Article pubs.acs.org/jmc
Identification of the Molecular Basis of Inhibitor Selectivity between the Human and Streptococcal Type I Methionine Aminopeptidases Tarun Arya,† Ravikumar Reddi,† Chandan Kishor,† Roopa Jones Ganji,† Supriya Bhukya,† Rajesh Gumpena,† Sheena McGowan,*,‡ Marcin Drag,*,§ and Anthony Addlagatta*,† †
Centre for Chemical Biology, CSIRIndian Institute of Chemical Technology, Hyderabad 500 007, India Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, Victoria 3800, Australia § Division of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland ‡
S Supporting Information *
ABSTRACT: The methionine aminopeptidase (MetAP) family is responsible for the cleavage of the initiator methionine from newly synthesized proteins. Currently, there are no small molecule inhibitors that show selectivity toward the bacterial MetAPs compared to the human enzyme. In our current study, we have screened 20 α-aminophosphonate derivatives and identified a molecule (compound 15) that selectively inhibits the S. pneumonia MetAP in low micromolar range but not the human enzyme. Further bioinformatics, biochemical, and structural analyses suggested that phenylalanine (F309) in the human enzyme and methionine (M205) in the S. pneumonia MetAP at the analogous position render them with different susceptibilities against the identified inhibitor. X-ray crystal structures of various inhibitors in complex with wild type and F309M enzyme further established the molecular basis for the inhibitor selectivity.
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INTRODUCTION Methionine aminopeptidases (MetAPs) are metalloenzymes with unique pita-bread structure that specifically cleave methionine residues from the amino terminus of a nascent polypeptide chain.1,2 In every living cell, ribosome-based biosynthesis of proteins is initiated by methionine, coded by AUG on the mRNA. MetAPs cleave the initiator methionine from 60% to 70% of all newly synthesized proteins.3 MetAPs are typically classified into two categories, type I (MetAP1) and type II (MetAP2), which differ by the presence of a 60 amino acid insertion within the catalytic domain of the latter.4 Both isoforms are present in eukaryotes, while prokaryotes have only MetAP1.5 Type I MetAP is further subclassified into types Ia−d. MetAP1a is present in all prokaryotes universally, while type Ic is an additional enzyme found only in actinobacteria including Mycobacterium tuberculosis.6 Types Ib and Id MetAPs are present in eukaryotes. Since the removal of methionine is an essential process for both prokaryotes and eukaryotes, numerous drug discovery efforts have been reported targeting these enzymes in search of antimicrobial and anticancer agents. To date, such studies have met with limited success3,7−15 and currently there has not been a single molecule identified that selectively targets bacterial MetAPs over the human enzymes. This is likely due to the high similarity between the active sites of bacterial and human MetAPs. There are at least 60 crystal structures of MetAPs deposited in the Protein Data Bank (PDB);16 however, there has been limited exploitation of these structures to define the © 2015 American Chemical Society
differences between prokaryote and mammalian MetAP enzymes to allow for the design of selective inhibitors. Recently, we reported a new genetic variant of the MetAP type Ia family from Streptococcus pneumonia (SpMetAP1a).17 Two small insertions (4 (insert 1) and 27 (insert 2) amino acids) were identified on the surface of the enzyme that is present in all the streptococcal MetAPs. In addition, the enzyme was crystallized in an inactive conformation where the active site was blocked by a surface loop. This was the first example of a MetAP crystallizing in an inactive form.17 To identify specific and selective inhibitors of SpMetAP1a, we have screened a library of 20 α-aminophosphonic acid derivatives against SpMetAP1a and MetAPs from human (HsMetAP1b), M. tuberculosis (MtMetAP1c) and Enterococcus faecalis (Ef MetAP1a). The α-aminophosphonates have been shown to act as potent and selective inhibitors against other metalloaminopeptidases.18−20 Previous studies using fluorogenic substrates with natural and unnatural amino acids against both human and E. coli MetAP enzymes have shown that only amino acids with hydrophobic side chains bind to the S1 pocket.21 Therefore, in our current study, we selected 20 compounds that contained hydrophobic side chains. Here we present the results of this study and the identification of a selective inhibitor of SpMetAP1a. Excitingly, this compound showed good selectivity over other MetAPs including the human Received: November 19, 2014 Published: February 20, 2015 2350
DOI: 10.1021/jm501790e J. Med. Chem. 2015, 58, 2350−2357
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Journal of Medicinal Chemistry
Figure 1. (a) Crystal structure of SpMetAP1a showing the loop (blue) in the inactive conformation covering the active site (green oval) (PDB code 4KM3). Two inserts identified unique to this family of protein from Streptococci are depicted in red. (b) Crystal structure of HsMetAP1b in complex with methionine (green) (PDB code 4UJ6). 2Fo − Fc electron density map is drawn at 2.5σ. S1 pocket residues are shown as gray sticks. For the sake of clarity, metal binding residues are omitted from the diagram. Metal ions in the active site are shown as blue spheres. Note that F309 side chain is in proximity to the Cβ atom of substrate methionine.
Figure 2. Sequence alignment of HsMetAP1b, MtMetAP1c, SpMetAP1a, and Ef MetAP1a. Residues highlighted in blue are metal binding where the backbone of the substrate peptide docks. Those highlighted in red are side chain recognition amino acids in the S1 pocket. Residue position marked with asterisk in the S1 pocket has an unusual variation. It is phenylalanine in the human and Mtb MetAP enzyme, while it is a methionine in SpMetAP1a and isoleucine in Ef MetAP1a. Also note that this residue is very close to the Cβ of the substrate methionine (Figure 1b).
HsMetAP1b enzyme. We have used bioinformatics, site-directed mutagenesis, biochemistry, and X-ray crystallography studies to elucidate the molecular basis of the selectivity. We have identified a methionine (205Met) in the active site of the SpMetAP1a and a phenylalanine (309Phe) in the human enzyme at the corresponding positions that are responsible for the different inhibitor specificities. This methionine is conserved across all of the MetAPs in the Streptococci bacteria, providing the first pathway to rational drug design targeting the bacterial MetAPs.
unique pita-bread fold and a buried active site in the center of the protein (Figure 1a). To understand the atomic detail of the active site and binding of a substrate with a P1 methionine, we solved the crystal structure of HsMetAP1b bound to a free methionine residue (Figure 1b). The structure confirms previous studies that have shown that the MetAP1b active site can be subdivided into a highly conserved metal-binding region, where the peptide backbone of the substrate docks, and a less conserved S1 pocket that accommodates the methionine side chain (Figure 1b, discussed later). To investigate the degree of conservation of this pocket, we performed a sequence and structural alignment of four selected MetAP enzymes; SpMetAP1a, HsMetAP1a, MtMetAP1c, and Ef MetAP1a. In analyzing this alignment, we
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RESULTS Methionine versus Phenylalanine in the S1 Pocket of the MetAP1a Enzymes. MetAPs are metalloenzymes with a 2351
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Met structure; the M309 side chain pushes the H310 by tilting it away from the S1 pocket (Figure 3b). Selective Inhibition of SpMetAP1a by β-Branched αAminophosphonates. We screened a 20-compound library of α-aminophosphonates against our four selected enzymes (Supporting Information Table 3). From our initial screen, we selected compounds that inhibited at least one of our selected MetAP enzymes and calculated a Ki against each enzyme (Table 2). The results showed that all compounds acted as competitive inhibitors (if inhibitory) and showed inhibition in the micromolar range. Compound 15 (1-amino-1-cyclohexylmethanephosphonic acid) selectively inhibited SpMetAP1a (Ki = 7.9 μM) and not the human M. tuberculosis and E. faecalis enzymes up to 500 μM concentration (Table 2). In addition to 15, compound 16 inhibited SpMetAP1a (18.89 μM) and Ef MetAP1a (52.19 μM), while the human and M. tuberculosis MetAPs were not affected up to 500 μM. In contrast, compound 2, the aromatic analog of 15, inhibited all MetAPs to different potencies while 3, the aromatic analog of 16, inhibited none of the enzymes tested (Table 2). Similarly, 19 (1-amino-2-methylpentylphosphonic acid) was selective against the streptococcal protein compared to the HsMetAP1a (∼15-fold) and MtMetAP1c (∼29 fold) while Ef MetAP1a was not inhibited (Table 2). Another noncyclic molecule 1-amino-2-propylpenthanephosphonic acid (20) showed the opposite behavior with improved selectivity toward human and M. tuberculosis enzymes compared to the streptococcal MetAP. Compound 18 inhibited all the tested enzymes (Table 2). The inhibition profiles of F309M were reversed compared to the wild type HsMetAP1b. The alteration of the conserved phenylalanine generated a protease that behaved like the SpMetAP1a, suggesting that this residue was acting as a molecular switch with regard to selectivity. Compounds 15 and 16, which did not inhibit HsMetAP1b (Table 2), inhibited the F309M (6.01 and 9.5 μM respectively, Table 2) and were comparable to SpMetAP1a (7.14 and 17.35 μM, respectively, Table 2). Similarly, compounds 2 and 19 also showed improved inhibition and 20 reduced activity against F309M in accordance with SpMetAP1a (Table 2). Selectivity Is Controlled via a Single Amino Acid Switch. To understand the molecular basis for the selective inhibition of SpMetAP1a and the F309M human mutant by 15,
focused on the nine amino acids that form the S1 pocket of the MetAPs22 (Figure 2). Between HsMetAP1b and SpMetAP1a, five of the nine amino acids were different. We identified that one of these changes was in position 309 (HsMetAP1b numbering) where the human enzyme has a rigid aromatic phenylalanine residue but the SpMetAP1a contained a flexible and linear methionine. Further analysis of the (total) 23 streptococcal MetAP enzymes shows that there is strict conservation of a methionine at this position (Figure 4s in Supporting Information). To investigate the functional role of the position 309 residue, we constructed a F309M mutant in the HsMetAP1b protein. This would effectively introduce a residue that is only conserved in the bacterial enzymes to the human protease scaffold. The F309M enzyme was active; however, it showed a slightly reduced catalytic turnover and overall efficiency compared to the wild type HsMetAP1b (>10-fold reduction, Table 1, Figure 2s in Table 1. Kinetic Parameters for Enzymes Used in This Study HsMetAP1b F309M SpMetAP1a MtMetAP1c Ef MetAP1a
Km (μM)
kcat (min−1)
kcat/Km (μM−1 min−1)
386.3 ± 20.3 401.9 ± 12.2 543.4 ± 11.3 980.0 ± 0.2 249.0 ± 35.9
2.9 ± 0.004 0.2 ± 0.006 0.3 ± 0.003 10.6 ± 0.70 0.3 ± 0.128
7.7 × 10−3 0.6 × 10−3 0.6 × 10−3 10.8 × 10−3 1.0 × 10−3
Supporting Information). To confirm that no gross structural changes had occurred in the point mutant F309M, we solved the X-ray crystal structures of HsMetAP1b and F309M to 1.56 and 1.87 Å, respectively (Figure 3 and Supporting Information Table 2). The overall structures were similar to rmsd of 0.09 Å for all Cα-atoms. In addition, the conformation of the methionine residue bound in the active sites of both the wild type and the mutant enzymes was similar, making identical contacts with common neighbors (Figure 3). In the HsMetAP1b-Met structure, two carbon atoms of F309 make hydrophobic contacts with the product methionine (3.5 and 4.1 Å) while only the Cε atom of the M309 in F309M-Met structure is in close contact (3.9 and 4.2 Å). In the HsMetAP1b-Met structure, F309 and H310 are involved in a π−π contact (3.5 Å) and in the F309M-
Figure 3. (a) Crystal structure of F309M in complex with methionine (brown) (PDB code 4U75). (b) 2Fo − Fc electron density map is drawn at 1.6σ. (b) Structural alignment of HsMetAP1b-Met (green) and F309M-Met (brown). There is no difference in the substrate methionine confirmation. The F309 and M309 in respective structures align well. In the wild type enzyme due to π−π interaction, F309 and H310 align with each other. Since π−π interaction is not possible, to avoid the close contact with M309, H310 tilts away from the active site in F309M structure. 2352
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Ki [μM] compd
R
HsMetAP1b
SpMetAP1a
F309M
MtMetAP1c
Ef MetAP1a
2 15 16 18 19 20
phenyl cyclohexyl cyclohexylmethyl 2-cyclopentylethyl 1-methylbutyl 1-propylbutyl
111.6 ± 16.1 >500 >500 9.9 ± 0.5 54.8 ± 10.6 4.9 ± 0.4
1.8 ± 0.2 7.9 ± 0.7 18.9 ± 1.64 6.5 ± 0.9 3.6 ± 0.5 78.7 ± 9.6
6.6 ± 0.9 6.0 ± 0.8 9.5 ± 1.1 5.5 ± 0.7 3.9 ± 4.4 61.3 ± 7.3
132.5 ± 18.3 >500 >500 6.1 ± 1.0 107.3 ± 11.7 9.5 ± 1.3
10.6 ± 1.9 >500 52.2 ± 4.5 17.5 ± 4.3 >500 >500
Figure 4. (a) Crystal structure of F309M in complex with 15 (brown) (PDB code 4U71). The α-cyclohexyl group binds very tightly in the hydrophobic S1 pocket, forming very short distances (3.1 Å) with Cε and Sδ atoms of M309. (b) Crystal structure of F309M in complex with compound 16 (chocolate) (PDB code 4U70). Note that the side chain is elongated in this structure by one carbon in the form of Cβ-atom. Because of this, cyclohexyl ring escapes the tight interaction with some S1 pocket residues. (c) Crystal structure of HsMetAP1b in complex with 18 at 1.85 Å (PDB code 4U6C). The side chain is a γ-branched cyclopentyl group, which is smaller than the cyclohexyl moiety.
Ef MetAP1a is a β-branched side chain, which is bulkier and therefore is likely to oppose the binding of 15. The crystal structure of F309M in complex with 16 (F309M16) was determined at 1.6 Å (Figure 4b). Similar to 15, compound 16 inhibits the SpMetAP1a with high affinity but does not inhibit HsMetAP1b and MtMetAP1c and moderate inhibition was noted with Ef MetAP1a. While 15 is a β-branched compound, 16 is γ-branched and therefore longer and more flexible. In the F309M-15 and F309M-16 structures the cyclohexyl ring adopts a stable “chair” conformation. The M309-Cε-atom rotates away by about 62° in F309M-16 compared to that in F309M-Met structure. Structural alignment of HsMetAP1b-Met and F309M-16 suggests that the distance between F309 and cyclohexyl ring of 16 would be only 3.2 Å and likely result in steric clashes. This provides the molecular basis for the lack of inhibition of HsMetAP1b and MtMetAP1c by compound 16. Compound 18 inhibits all the MetAPs tested including F309M (Figure 4c). The side chain is a γ-branched cyclopentyl group, which is smaller than the cyclohexyl moiety. The crystal structure of HsMetAP1b in complex with 18 at 1.85 Å explains why a phenylalanine, methionine, or isoleucine (at the analogous F309 position in HsMetAP1b) would not induce any serious steric clashes to accommodate this molecule. The shortest distance between the inhibitor and the F309 is approximately 3.4 Å. Compound 2 (1-amino-1-phenylymethylphosphonic acid) is an aromatic analogue of 15 and inhibits all MetAPs tested and shows a preference to those that have methionine (SpMetAP1a,
the crystal structure of the complex of F309M-15 was determined at 1.8 Å resolution (Figure 4a). As expected and was determined in all of our bound structures, the αaminophosphonate group docks to the metal center in addition to forming hydrogen bonds with the two histidine side chains located at the entrance to the active site (Figure 4a). The αcyclohexyl group binds in the hydrophobic S1 pocket. The Cεcarbon atom of M309 makes very short contact (3.1 Å) with cyclohexyl group, while Sδ-sulfur atom is placed at 3.7 Å. It is important to note that both these distances are shorter than total van der Waals radii of the atoms involved (M309Cε−H···H− Ccyclohexyl 5.8 Å and M309Sδ···H−Ccyclohexyl 4.7 Å, respectively). In spite of these short nonclassical interactions, this molecule selectively inhibits the F309M mutant and the SpMetAP1a. To understand why compound 15 does not inhibit the HsMetAP1b, a structural alignment of the F309M-15 and HsMetAP1b-Met was compared and analyzed. If compound 15 were to bind to the HsMetAP1b in its observed conformation, the aromatic ring of F309 and the cyclohexyl group of the inhibitor would only be 2.1 Å apart. When compared to the F309M-Met structure, the Cεatom in the F309M-15 structure rotates away by about 55° from the center of S1 pocket, thereby making way for the inhibitor. On the other hand, since the F309 is a rigid planar ring with limited conformational flexibility in the wild type HsMetAP1b, binding of compound 15 is difficult. Similarly, in the MtMetAP1c the corresponding position is occupied by a phenylalanine, and therefore, no inhibitory activity was seen. Isoleucine in 2353
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Figure 5. (a) Crystal structure of HsMetAP1b in complex with 2 (green), aromatic equivalent of compound 15 (PDB code 4U6E). Since the side chain is planar, it slides into the S1 pocket of the wild type enzyme with less steric clashes compared to 15. (b) Crystal structure of F309M in complex with 2 (magenta) (PDB code 4U73). Methionine sulfur atom is well acknowledged to interact with the aromatic ring and the 4.1 Å could be stabilizing, therefore better inhibition with SpMetAP1a and F309M mutant compared to HsMetAP1b.
Figure 6. (a) Crystal structure of HsMetAP1b in complex with compound 19 (green) (PDB code 4U69). The longer chain binds in the S1 pocket, while the one carbon stretch points out toward the entrance of the active site. (b) Crystal structure of F309M in complex with compound 19 (brown) (PDB code 4U6Z). (c) Structural comparison of HsMetAP1b and F309M in complex with 19. Because of the flexibility, the side chain of the M309 moves away from the S1 pocket. This results in the extra space, and hence, both branches of the inhibitor bind in the S1 pocket. (d) Crystal structure of HsMetAP1b in complex with compound 20 (green) (PDB code 4U1B). Here, one branch is well placed in the S1 pocket while the other branch is solvent exposed. (e) Crystal structure of F309M in complex with compound 20 (brown) (PDB code 4U6W). (f) Structural comparison of the mode of binding of HsMetAP1b and F309M in complex with 20. The M309 side chain is disordered, while both branches of the inhibitor bind in the S1 pocket of the F309M structure.
between the Sδ of M309 and the inhibitor aromatic ring in F309M-2. The lower affinity of the 2 to HsMetAP1b and MtMetAP1c could be due to the tight fit of the two aromatic rings. The methionine sulfur atom is well placed to interact with the aromatic ring and therefore induces improved inhibition of SpMetAP1a and F309M.23 β-Branched Compounds Occupy Different Positions Based on the Composition of the S1 Pocket. Compound 19
F309M) and isoleucine (Ef MetAP1a) compared to phenylalanine (Table 2). To understand the molecular basis for the differential preference, we determined the crystal structures of HsMetAP1b-2 and F309M-2 complexes at 1.85 and 1.8 Å, respectively (Figure 5a and Figure 5b). In both structures the orientation of the aromatic ring is similar. The shortest distance between the F309 and the inhibitor aromatic ring is 3.7 Å in HsMetAP1b-2 (through an edge−π interaction), while it is 4.1 Å 2354
DOI: 10.1021/jm501790e J. Med. Chem. 2015, 58, 2350−2357
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the compound.26 Similarly, a single amino acid exchange inverts the susceptibility of two related receptor tyrosine kinases for the ATP site inhibitor STI-571.27 However, many of the previous studies regarding differential susceptibilities as a result of a single amino acid change have been between closely related isoforms from the same organism. In our current study we have shown that a single amino acid is responsible for differential susceptibilities between the streptococcal and human enzymes. A Single Amino Acid Responsible for Selectivity. We have established that the position occupied by F309 in the human enzyme is responsible for the specificity of the MetAP. The presence of the phenylalanine in the human enzyme effectively renders the enzyme resistant to compounds 15 and 16, which inhibit the streptococcal enzyme in the low micromolar range. Mutation of this single amino acid to a bacterial-like residue completely reverses the selectivity of the enzyme toward the inhibitors. Crystal structures of various complexes with wild type and the F309M human enzyme suggest that the flexible nature of the methionine side chain allows inhibitor-induced conformational rearrangement of the active site, a property that is common to proteases. The conformational flexibility of the Cε-methyl group of methionine is approximately 2.5 Å in all directions, while that for phenylalanine is only 0.5 Å (Figure 7). This restricts the capacity of the human enzyme to
displayed better affinity toward SpMetAP1a and F309M compared to either HsMetAP1b or MtMetAP1c (Table 2). Conversely, 20 displayed better affinity toward HsMetAP1b or MtMetAP1c than SpMetAP1a and F309M. However, both compounds did not inhibit the Ef MetAP1a enzyme. To understand the structural basis for this unusual behavior, we have determined crystal structures of HsMetAP1b and F309M in complex with 19 and 20 (Figure 6). While compound 20 is symmetrically β-branched with aliphatic chains of three carbons each, compound 19 is asymmetric with one and three carbons. In the crystal structure of HsMetAP1b with 19 (HsMetAP1b-19, Figure 6a), the longer chain binds in the S1 pocket while the one carbon stretch points out toward the entrance of the active site, which is solvent exposed. Similarly in HsMetAP1b-20 structure, one of the branches is placed in the S1 pocket and the other points out into solvent exposed region (Figure 6c). In contrast, both branches of 19 and 20 bind in the S1 pocket in their respective structures of F309M mutant (Figure 6d, Figure 6e, and Figure 6f). This is possible because of the flexible M309 methionine that can change its conformation when required. In the F309M-20, M309 adopts two different conformations to accommodate the larger branching. To understand the structural reason for the inability of 19 or 20 to inhibit Ef MetAP1a, we have carried out simple mutation in coot for F309I of the HsMetAP1b followed by docking of 19 and 20. The β-branching of the isoleucine residue in the S1 pocket prevents the occupation of another β-branched compound. The higher affinity of the 19 toward F309M and SpMetAP1a suggests that it is better for the entire molecule to be buried in the hydrophobic S1 pocket (Figure 6c). However, this should not happen at the cost of strain on the protein, which is the case in F309M-20 structure and hence the lower potency observed toward F309M and SpMetAP1a enzymes (Figure 6f). Calculation of Ligand Efficiency of Inhibitors of SpMetAP1a. Ligand efficiency (LE) has emerged as an important selection criteria of promising lead compounds in drug discovery. LE is most commonly defined as the ratio of the free energy of binding over the number of non-hydrogen atoms in a molecule (−ΔG/HA or 1.4(−log IC50)/N, where HA and N indicate the number of non-hydrogen atoms).24,25 In the fragment based screening, compounds with LE of 0.3 are considered as good starting point. In the present study, all compounds that showed the inhibition against the SpMetAP1a displayed LE values in the range of 0.44−0.69, placing them as potential starting fragments for further drug discovery (Table 3).
Figure 7. Comparison of various conformations adopted by of F309 (pink) and M309 (green) in different complexes of respective complexes. Note that M309 is more flexible compared to F309. Specifically, the Cε-methyl group adopts conformations in all 360° accommodating inhibitors of various sizes and shapes unlike the wild type enzyme.
adapt to the inhibitor, and in contrast, the available conformational freedom allows methionine to adjust as required. Compound 15 and the methionine in F309M were in close proximity (3.1 and 3.7 Å) but did not limit the inhibition (Table 2 and Figure 4). We suggest that in the absence of any classical hydrogen bonding or hydrophobic interactions, the polarization of Cε-atom of methionine promotes an electrostatic interaction with the inhibitor. In the case of human wild type enzyme, the rigid aromatic phenylalanine ring would be only 2.1 Å from the inhibitor, inducing steric clashes with the compound. In addition to the F to M variation in the S1 pockets of human and Pneumonial MetAPs, there are four other residue positions that vary between the two species (Table 1s in Supporting Information). Two residues (P192 and T231 in human; Q57 and V100 in SpMetAP1a) are close in space to each other and also are in proximity to the amino terminus of the substrate/inhibitor (Figure 1b). In human MetAP1b structure T231 forms hydrogen bond (2.9 Å) with the amino terminus, while the P192 is rigid and contributes only to the hydrophobicity of the overall electrostatics of the S1 pocket. It is possible in the case of SpMetAP1a that V100 may move or adopt a different side chain conformation such that some space is formed to accommodate Q57 side chain that will form hydrogen bond with the amino terminus. Y195 and F198 in human and V60 and Y67 in SpMetAP1a at analogous positions are placed near the side chain
Table 3. Ligand Efficiency of Inhibitors against SpMetAP1a compd
LE (kcal mol−1 HA−1)
2 15 16 18 19 20
0.67 0.59 0.51 0.56 0.69 0.44
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DISCUSSION Single amino acid differences in the active sites of closely related isoforms of proteins can confer opposite susceptibilities to substrate/inhibitors. For example, the SC-58125 compound behaves differently with human COX-1 and COX-2 but mutation of a single amino acid in the active site can reverse the targeting of 2355
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Journal of Medicinal Chemistry
Mutagenesis, Expression, and Purification of Proteins. Expression and purification methods for SpMetAP1a,17 Ef MetAP1a,30 HsMetAP1b,31 and MtMetAP1c6 were reported earlier. Briefly, all proteins were purified using the Ni-NTA column at better than 95% purity judged by SDS−polyacrylamide gel (Figure 1s in Supporting Information). Note that human MetAP1b used in this study was a Δ89truncated enzyme. Site directed mutagenesis method was used to construct the HsMetAP1b (F309M) mutant by using the following nucleotides pairs: 5′-CACAAGCTTATGCATACAGCTCCCAATGTACCCCAC-3′; 5′-GGGAGCTGTATGCATAAGCTTGTGGATTCCATGCCC-3′. This mutant is referred to as F309M. Enzyme Kinetics. The protein concentration was fixed for each of the enzyme reactions after standardizing their reaction rates to be similar. Active enzyme concentrations of 15 μM for SpMetAP1a, 12 μM for Ef MetAP1a, 4 μM for HsMetAP1b, 8 μM for MtMetAP1c, and 15 μM for F309M mutant were used. Activity was monitored using a chromogenic substrate as detailed in Supporting Information. Kinetic parameters (Km and kcat) were determined using standard Michaelis− Menten equations and are detailed in Supporting Information. IC50 values were determined using compounds in a concentration range of 1−500 μM. Ki values were determined by a Dixon plot using Met-pNA as a substrate. Both assays are detailed further in Supporting Information (Figure 3s). Protein Crystallization, X-ray Data Collection, and Refinement. Crystallization of HsMetAP1b was performed as per.31 For inhibitor-bound structures, a final concentration of 2 mM α-aminophosphonate compound and 1 mM freshly prepared CoCl2 were added to the crystals in the cryoprotectant solution (6% PEG 10,000 25% glycerol and 100 mM HEPES, pH 6.0) and soaked for 24 h at 25 °C. Crystals were vitrified in liquid nitrogen stream for data collection. X-ray diffraction data were collected on our home source (Rigaku Micromax 007 rotating anode X-ray generator and R-axis IV++ detector system). Diffraction data were processed using HKL3000.32 Since all the structures were isomorphous with the HsMetAP1b structure (2B3K), refinement was initiated without molecular replacement after transferring the Rfree flag from the original structure. Structure refinement and modeling were carried out using Refmac533 and Coot.34 Data collection and refinement statistics can be found in Supporting Information Table 2.
of the substrate/inhibitor and make long distance hydrophobic contacts. These differences do not seem to be important in inhibitor selectivity but will be verified through our future experiments. Active Sites of MetAPs Are Conserved within a Genus but Different between Them. HSSP alignment based on the human and M. tuberculosis MetAPs suggests that the active sites of related proteins are highly conserved (Table 1s in Supporting Information). However, equivalent alignments using SpMetAP1a and Ef MetAP1a structures show divergent active sites. Of the 2500 MetAP sequences found in the HSSP file associated with SpMetAP1a (PDB code 4KM3), about 20 bacterial families are identified and sequence conservation is weak across the alignment. In contrast, alignment of all 23 Streptococci MetAPs shows that the S1 pocket is highly preserved within the genera (Figure 4s in Supporting Information). Therefore, it appears that conservation of the S1 pocket of MetAP enzymes is limited to the genus and suggests that drug discovery programs against microbial MetAPs should be customized for each family rather than expecting broad specificity from inhibitors. This is supported by our data for MtMetAP1c that has a specificity profile more similar to humans than other bacterial proteases tested. Human infection with S. pneumoniae predominantly causes pneumonia and other pneumococcal diseases; however, it can also cause other invasive diseases such as meningitis and bacteremia. In 2011, there were nearly 11 500 cases of invasive pneumococcal disease in the USA caused by pneumococcal bacteria resistant to one or more antibiotics.35 The prevalence of drug-resistant S. pneumoniae varies globally. Other pathogenic Streptococci include the group A Streptococci that can cause infections such as strep throat, scarlet fever, impetigo, and cellulitis. In our current study we have identified that the MetAP1a enzyme from Streptococci is an attractive drug target for the development of new agents to combat this pathogenic bacteria. We have provided crucial and extensive structure− activity relationship data from which a drug design program can begin.
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S Supporting Information *
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Bioinformatics table, SDS−PAGE gel, enzyme kinetics, and Xray crystallographic table. This material is available free of charge via the Internet at http://pubs.acs.org.
CONCLUSIONS For the first time, we have identified molecules that selectively target a streptococcal MetAP over a human homolog. We show that a single amino acid in the S1 pocket determines the sensitivity of different MetAP enzymes against the small molecules tested. We have also identified that β-branched amino acid analogs are good starting compounds for inhibitors of streptococcal MetAPs. Our results highlight the importance of the composition and architecture of the S1 pocket and suggest that inhibitor design should consider carefully the substrate binding positions that are particular to any given bacteria. This work provides the first step in a pathway to achieve species/genus selectivity of MetAP inhibitors.
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
Corresponding Authors
*S.M.: e-mail,
[email protected]; phone, +613 99029309; fax, +613 9902 9500. *M.D.: e-mail,
[email protected]; phone, +48-74-3204526; fax, +48 71 320 2427. *A.A.: e-mail,
[email protected]; phone, +91 40-27191860; fax, +91 40-27160387. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Thanks are given to Council of Scientific and Industrial Research (CSIR) and University Grants commission (UGC), New Delhi, India, for research fellowships to T.A., R.R., C.K., R.J.G., and R.G. S.M. is an Australian Research Council Future Fellow (Grant FT100100690). The work was also supported by a statutory activity subsidy to M.D. from the Polish Ministry of Science and Higher Education for the Faculty of Chemistry at Wroclaw University of Technology. A.A. acknowledges the financial
MATERIALS AND METHODS
All chemicals including substrates used in this study were purchased from Sigma-Aldrich, USA. Enzymes for molecular biology were purchased from Fermentas (Canada). Bioinformatic Analysis. Sequence and structural alignment analysis was performed as described in the Supporting Information. α-Aminophosphonates. All α-aminophosphonates used in this study were obtained as described earlier.28,29 Purity of all compounds is better than 95% as determined by 31P NMR. 2356
DOI: 10.1021/jm501790e J. Med. Chem. 2015, 58, 2350−2357
Article
Journal of Medicinal Chemistry
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support for research from Department of Science and Technology (Grant SR/SO/BB-55/2008), Department of Biotechnology (Grant BT-BRB-TF-2-2011), and Council of Scientific and Industrial Research (SMiLE), New Delhi, India.
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ABBREVIATIONS USED MetAP, methionine aminopeptidase REFERENCES
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