Chapter 11
Molecular Design of Fluorine-Containing Peptide Mimetics 1
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Piotr Cieplak , Peter A. Kollman, and Jan P. Radomski 1
Quantum Chemistry Laboratory, Department of Chemistry, University of Warsaw, Pasteura 1, Warsaw 02-093, Poland Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143 Interdisciplinary Center for Modeling, University of Warsaw, Banacha 2, Warsaw 02-097, Poland 2
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The free energy perturbation (FEP) and free energy derivatives (FED) methodology with molecular dynamics simulations has been applied to study possible improvement of the inhibition properties of the JG-365 inhibitor toward HIV aspartic protease. Our study concerns the assessment of the effect of replacement of some peptide bonds in JG-365 by trans-ethylenic or fluoroethylenic units. According to our free energy perturbation simulations such replacement could be beneficial for two of those peptide bonds but not for the others. The results of application of the free energy derivative technique confirms the FEP results and also suggest other possible modification of the JG-365 inhibitor which could lead to increased potency. Our results are only predictive in nature, since the proposed pseudopeptidic inhibitors have not yet been synthesized. Finding therapeutics against diseases like AIDS, malaria, cancer and many others, is an important but very difficult task. Theoretical methods and the use of computational techniques can be very helpful in the process of developing new drugs. The main effort in this area is focused on designing new enzyme inhibitors. Theoretical methods are used for predicting binding trends in enzyme - inhibitor complexes. Although not all the effects accompanying such a binding could be taken into account in computer simulations, some insights could be gained if the free energy differences could be calculated. Free energy is the main quantity used to describe the general trends in any chemical processes and since it is a state function, only the knowledge of the initial and the final states is needed. In this article we present the results of applying free energy perturbation (FEP) (1,2) and free energy derivative (FED) (3,4) methodologies to designing peptide mimetic inhibitors of the HIV protease enzyme. The first method provides appropriate free energy differences of binding and the latter one point out at the places in the inhibitor where the chemical modifications could be performed in order to increase binding properties. In recent years significant work has been directed toward the inhibition of theHIVreverse transcriptase and the HIV aspartic protease as a most accessible target for chemotherapeutic intervention infightingthe AIDS disease (5,6). In our study we have been focused on inhibition of the HIV aspartic protease. TheHIVprotease is responsible for cleaving a polypeptide precursor into the functional proteins, which are essential for virial replication (6). It is estimated that more than 160 structures of different inhibitors withHIVprotease 0097-6156/96/0639-0143$15.00/0 © 1996 American Chemical Society
In Biomedical Frontiers of Fluorine Chemistry; Ojima, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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complexes have been determined by X-ray crystallography. These structures could be used as a starting point in any further molecular modelling. A summary of a variety of molecular dynamics and free energy calculations for some of those structures are presented in Ref. (7). Our calculations, partially described in Ref. (8), have been performed for one of such structure, solved by Wlodawer et al (9-11), containing the potent JG-365 inhibitor (0.24nM inhibition constant). This is hydroxyethylamine (HEA) analog of the ACE-SerLeu-Asn-Phe-Pro-Be-Val-OMe peptide (Fig.l). The hydroxyethylamine (HEA) analog of the Phe-Pro peptide bond resembles the tetrahedral intermediate for the peptide bond hydrolysis reaction. It is well established that among the most effective protease inhibitors are modified peptides that act as transition state analogues (12). The JG-365 is an example of such inhibitor and has been used as our lead compound in molecular dynamics simulations. The purpose of those calculations was to suggest chemical changes within the inhibitor which could possibly improve its binding. We propose that this goal could be achieved by replacing some of the peptide bonds with either ethylene or fluoroethylene units. JG-365 has seven peptide bonds and each of them could be replaced by its nonpeptidic isostere (see Fig.l). In the following discusion we will use the standard notation (13) for the inhibitor amino acids, i.e. P4 for SER, P3 - LEU, P2 - ASN, PI PHE, ΡΓ - PRO, P2' - ILE, P3* - VAL.
P4 P3 P2
P
1
P
r
p 3
,
JG-365
Figure 1 .Amino acid sequence in the inhibitor Why were peptidomimetics chosen as a target? The usefulness of peptide mimetics, (e.g. ethylenic or fluoroethylenic derivatives) have already been demonstrated (14-16). Why could peptidomimetics be a good choice for possible modification of the original inhibitor? One of the important arguments is that replacing the peptide bond by its isosteric ethylenic or fluoroethylenic unit does not change the already appropriate conformation of the inhibitor needed in the active site, and thus it fulfills conformational constraints. The fluoroolefin unit, as an excellent steric and electronic mimic for the peptide bond, was recently used in organic synthesis as a tool for controlling peptide conformations (17,18). The synthesized peptide mimetics exhibited inhibitory effects on the cyclophilin enzyme. In another case, the ethylene unit was incorporated into the met-enkephalines and the resulting derivative exhibited biological activity (19-22]). It was found that such a modification prevented premature degradation of pseudopeptide bonds by amino-peptidase
In Biomedical Frontiers of Fluorine Chemistry; Ojima, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
11. CIEPLAK ET AL.
Design of Fluorine-Containing Peptide Mimetics
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and it was assumed that the increased lipophilicity of the peptide should facilitate its passage through the blood-brain barrier and membranes. Incorporation of the olefinic bond into substance Ρ also increased its biological potency compared to the unmodified compund (21). The dipole moment of the trans ethylene (-0.1D) and trans fluoro-ethylene units (~1.5D) have different values, and orientation than the peptide bond itself (~4.5D), (see Fig.2). Thus, one might observe a totally different pattern of intermolecular interactions with the enzyme. Replacing the strongly interacting peptide bond for another chemical unit which does not form strong hydrogen bonds could be advantageous in some cases. This is especially important when one must compare the two states of the inhibitor, one in the enzymatic site and the other in aqueous solution.
4.5 D
0.1 Η
\ -0.3 C
/
/ C 0.1 \ Η 0.03
-0.1
0.3 C Z±I C -0.2
/
\ Η 0.1
Figure 2 Charge distribution and dipole moments for peptide bond and for ethylenic and fluoroethylenic units. Olefin and fluoro containing molecules were also used in studies devoted to inhibiting HIV protease. For example difluoroketonesHIV-1protease inhibitors exhibit nanomolar activity (23). Keenan et al (24) attempted to synthesize the olefm-hydroxyethylene peptide mimetics. They showed that replacing the peptide bond by an ethylene unit in the P2-P1 sites decreases the association of the inhibitor from K—37 nM to 3.6 μΜ. This was expected, since that peptide bond is engaged in holding the inhibitor in the active site. That also means that replacing the peptide bond between P2-P1 into a weaker interacting unit should be unfavorable. This example shows using a more weakly interacting peptide mimetic unit to replace the peptide bond could serve as a probe for testing the importance of the hydrogen bond pattern in the active site. Peptidomimetics have attained a prominent position in rational drug design (16). Olson et al (16) proposed some general criteria which should be fullfiled in designing peptide mimetics. These can be summarized as follows: a) replace as much of the peptide backbone In Biomedical Frontiers of Fluorine Chemistry; Ojima, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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as possible by a nonpeptide framework, b) maintain the peptide side chain pharmacophore groups as in the peptide, since they are most likely to be recognized by a receptor, c) do not change or restrict conformational flexibility too much in the first iteration step of generating mimetics, d) select appropriate targets based on availability of a pharmacophore hypothesis. In our studies we propose to modify the very potent JG-365 inhibitor by replacing peptide bonds by ethylene or fluoro-ethylene units. Such modifications satisfy all requirements for peptide mimetics mentioned above. We expect that such modified inhibitors could have many desired properties, for example, the increased resistance to enzymic degradation by amino-peptidases and thus longer duration of their inhibition action. The Free Energy Perturbation Method To study the JG-365 inhibitor and theHIV-asparticprotease complex as well as the inhibitor itself in water solution we used the molecular dynamics method (25), the AMBER program (26) and the empirical potential function (27£8) for energy calculation: (1)
K*i = Σ W - r f + Χ Κ (Θ - e f + X \ bonds angles dihedrals eq
y ^ _ h i
