ECE-2 is a recently identified member of M13 family of zinc metallopeptidases of which NEP is the best characterized. Until recently most of the structural information about these enzymes was based on the studies carried out on the bacterial homolog-thermolysin (TLN). Although there is very limited homology between the primary sequences of TLN and NEP, these two enzymes have similar catalytic properties and are inhibited by the same type of molecules such as phosphoramidon and thiorphan. In addition they both contain the highly conserved consensus sequences, HExxH and ExxxD, which contain residues critical for Zn2+
coordination and catalysis. Based on early crystallographic studies Matthews proposed a mechanism for the TLN-catalyzed cleavage of peptides 31
. According to this model the two His residues (H142 and H146) together with the E166 and a water molecule are involved in the tetrahedral coordination of the Zn2+
atom. The incoming substrate displaces the water molecule towards the catalytic E143 residue. The negative charge of E143 polarizes the zinc-coordinated water molecule and promotes its nucleophilic attack on the carbonyl carbon of the scissile peptide bond. Structural studies also indicated the importance of H231 and Y157 residues in the stabilization of the transition state.
Later studies in NEP using site-directed mutagenesis have identified corresponding residues involved in zinc coordination, catalysis and substrate binding and confirmed that the proposed catalytic mechanism was also valid for the mammalian enzymes 19-26
. Several studies also modeled the structure of NEP, as well as other family members using the crystal structure of TLN as a template. However, the availability of the crystal structure of NEP complexed with phosphoramidon provides a more suitable and reliable template for the modeling of metallopeptidases of this family and has therefore been used to model the structures of several members of M13 family, including endothelin-converting enzyme-1 (ECE-1), Kell blood group protein and neprilysin-2 (NEP-2) 32-34
. ECE-1 and ECE-2 share common substrates and approximately 60% sequence homology. The consensus sequences that are conserved among zinc metallopeptidases are identical between these two proteins, indicating significant structural similarities. Therefore the molecular model of ECE-1 could provide considerable information about the architecture of the ECE-2 active site32
. However, since the molecular model of ECE-1 was not deposited in the PDB, a detailed comparison of our model with that of ECE-1 was not possible.
There are a number of important physiological differences between ECE-1 and ECE-2, such as the pH dependency and sensitivity to inhibition by phosphoramidon 12
. This generic metallopeptidase inhibitor is ~100 less potent towards ECE-1 (IC50
=3.5μM) than ECE-2 and NEP (IC50
=2-4nM) suggesting that despite a number of similarities, some aspects of catalysis and inhibitor binding to the active site of each of these peptidases could be quite distinct. In addition, the detailed information about the binding pocket of each of these peptidases will be useful to design specific and selective inhibitors of ECE-1 and ECE-2. Because of the involvement of ET in various cardiovascular, renal, pulmonary and central nervous system diseases 35-37
much effort has been put towards developing inhibitors of this pathway as potential therapeutic agents. In this light, inhibitors of ECE, which catalyze a rate-limiting step in ET production, have received a great deal of attention. In addition, double inhibitors of ECE and NEP that would interfere with the production of ET, a vasoconstrictor, as well as with the degradation of atrial natriuretic peptide (ANP), a potent vasorelaxant, have also been designed as potential therapeutics for the treatment of hypertension 38
. However, most of these studies did not differentiate between ECE-1 and ECE-2 and focused on the inhibition of ECE-1 activity without addressing the effect of these inhibitors on ECE-2. To date only a trisubstituted quinazoline, PD069185 (6
) () and its analogs, have been tested and reported to be selective for ECE-1 over ECE-2 39
In order to help us identify specific inhibitors of ECE-2, we generated a 3D molecular model of ECE-2 based on the x-ray structure of NEP. A comparison of the active sites of these two enzymes revealed that the residues involved in catalysis as well as the residues in the vicinity of the substrate/inhibitor binding pocket that interact with the substrate are highly conserved. However, we identified two amino acid differences in the active sites of NEP and ECE-2, which could potentially account for pharmacological differences between NEP and ECE-2.
One of the residues, F544 of NEP within the conserved consensus sequence 542
is substituted by Y563 in ECE-2 and Y552 in ECE-1. Based on earlier mutagenesis studies as well as crystal structure, both N542 and A543 of NEP were shown to form hydrogen bonds with phosphoramidon 11, 25
. Sansom and colleagues 40
also demonstrated the importance of Y552 within this sequence, for the catalytic activity of ECE-1. Although in the ECE-2 model Y563 was not found to directly interact with the phosphoramidon, it was found to hydrogen bond to N561 and thus affect inhibitor binding. Y563 is also in a position to interact with the catalytic E603. While the distance between these two residues suggests only a weak interaction, even a slight movement of the catalytic Glu could have a drastic effect on the catalytic activity of the enzyme. Indeed, we found that the substitution of the Tyr 563 residue with Phe in ECE-2 resulted in approximately six-fold lower catalytic activity for the enzyme and more than sevenfold decrease in inhibitor potency. The fact that this mutation did not influence substrate binding but affected inhibition by the transition state analog-phosphoramidon, leads us to hypothesize that Y563 may be involved in the catalytic activity, and in the stabilization of the transition state of the enzyme, by participating in the positioning and orientation of E603. Considering that this substitution is very conservative, such small, albeit significant change in enzyme properties demonstrate the importance of this residue for the catalytic activity of ECE-2.
Another difference we found in the active site of these two enzymes is that the Arg 110 residue in NEP is substituted by Trp in ECE-2. R110 is one of the residues which along with R102 and D107 form the S2' subsite of NEP. In ECE-2 this subsite is large, similar to NEP, and can accommodate bulky side-chains. The presence of Trp in place of R110 could result in a wider pocket with reduced specificity. We observed that mutation of Trp 148 back to Arg slightly but significantly increased affinity for the substrate however it did not affect the inhibitor potency. The change in affinity may be explained by the fact that the McaBk2 peptide has a Lys residue in the P2' position and its long side chain is held tighter in the S2' pocket with the reduced volume. The more compact Trp side chain of the inhibitor phosphoramidon in the S2' pocket is not affected by this change. Testing additional substrates with different residues in P2' position is needed to address this notion.
After confirming the validity of the 3D molecular model of ECE-2 by site-directed mutagenesis we used the model to screen a library of approximately 13000 small drug-like molecules. Initially the structures of the compounds were docked into the active site of the ECE-2 model. Compounds that displayed highest binding were tested experimentally using in vitro
an enzyme assay. In the experimental screening we identified two compounds, 1
, that inhibited ECE-2 activity with micromolar potency and were ~ 10 times more selective for ECE-2 as compared to NEP. We found that these compounds also inhibit ECE-1 activity with an affinity similar to that of ECE-2 (Gagnidze and Devi, unpublished). This is not surprising since the homology between ECE-2 and ECE-1 is higher than that between ECE-2 and NEP and that Y563 of ECE-2 is also conserved in ECE-1. Furthermore, Y563 in ECE-1 has been shown to play a role in the catalytic activity of the enzyme40
An examination of the docking of these compounds to the active site of ECE-2 revealed that they bind in close proximity to the Zn2+ atom and are in a position to form hydrogen bonds with the His residues that coordinate Zn2+ as well as the catalytic Glu603. Thus they are in a position to affect the hydrolysis of the substrate (). In order to identify compounds with higher inhibitory potency towards ECE-2 we searched for analogs of 1 and found three compounds that were commercially available. Two of these three analogs, 4 and 5, were ~5-6 times less effective compared to 1. These two compounds have additional CH3 groups in the P1' position and this may interfere with the stringent specificity of S1' site that is characteristic of these metallopeptidases. An optimal way to increase the potency of these inhibitors would be through chemical optimization using the structures of 1 and 2 as a lead. Additional analyses are also needed to ensure the selectivity of these compounds for ECE-2 and the chemical optimization may be a useful strategy to achieve this goal as well. Information from these studies will be useful in the identification of specific inhibitors of ECE-2 that will serve as hitherto unavailable tools for studies examining the physiological role of this peptidase in vivo.