting in repulsion of this fluorophenyl group. Glutamate (E61) side chain of EfMetAP1b is deprotonated at pH 7.5 while the lysine (K98) side chain in the MtMetAP1c is protonated which leads to electrostatic repulsion with fluorophenyl group of MB07. It is surprising that MB09, despite its structural similarity to MB07, has a distinct activity profile. One possibility is that the π-systems differ in the inhibitors. The fluorine atom having electronwithdrawing nature may pull electrons from the aromatic ring making it electron deficient in MB07. On the other hand the –OH group of Y196 (in HS1) forms a strong hydrogen bond with Q128 through a water molecule, making the aromatic ring electron rich. This promotes favorable π-π interaction between MB07 and enzyme. In contrast, the electron-donating methoxy group in MB09 makes the aromatic ring electron rich resulting in unfavorable interaction. Though the morpholino group in MB08 cannot form π-π interaction, it can interact with other parts of the proteins (Ala312 in HsMetAP1b or Ser177 in EfMetAP1a) (Figure 5). Effect of substitutions at R2, R3 and R4 Due to the presence of two nitrogen atoms in the B-ring and the possibility of rotation between the two aromatic rings (A and B) of the pyridinylpyrimidine, R1 and R3 can be interchanged during the binding of inhibitors to the enzymes. However, as there is limited space in the protein structure near the R3 binding region (HS3), a smaller substituent is always preferred. Residues near R3 binding region (HS3) are variable between the enterococcus (V166), tuberculosis (T203) and human (C301) MetAPs. This variability is evident from the differential inhibition of these three enzymes. Compounds (MB10 and MB11) with bulky and rigid R2 group do not show inhibition against any of the enzymes. When the substituents are small and flexible, MetAPs from enterococcus and human are inhibited but not the one from tuberculosis. R3 substitution



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has much lower size limit in terms of inhibition, which is also evident from the structure. It is also clear that substitution cannot be placed at R4 (Figure 6). Conclusion MetAPs are essential enzymes, making them good drug targets for cancer and microbial infections. Due to the similarity in the active sites between enzymes from different species, it is challenging to design specific inhibitors. In this study, the differences in the entrance of the active sites of MetAPs from M. tuberculosis, E. faecalis and human have been identified in the form of three hotspots. Pyridinylpyrimidine based inhibitors were identified to target these three hot spots. Many of the 38 pyridinylpyrimidine molecules displayed differential activity against three MetAP enzymes. Crystal structures of four selected inhibitors and molecular modeling studies provided the basis for the differential activity of different inhibitors against the three MetAP enzymes. This is a first study dedicated to understand the fine differences between subclasses of MetAP1 family and successfully identify molecules that displayed selectivity. This understanding will help in designing better inhibitors with high affinity and selectivity against cancer and microbial diseases.


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MATERIALS AND METHODS All chemicals used for protein expression and purification were purchased from SigmaAldrich, USA. Enzymes for molecular biology were purchased from Fermentas. 38 pyridinylpyrimidinebased molecules were obtained from Maybridge (London) with 95% or better purity. No further characterization of the compounds was carried out. Sequence and structural alignment Sequences of EfMetAP1a (Uniprot ID: C7VIN6), HsMetAP1b (P53582) and MtMetAP1c (P0A5J2) were obtained from Uniprot.32 Multiple sequence alignment was performed by clustalW33 and figure was generated by ALINE software (Figure S1).34 Coordinates for these MetAPs were obtained from protein data bank (PDB) (EfMetAP1a: 3TB5, HsMetAP1b: 2G6P, MtMetAP1c: 1YJ3). Superimposition of the coordinates was carried out in Coot and PyMol.35, 36 Protein expression and purification Reported protocols were followed for the purification of EfMetAP1a,23 full length HsMetAP1b28 and MtMetAP1c3 using the clones reported therein. All proteins were purified using the Ni-NTA column. Purity was verified by SDS-polyacrylamide gel (Figure S2). Enzyme kinetic assays Enzyme kinetics were performed for each of the enzyme separately in 100 µL volume containing 25 mM HEPES (pH 7.5), 150 mM KCl, active enzyme (12 µM for EfMetAP1a, 4 µM for HsMetAP1b and 8 µM for MtMetAP1c), CoCl2 (three molar equivalents with respect to the corresponding enzyme). Note that these three enzymes displayed activities to different extents. Enzyme concentrations were varied so that they display normalized activity. After incubation of the above reaction mixture for 30 minutes at 30 °C, 200 µM of the substrate, methionine para-



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nitroamilide (Met-pNA) was added to makeup the 100 µL final volume. Formation of pNA was monitored continuously at 410 nm wavelength in the microplate multimode reader (TECAN). IC50 determination All compounds were dissolved in dimethylsulfoxide (DMSO) to a stock concentration of 10 mM and were screened against EfMetAP1a, HsMetAP1b and MtMetAP1c in the concentration range from10 nM to 64 µM. IC50 was determined from the graphs plotted using percentage inhibition verses logC value (for the inhibitor concentration) using Sigmaplot 11.2 (Table 1). Protein Crystallization, X-ray data collection and refinement Crystallization of HsMetAP1b was carried out as reported earlier.21 Briefly, diffraction quality crystals were obtained by mixing 5 µL of apo protein (in 25 mM HEPES at pH 8.0, 5 mM methionine, and 150 mM KCl) with 5 µL reservoir solution (12%–16% PEG monomethyl ether 2000, in 100 mM HEPES at pH 5.4–6.2) in a hanging drop that was incubated at 25 °C. Rod shaped apo crystals were transferred into 10 µL cryo-protectant solution (16% PEG monomethyl ether 2000, 25% glycerol, and 100 mM HEPES at pH 6.0), and 2 mM of pyridinylpyrimidine (final concentration) dissolved in DMSO and freshly prepared CoCl2 (1 mM, final concentration) were added and incubated for 24 h. Crystals were then directly frozen in liquid nitrogen for data collection. X-ray diffraction data were collected on beamline 8.2.2 at the Advanced Light Source (ALS) using a radiation of wavelength 1.00 Å. Diffraction data were processed by HKL3000 and Scalepack.37 Using the coordinates of the native HsMetAP1b (PDB code 2B3K) with all water molecules removed, structure refinement and modeling were carried out using respectively Refmac5 and Coot (Table 2).35, 38 Molecular Modeling


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The pyridinylpyrimidine moiety was fixed with the third cobalt ion. Multiple rotomers of the substituents at R1, R2 and R3 were explored to find the best fit with least steric clashes and chemically sensible interactions such as hydrogen bonds and π-π interactions. For this ‘.cif’ files required for each molecule were generated on the PRODRG server and imported into the coot.35, 39

Multiple conformations for the substituents were explored.

Supporting Information Available: Sequence alignment and SDS-page gel are submitted as supporting information. This material is available free of charge via the Internet at http://pubs.acs.org. PDB ID Codes: 4IKR, 4IKS, 4IKT and 4IKU Corresponding Author: *For A.A.: Phone, 91-040-27191860; Fax, +91 40 27160387; E-mail, [email protected]; For J.O.L.: phone, +1 410 955 4619; Fax, +1 410 955 4620; E-mail, [email protected].

Acknowledgment: Thanks to University Grants commission (UGC), Council of Scientific and Industrial Research (CSIR), New Delhi, for research fellowships to CK, TA, RR, RG and VS. AA acknowledges the financial support for research from Department of Science and Technology (SR/SO/BB-55/2008), Department of Biotechnology (BT-BRB-TF-2-2011), and Council of Scientific and Industrial Research (SMiLE project) New Delhi, India. Abbreviations Used: MetAP, methionine aminopeptidase



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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1: IC50 (µM) of pyridinylpyrimidine derivatives against recombinant EfMetAP1a, HsMetAP1b and MtMetAP1c

Comp

R1

R2

R3

R4

ound

Cl

MB01

CH3

H

EfMetAP1a

HsMetAP1b

MtMetAP1c

(HsMetAP1b/E

(HsMetAP1b/Mt

fMetAP1a)

MetAP1c)

5.55±0.07

0.59±0.04

(0.11)

4.27±0.33 (0.14)

MB02

Cl

CH3

H

>64

>64

>64

MB03

Cl

CH3

H

>64

>64

>64

MB04

Cl

CH3

H

2.21±0.09

0.38±0.02

4.70±0.03

(0.17)

Cl

MB05

CH3

H

40.0±1.62

(0.08)

3.15±0.15

>64

0.64±0.07

4.36±0.19

(0.08)

MB6

F F F

H

OH

H

4.92±2.79

(0.15)

(0.13)

MB07

Cl

CH3

H

>64

2.21±0.91

>64

MB08

Cl

CH3

H

7.23 ± 1.75

0.77±0.07

>64

(0.11)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Cl

MB09

MB10

F F F

MB11

Page 22 of 31

CH3

H

>64

>64

>64

H

H

>64

>64

>64

H

H

>64

>64

>64

CH3

H

10.5±1.5

0.68±0.04

3.10±0.18

F F F

Cl

MB12

(0.14) NH2

MB13

H

H

3.71±0.22

(0.22) 0.72±0.07

(0.196) MB14

MB15

O

Cl

(0.21)

H

CH3

H

>64

7.46±0.83

>64

H

CH3

H

3.65±0.59

0.63±0.03

6.08±0.20

(0.17) MB16

CH3

3.41±.09

H

SH

H

7.92±0.84 (0.07)


(0.10) 0.59±0.08

>64

Page 23 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


MB17

MB18

CH3

Cl

H

H

>64

7.23±0.02

>64

H

OH

H

3.27±0.31

0.75±0.05

>64

11.9±3.8

8.32±1.87

0.85±0.03

(0.07)

(0.10)

2.32 ± 0.37

0.66±0.06

(0.23) OH

MB19

MB20

NH2

H

H

(0.29)

1.67±0.32 (0.39)

MB21

CH3

OH

H

>64

>64

>64

MB22

CH3

OH

H

4.80±0.89

0.55±0.042

>64

(0.11) H

MB23

MB24

CH3

OH

H

>64

>64

>64

H

3.10±0.67

0.61±0.04

>64

0.59±0.04

>64

0.43±0.03

>64

(0.20) MB25

OH

H

14.2±1.17 (0.04)

MB26

OH

H

15.5±2.18 (0.03)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

H

MB27

10.6±1.75

Page 24 of 31

2.77±0.29

> 64

0.72±0.03

>64

1.17±0.03

4.46±0.35

(0.26)

OH

MB28

H

6.99±1.13 (0.10)

H

MB29

4.33±0.43 (0.27)

Cl

MB30

CH3

H

7.77±1.08

(0.26) 4.65±0.58

(0.60) Cl

MB31

CH3

H

6.50±1.13

(0.45) 1.44±0.15

(0.22) MB32

NH2

H

OH

H

3.11±0.40

10.34±0.40

2.41±0.29 (0.60)

1.03±0.09

>64

0.95±0.14

2.34±0.20

(0.33) MB33

OH

H

H

1.56±0.32 (0.61)

MB34

H

OH

H

16.9±4.64

(0.40) 1.07±0.07

>64

(0.063) MB35

Cl

CH3

F F F

>64

>64

>64

MB36

Cl

CH3

F F F

>64

>64

>64

XC22

Cl

CH3

7.38±1.39

0.67±0.03

8.40±0.30

H

(0.09)


(0.08)

Page 25 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

shoc1


H2N O NH

Cl

CH3

H

5.64±0.70 (0.31)


1.79±0.08

6.63±0.13 (0.26)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 31

Table 2: Crystallographic Table Cell parameters

MB01

MB17

XC22

shoc1

Space group

P21

P21

P21

P21

a (Å)

47.34

47.22

47.18

47.49

b (Å)

77.42

77.33

77.37

77.38

c (Å)

48.02

47.15

47.69

48.09

β (deg)

91.03

92.36

91.73

90.76

Resolution range

50.0-1.80 (1.80-

50.0-1.70 (1.70-

50.0-1.60 (1.60-

50-1.30 (1.30-

(Å)

1.86)

1.76)

1.66)

1.33)

reflections

888682

580930

565733

817480

Total

30452 (2182)

34271 (2406)

39219 (2308)

75896 (4103)

Completeness (%)

92.4 (66.7)

92.0 (65.1)

86.6 (51.2)

89.3 (48.4)

I/σ (I)

22.63 (2.68)

24.4 (2.92)

27.97 (2.66)

37.29 (2.47)

Rsym (%)

3.30 (24.4)

5.1 (30.0)

3.9 (5.4)

3.3 (26.8)

R (R-free) (%)

18.04 (21.12)

18.44 (21.55)

19.59 (22.03)

18.89 (29.28)

∆bonds (Å)

0.02

0.01

0.02

0.02

∆angles (deg)

1.97

1.38

1.97

1.96

Data collection

(Highest res. shell) Collected

Unique (High. Res.)

Refinement statistics


Page 27 of 31

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Figures

Figure 1: a). Stereo diagram of the active site bound with a pyridinylpyrimidine molecule. The surface diagram represents active site where two metal ions bind for catalysis. The A-ring of the pyridinylpyrimidine points inside the S1 pocket while the third metal ion (blue sphere) sits in the S1’ pocket and B-ring in the S2’ region. b). Representation of the active site MetAP with the alignment of three MetAP enzymes (pink: EfMetAP1a (PDB ID: 3TB5), green: HsMetAP1b (PDB ID: 2GP6) and cyan: MtMetAP1c (PDB ID: 1YJ3)). All MetAPs share similar catalytic domain represented in gray surface. MetAP1b and MetAP1c possess additional residues on the amino-terminus that form the roof to the entrance of the active site. Three hotspots are shown as



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Page 28 of 31

HS1 (E61 of EfMetAP1a, Y196 of HsMetAP1b and K98 of MtMetAP1c), HS2 (M179 of EfMetAP1a, N314 of HsMetAP1b and V216 of MtMetAP1c) and HS3 (V166 of EfMetAP1a, C301 of HsMetAP1b and T203 of MtMetAP1c) and three possible substituents on the B-ring of pyridinylpyrimidine as R1, R2 and R3. R4 is substituent on the A-ring. In both the figures, blue sphere represents the third cobalt ion while the red spheres represent the water molecules coordinating this metal center.

Figure 2: a) Omit maps of XC22 (PDB ID: 4IKT) in complex with human MetAP at 3.8 σ and b) MB17 (PDB ID: 4IKS) at 3.6 σ held to the third metal ion (blue sphere) which in turn is coordinated by three water molecules (red spheres). c). Alignment of the XC22 (yellow sticks)


Page 29 of 31

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and MB17 (cyan) molecules in complex with HsMetAP1b. Note that roof region provides extrahydrophobic protection from bulk solvent for the XC22 which is absent in MB17 structure that is reflected in two alternate conformation both of which are exposed to solvent.

Figure 3: a) Stereo diagram of the omit map of shoc1 (PDB ID: 4IKU) at 4 σ. The phenyl group in the R1 substituent folds back onto the B-ring to form π-π interactions. B). Alignment of the crystal structures of HsMetAP1b in complex with shoc1 and MB30 (PDB ID: 2G6P). With additional formamide group, shoc1 make hydrogen bond contacts to the HS1 region of the protein which is not possible in the MB30. Blue sphere indicates 3rd cobalt ion while red spheres for water molecules.



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Page 30 of 31

Figure 4. Stereo representation of the crystal structure of MB01 (PDB code: 4KIR) in complex with HsMetAP1b. The omit map at 4.0 σ is shown as green mesh around the MB01 molecule. Enzyme is shown in the gray surface diagram.

Figure 5: Alignment of models of MB07 (cyan), MB08 (gold) and MB09 (pink) in the active site of HsMetAP1b. Note that only the best fit of the multiple conformations is represented for each molecule. MB07 forms π-π interactions with Y196 of HS1 while MB08 extends to interact with A312 (not shown). MB09 experiences steric clashes with the protein in the current representation.


Page 31 of 31

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Figure 6: Proposed model of inhibitors that are favored by MetAPs. Table of Contents Graphics


Identification, Biochemical and Structural Evaluation of Species

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