A poke in the eye: inhibiting HIV-1 protease through its flap-recognition pocket

Abstract

A novel mechanism of inhibiting HIV-1 protease (HIVp) is presented. Using computational solvent mapping to identify complementary interactions and the Multiple Protein Structure method to incorporate protein flexibility, we generated a receptor-based pharmacophore model of the flexible flap region of the semiopen, apo state of HIVp. Complementary interactions were consistently observed at the base of the flap, only within a cleft with a specific structural role. In the closed, bound state of HIVp, each flap tip docks against the opposite monomer, occupying this cleft. This flap-recognition site is filled by the protein and cannot be identified using traditional approaches based on bound, closed structures. Virtual screening and dynamics simulations show how small molecules can be identified to complement this cleft. Subsequent experimental testing confirms inhibitory activity of this new class of inhibitor. This may be the first new inhibitor class for HIVp since dimerization inhibitors were introduced 17 years ago.

Figure 1

a) When a monomer closes, it places its flap tips within the flap-recognition site of the other monomer. The right monomer (gray with surface) is the apo, semi-open state that shows the pre-existence of the site. The left monomer (yellow) is in the bound, closed state. I50 and G51 are shown in stick representation in direct contact with the “eye”. b) The individual residues within the new eye site are each colored individually and labeled to show their placement within the cleft. G78 and V56 are not visible in this view.

Figure 2

a) MPS pharmacophore model mapping the eye region of the semi-open conformation. Elements are color-coded according to chemical functionality: red for hydrogen-bond donor, blue for hydrogen-bond acceptor, cyan for hydrophobic, and green for aromatic. b) Close-up view of model with a 90° rotation as in Fig 1b. Compound 1 (2,2,4-trimethyl-1,2-dihydroquinolin-6-yl benzoate), identified through the virtual screen, is shown overlaid with MPS pharmacophore model to demonstrate the agreement between its chemical scaffold and the pharmacophore elements.

Figure 3

a) The stability of compound 1 in the eye site is demonstrated by the RMSD to its starting position. Each of the five independent LD is given. Several events are seen where the ligand dissociates and rebinds again in the same pocket (temporary spikes up to 10 Å). In the second simulation (purple line), 1 starts in the flap-recognition pocket of one monomer, disassociates into the central active site, then binds in the eye site of the other monomer. b) Representative structures from 2-3 ns of Run 2 show the migration. An early snapshot is shown with a blue backbone and yellow inhibitor; a late structure is shown with a red backbone and gray inhibitor. The transparent inhibitors in the central pocket are actual positions sampled during the migration. c) A close-up, top view of the flap region shows how the flap tips pack against the inhibitor. The handedness stays the same, but the packing shifts from right (blue) to left (red) with the migration of compound 1.

Figure 4

a) Overlay of snapshots taken every 0.5 ns across the third LD simulation. A large degree of sampling is seen in the flap region. b) However, some order is apparent in the correlated dynamics (strong positive correlations are in red and yellow, pronounced anti-correlated motion is dark blue). Over the entire simulation, the strongest positive correlations between the monomers (upper left and lower right regions) are the flap tips (noted white circles) and the C-terminal β-sheets that comprise much of the dimer interface (marked with white triangles). c) The strength of the correlation between the flaps varies over the course of the simulation, periodically showing very strong correlations. The periods of strong coupling lasted a maximum of 1.75 ns in Run 1, 1.25 ns in Run 2, and 2.5 ns in Runs 3-5.

Figure 5

The average structure from the 10-ns MD simulation is shown in yellow; compound 1 is colored by atom type. The semi-open, apo form is shown in blue, and the closed, bound form is shown in red (co-crystallized inhibitor in the central pocket is not shown for clarity). The MD samples a closed conformation that differs from traditional bound crystal structures. The flaps are lowered, as is appropriate for a closed structure, but the elbows are displaced slightly outward as in the apo state. The handedness of the flaps matches the apo state, not the bound state.

Figure 6

Compound 2 is a para-methoxy derivative: 2,2,4-trimethyl-1,2-dihydroquinolin-6-yl 4-methoxybenzoate. Its IC₅₀ was measured to be 18 ± 3 μM. The activity of HIVp was monitored using a FRET-based assay; upon cleavage of the quenched peptide substrate, fluorescence is recovered. Inhibition is measured as a result of the time-dependent decrease of fluorescence intensity that is linearly related to substrate cleavage. Pepstatin A is shown as a control (IC₅₀ = 3.8 ± 0.5 μM under the assay conditions used).