CARACTERISTICAS SOCIOECONÓMICA DE LOS POBLADORES

The basis for much of this work was the structure of the reduced peptide inhibitor MVT-101 complexed with the protease.^^^ MVT-101 was chosen as it is highly congruent to the substrate and consequently to our inhibitor. The hydroxyethylene inhibitors are also congruent to the substrate but the hydroxy group is hydrogen bonded to the active site aspartates. This may induce a bias into the starting active site structure that may affect the outcome of subsequent calculations. The sequence of inhibitor 100 was built onto the peptide backbone of MVT-101 (see Figure 2.5.2).

HgC y 3 H O CH

N

Figure 2.5.2: MVT-101

This gave an approximation of the bound conformation of the inhibitor in the active site. A substructure minimisation in the active site of the HIV-1 protease allowed the altered side-chains to relax into the enzyme subsites and adopt reasonable bound conformations. The bound conformation of compound 1001 is shown in Figure 2.5.3.

Figure 2.5.3: The modelled bound conformation of compound 1001 118

The interactions between the P3 residue (the Boo group) and the residues of the S3

pocket were examined in this model structure to identify interactions important for binding. A conformational search was then carried out on a substructure consisting of the Boo group and those residues having one or more atoms within 6 Â of the atoms of the Boo group. This was designed to identify the low energy conformations of the Boo group in the S3 pocket and the interactions made with the selected side- chains.

A further set of calculations were carried out in which the methylene of the reduced peptide moiety was replaced by PO(OCH3), to give the full sequence of our target compound. This was carried out using the INSIGHT program to take advantage of the generalised force-field available in INSIGHT (GFF91^®^>^^^) that allows calculations to be carried out on systems for which high-quality bond parameters are not available. The differences in the side-chain conformations between the structures with and without phosphorus were found, as expected, to be very small (the two structures overlaid with an r.m.s. displacement of around 0.1 Â).

The number of interactions found in either case between the Boc residue and the enzyme were found to be limited. The main contacts made by the Boc group at P3

are with residues Val 82B, Gly 48A and Arg 8B of the S3 pocket (A represents amino acids from one of the monomers, B from the other). All these contacts are non-polar and are not expected to contribute greatly to the binding energy. The major polar interaction in the S3 pocket, with Asp 29A, was not always observed with the Boc group, which probably reflects deficiencies in the methodology rather than an actual lack of interaction. Thus, it is not expected that this residue will add greatly to the potency of our compounds. The interactions identified compare reasonably well with the interactions identified in the published X-ray structures of inhibitors co­ crystallised with the protease and are illustrated in Figure 2.5.4

Figure 2.5.4: interactions in the F3 pocket

The observed improvement in the binding of the Phe-Phe and Cyc-Phe scissile bond analogue containing inhibitors over those containing Phe-Pro was investigated. The possible interactions of the different P^' side chains with the S^’ pocket were examined using two different crystal structures. The model structure of Weber^^® which has a Tyr-Pro containing substrate was used initially. The coordinates for the structure of the protease co-crystallised with a proline-containing hydroxyethylamine inhibitor, JG-365^®® (Figure 2.5.5) were then made available.

H H N OH _N H ‘CONN. Figure 2.5.5: JG'365

The Pro residue in the model structure forms van der Waals contacts with only 3 residues, Leu 23A, Val 82A and lie 84A and packs against Asp 25A. In the JG-365 structure the Pro residue is in contact with residues Pro 81 A, Val 82A, lie 84A and Gly 49B. In contrast the P-j Phe side-chain in the symmetric A-74707^^^ forms contacts with Leu 23A, Val 80A, Pro 81 A, lie 84A, Asp 28B and lie 50B. Thus, proline at P-j’ has less Interactions with the S^’ subsite than phenyalanine at P-j’, which is consistent with the usually less potent inhibition of proline-containing analogues.

This large alteration in the conformation of the active site on binding inhibitors of differing steric requirements has been observed before.^^^ should be noted that in spite of the deficiencies of the Weber model structure it is probably a better model for the interactions between our inhibitors and the enzyme than JG-365. This is due to the “bending” effect of hydroxyethylamine inhibitors discussed above, arising from water-bridged hydrogen bonding to the flap.

The procedure carried out using MVT-101 to build a model of our inhibitors was repeated for JG-365 and for the substrate Ser-Gln-Asn-Tyr-Pro-lle-Val from the model structure of Weber. The structures were subjected to substructure minimisation and the resulting interactions compared. Essentially the same results were obtained for the interactions with the P3 pocket. The interactions between the Pro residue and

the S^’ pocket were found to be different for the JG-365 based structure than for the model substrate structure, probably due to the bridging water mediated bending of the inhibitor backbone. Which one represents a better model for the interactions actually present in the enzyme-inhibitor complex is not known and awaits an X-ray crystal structure.

The configuration at phosphorus was investigated by building both enantiomers at phosphorus into the active site (on the inhibitor with the (R) configuration at carbon) and minimising the structures as above. Subsequently, molecular dynamics simulations were carried out on a substructure consisting of residues 22-27 of each monomer and the central residues (Phe-\j/[POOGH3]-Pro) of the inhibitor in both configurations at phosphorus. It was found that the (8) configuration (see Figure 2.5.6) disrupted the coplanarity of the active site aspartates, due to the intruding methyl group. This disruption of the active site hydrogen-bonding network is an unfavourable process and is consistent with the less potent inhibition observed with the inhibitors having an (8) configured phosphorus atom.

O: O: OH O HoC- O: (S ) (R) OH O' O:

Figure 2.5.6: The epimers differing at phosphorus

The (R) configured phosphorus has the phosphoryl oxygen pointing toward the aspartates and allows them to retain their hydrogen bonding network and remain coplanar. Given that this analysis is correct it becomes difficult to account for the equivalence in binding potency of inhibitors 2002 and 2004 (see above). The

explanation may lie in the extensive cooperativity of the residues of the active site, allowing 2 different configurations at phosphorus to form interactions of equivalent

strength with the enzyme.

All the above work is based on the assumption that our inhibitors bind In the same way as substrates and other substrate-based inhibitors such as M V T - 1 0 1 . This could

be demonstrated in two ways. Firstly, if it could be shown by kinetic analysis that these are competitive inhibitors then they must bind in the active site, in competition with the substrates. Alternatively, if the P-j ’ residue were of the D-configuration then

an active-site directed inhibitor would bind more weakly, by around an order of magnitude, than an inhibitor of the L-configuration at p^’,390 It was also assumed that our inhibitors only have one binding mode in the active site, or, if there were two, that one represents only a small fraction of the total. This can only be demonstrated unambiguously with a highly refined X-ray crystal structure determination.