Inhibitor design by wrapping packing
defects in HIV-1 proteins
Ariel Fernández 1,2, Kristina Rogale 2,3, Ridgway Scott 2,4 and Harold A. Scheraga 5
(1)
(2)
Institute for Biophysical Dynamics, The University of
(3) Program in Applied and
Computational Mathematics,
(4)
Department of Mathematics and Department of Computer Science,
The
(5)
Baker Laboratory of Chemistry and Chemical Biology,
Two viral proteins, HIV-1 protease and HIV-1 integrase, have been targeted for inhibitor design in efforts to prevent full assembly and maturation of HIV-1 virions. The enzymatic mechanism of these proteins involves groups which serve as general acids or bases. Furthermore, catalytic activity requires that water be removed from the microenvironment surrounding the chemical reaction, or be severely constrained to serve as an activated nucleophile. Here we identify newly found structural features that favor water removal from polar catalytic regions. Thus, packing defects in the form of under-wrapped backbone hydrogen bonds, termed dehydrons, are strategically placed in the structure to induce dehydration of the enzymatic pathway. Their effectiveness arises as dehydrons become strengthened and stabilized upon water removal. Thus, packing defects act synergistically with polar active groups to enhance the enzymatic electrostatics. On the other hand, since dehydrons are inherently sticky, they constitute highly specific targets for inhibitor drug design, especially on the polar regions directly interacting with the substrate. We noticed that effective inhibitors attach to polar surfaces by wrapping the dehydrons, with a net effect of blocking the catalytic or mechanically active sites. The dehydrons are thus needed for functional reasons, which in turn make them suitable targets. The significant differences in success when targeting HIV-1 protease, FIV protease and HIV-1 integrase are rationalized here in terms of the dehydron distribution around the active sites, in turn revealing possible improvements in the targeting strategy. Thus, new principles of design optimization are proposed to create an inhibitor that can only be neutralized at the expense of the loss of catalytic function.
The removal of water molecules surrounding backbone hydrogen bonds is required to guarantee the structural integrity of soluble proteins (1-6) and also places constraints on the allowed conformational changes along folding pathways (7, 8). Backbone hydrogen bonds typically prevail provided nonpolar groups are clustered around them. This “wrapping” (6) provides an anhydrous microenvironment that makes it thermodynamically unfavorable to expose the backbone amide and carbonyl groups. Thus, soluble protein structure prevails by keeping its hydrogen bonds “dry in water”. On the other hand, the backbone hydrogen bonds which are intramolecularly under-dehydrated, the dehydrons (2), constitute structural markers for protein interactivity. This was demonstrated experimentally (9) as well as statistically, by examination of protein-protein interfaces and supramolecular protein assemblies (1,2). Dehydrons are inherently sticky (9), a property that finds an energetic and a thermodynamic basis: the partial amide and carbonyl charges are de-screened as surrounding water is removed, and in turn, water removal destabilizes the nonbonded state (or equivalently stabilizes the bonded state) by preventing the hydration of the amide and carbonyl groups.
Many enzymatic reactions involving nucleophilic attack on scissile bonds become more efficient when some surrounding water can be removed to enhance the electrostatic interactions. Occasionally, especially in hydrolysis, a few water molecules must be selectively confined to participate in the reaction. Since dehydrons favor removal of surrounding water without necessarily involving hydrophobic residues, it is expected that they could play a significant role in shaping the microenvironments at the active site. We shall explore this aspect in this paper, especially in connection with the possibility of designing inhibitors of catalytic function.
Many enzymes involve polar side-chain groups that can serve as general Lowry-Bronsted acids and bases as they interact with the substrate in a concerted or multi-step fashion. The aspartyl proteinase HIV-1 protease (10-12) and the HIV-1 integrase (13-15) are examples of such enzymes. These proteins have been targeted in inhibitor drug design geared at preventing the full assembly and maturation of HIV-1 virions (16,17) in AIDS therapy. Partial water exclusion from the microenvironment around the chemical reaction, be it hydrolysis, trans-phosphoesterification, proton donor-acceptor chemistry, etc., is important to ensure the efficiency of the enzymatic mechanism. In this regard, surface nonpolar groups flanking the active groups might be useful, but when the groups interacting with the substrate are themselves polar, an alternative structural feature, the dehydron, could become a primary contributor to shape the functional microenvironment.
This implies that dehydrons, which favor water removal, can act concurrently and synergistically with polar catalytic groups at the active sites by inducing the de-screening of the charges. At the same time, since the dehydron is sticky, it is expected that it should represent a highly specific and efficient target for inhibitor drug design, as the evidence presented here reveals. This novel structural marker may aid drug design and improve its efficiency, in particular optimizing the resilience of the inhibitor to resistant mutation.
Methods
To identify the dehydrons in a domain
fold, multi-domain chain or protein complex in one-chain or multiple-chain PDB
entries, we used a program called YAPView (http://sosnick.uchicago.edu/aifoldlab/YAPView/YAPView.html)
which detects dehydrons in PDB structure according to the following premises:
The extent of intramolecular hydrogen-bond desolvation in monomeric structure
is quantified by determining the number of nonpolar carbonaceous groups within
a desolvation domain. This domain is defined as two intersecting balls of fixed
radius centered at the a-carbons of the
hydrogen-bonded residues (2). If we examine complexes or multimers, the count
includes nonpolar groups from the monomer as well as those from its binding
partner(s). The statistics of hydrogen-bond wrapping vary according to the
desolvation radius adopted, but the tails of the distribution invariably single
out the same dehydrons in a given structure over a 6.0-7.4Ĺ range in the
adopted desolvation radius. In this work the value 6.2Ĺ was used for
consistency.
In most (~92% of PDB entries) stable protein folds, at least two thirds of the backbone hydrogen bonds are wrapped on average by r=27.1±7.5 nonpolar groups (or 14.0±3.7 counting only side-chain groups and excluding those from the hydrogen-bonded residue pair). Dehydrons are then identified as hydrogen bonds in the tails of the distribution, i.e. with 12 or less nonpolar groups in their desolvation domains (their r-value is no greater than the mean minus two Gaussian dispersions).
The dehydron is then identified as an insufficiently wrapped hydrogen bond. A more rigorous characterization takes into account the sensnitivity of the hydrogen-bond coulombic energy to removal of surrounding water or decrease in solvent polarizability (2). Thus, our adopted criterion introduces a sufficient condition for the existence of a dehydron. While being a satisfactory approximation, this criterion may miss the specific case where the number of nonpolar wrappers exceeds 12 but they are very unevenly distributed in space around the bond.
Results
Packing
defects as drug targets in HIV-1 protease
The HIV-1 protease, an aspartyl proteinase, is an obligatory homodimer in its active functional form (10-12), with two equivalent D25 residues acting in opposite ways as general acid and base to catalyze the hydrolysis of the substrate peptide bond. A thermodynamic factor favoring water exclusion becomes essential to ensure that the substrate is properly anchored and manipulated by the protease and the electrostatics of the nucleophilic attack are properly enhanced along the enzymatic pathway. Thus, it is not surprising to find a cluster of hydrophobic residues, L23, L24 and I84, surrounding the catalytic site D25. On the other hand, the substrate polypeptide must be anchored by the active residues D29 and D30, and dragged along with the aid of a gating mechanism that makes use of the flap 46-55 region (Fig. 1a). However, no hydrophobic patch is present either at the rim of the catalytic pocket or at the flap. Furthermore, since the active residues are themselves polar, removal of surrounding water cannot be prompted by their sole presence. The distribution of dehydrons in the protein (Fig. 1a) reveals how packing defects are strategically placed in the structure to favor the exclusion of surrounding water where needed for the enzymatic activity.
The dehydrons in the HIV-1 protease monomer are: (G49, G52), (G78, T80), (A28, R87); (D29, N88) and (T91, G94). Note that with the sole exception of R87, all dehydrons involve residues known to be poor wrappers (G, A, D, N, T or S) of the protein backbone (18). The dehydrons (G49, G52), (G78, T80) and (T91, G94) are determinants of the HIV-1 protease dimerization (cf. ref. 1), as revealed by comparing Figs. 1a and 1b. This is so since the nonpolar side chain groups of a monomer contribute to further and favorably desolvate the dehydrons of the other. This intermolecular wrapping is depicted in Fig. 1b by thin lines joining the a-carbons of the wrapper residues with the center of the hydrogen bonds which are being intermolecularly dehydrated. Thus, one functional role of these dehydrons arises as HIV-1 must dimerize to become an active catalyst.
It should be noted that there is a symmetry-breaking induced fit upon dimerization (Fig. 1c), as clearly determined by computing the wrapping of the PDB (protein data bank) entry 1a30 for the HIV-1 protease. This distortion takes place in one monomer as its flap wraps the (G49, G52) dehydron of the other monomer. Thus, one flap remains flexible in the dimer, as needed for processivity, while the other becomes rigidified.
The flexibility of the 46-55 flap requires that its backbone hydrogen bond be partially exposed to water. Thus the under-wrapping of the dehydron (G49, G52) appears as a necessary design feature. On the other hand, the feature that confers flexibility also confers stickiness (9) to the flap, as mechanistically needed to drag the substrate peptide chain. This stickiness suggests a strategy for inhibitor design by wrapping the packing defects.
The functional role of the protease dehydrons can be clearly delineated: (G49, G52) confers flap flexibility and is a dimerization inducer (Figs. 1a,b); (G78-T80) induces dehydration at the catalytic core (Fig. 1a); (A28, R87) provides stickiness to the substrate-harnessing region, and acts as a dimerization inducer (Figs. 1a,b); (D29, N88) provides stickiness to the substrate-harnessing region (Fig. 1a); and (T91, G94) is a dimerization inducer (Figs. 1a,b). The spatial orientation of the peptidic substrate chain can be clearly delineated by the prevailing dehydrons in the dimeric structure, as shown in Fig. 1b. It is also worth indicating that all residues whose site mutation impacts drug resistance and substrate specificity (marked in red in Fig. 1a) (16) are actually wrappers of the dehydrons in HIV-1 protease, and thus aminoacid substitution at such sites modulates the sensitivity of the protein to removal of surrounding water (2,19).
As shown before, dehydrons constitute highly specific sites for protein-ligand association (1,2). The fact that they occur precisely at the active region for substrate harnessing and at the flap region in the HIV-1 protease makes this molecule an ideal target for inhibitor design. A novel underlying inhibitor strategy is suggested by the thermodynamically and energetically favorable wrapping of packing defects. Thus, the inhibiting EDL-tripeptide (20) wraps all three catalytically important dehydrons in HIV-1 protease: (G49, G52), (A28, R87) and (D29, N88). This intermolecular desolvation is shown in Fig. 1d. In this case, the functionality of the dehydrons, as inducers of local water removal or by conferring flexibility to the flap, is precisely the property that enables effective drug design. All three dehydrons occur at catalytically active sites, and because they are inherently sticky (9) and possess intersecting desolvation domains (Methods), such sites can be simultaneously blocked with a single inhibitor, as shown in Fig. 1d.
Impairing FIV protease
by wrapping its unfavorable protein-water interfaces
Given their lower specificity and affinity, drug inhibitiors of the homologous FIV protease, the feline immunodeficiency virus protease, have not been nearly as successful as those for HIV-1 protease (16, 21). The dehydrons in the monomeric state (pdb.4fiv) are displayed in Fig. 2a. They are: (G58, G61), (G52, N67) and (Q54, G65) in the flap region; (D30, A33) and (A33, R104) in the catalytic region; (D34, D105) in the substrate-harnessing region; and (R104, I108) and (I108, N111) serving as dimerization inducers. With the sole exception of dehydron (G52, N67), all dehydrons can be clearly assigned functional roles in strategically inducing water exclusion: (G58, G61) and (Q54, G65) confer flexibility to the flap; (D30, A33) acts jointly with the nonpolar residues L28, L29 and L101 to ensure the dehydration of the catalytic site D30 (marked in black in Fig. 2a); (D34, D105) and (A33, R104) favor water removal as they harness the substrate polypeptide chain; while all dehydrons except (G52, N67) act as anchors in the dimerization of the protease (Fig. 2b). All residue sites assumed to affect substrate and inhibitor specificity (16) are marked in red in Fig. 2a if they are engaged in the wrapping of a dehydron, and in yellow otherwise. With the exception of I98 and Q99, which represent catalytically active sites (16), amino-acid substitution in the remaining residues marked in red in Fig. 2a affect the enzymatic activity by modulating the sensitivity of the active site to water removal, as inferred from the distribution of dehydrons along the flap and catalytic rim (Fig.2a).
The dimer active form of the FIV protease, with its equivalent catalytic D30 residues playing opposing roles as general acids as bases contains only one dehydron, (G52, N67) which is of no direct relevance to enzymatic activity, unless some hitherto unknown allosteric pathway is invoked. The highly exposed nonpolar residue L10 acts as the major intermolecular wrapper, as shown in Fig. 2b. The lack of dehydrons around the active site and flap in the FIV protease dimer poses a major difficulty in designing specific inhibitors, as the thermodynamically unfavorable protein-water interface is restricted to the localized region with exposed nonpolar residues L28, L29 and L101 around the catalytic D30. No dehydron on the substrate-harnessing track or on the flap contributes to the purported inhibitor binding or pivoting and thus, the restricted nature of the binding hot spot reduces the inhibitor specificity.
Novel drug epitopes
for HIV-1 integrase
HIV-1 integrase (pdb.b9f), the enzyme that integrates viral DNA with the host-cell DNA, is an important inhibitor target in antiviral therapy because it has no equivalent counterpart in the human host cell. Its catalytic residues D64, D116 and E152 form a triad of general acid/bases (15, 17) which has been targeted by drug designers with low or moderate success. The integrase inhibitor 5CITEP and similar drugs (15) bind longitudinally along the major groove of the integrase, parallel to the major 146-164 helix (Fig. 3a,b). The functionally active residues along this groove are essentially polar: Q148, E152, N155, K156 and K159. Thus, the removal of water surrounding this region is not “conventionally” induced by the presence of hydrophobic patches on the protein surface, and can only be fostered by the presence of the dehydron (E152, K156). This dehydron actually serves as the epitope for inhibitor binding (15, 17, 22). The strategic location of this dehydron fully justifies the docking mode of the inhibitor.
The distribution of dehydrons in the integrase clearly marks two tracks along which water removal is favored. While the host DNA is docked along the (E152, K156) dehydron, the viral DNA is anchored by two dehydrons, (N117, N120) and (S119, T122), which, with seven nonpolar wrapping groups each, constitute the most under-wrapped hydrogen bonds in the PDB.
The two dehydrons (N117, N120) and S(119, T122) have not been targeted by any drug inhibitor so far, although their functional role appears to be paramount to properly orient and attach the viral DNA (compare with ref. 22, Fig. 4), as Fig. 3c reveals. Thus, rather than merely targeting the (E152, K156) dehydron, we propose an extended inhibitor-binding epitope defined by the three dehydrons (E152, K156), (N117, N120) and (S119, T122) around the catalytic triad. This increase in the pivoting binding surface for the inhibitor beyond the (E152, K156) dehydron ensures a higher specificity and affinity.
Discussion
This paper addresses the problem of elucidating how specific enzymes may induce the removal of water surrounding catalytically competent polar residues. Surrounding the catalytic polar residue with nonpolar residues to create a thermodynamically unfavorable protein-water interface seems an obvious possibility, as such surface organizations de-screen the catalytic site while raising its self-energy. In this way, the presence of vicinal hydrophobes strengthens the intermolecular electrostatic interactions involved in enzymatic activity. However, when several spatially adjacent polar groups are needed for the activity, an alternative factor inducing water removal is naturally designed to functionalize the polar groups. This factor is the dehydron, a pre-formed under-wrapped hydrogen bond in the enzyme backbone. The dehydron is strengthened and stabilized by water removal and thus acts synergistically with the enzyme intermolecular electrostatics which engages the polar residues paired by such hydrogen bonds. Thus, dehydrons induce water removal, thereby causing also the de-screening of the side-chain charges, which in turn is required for their effective participation in catalytic activity.
The three HIV-1 and FIV viral protein enzymes investigated in this work reveal different problems faced by inhibitor designers in trying to block catalytic activity in antiviral therapies. Thus, the functionality of dehydrons as modulators of the solvent environment (dehydrators) for the enzymatic activity makes them also good candidates for drug targeting. This is so because dehydrons are inherently sticky and have been evolutionary placed at the active site of the enzyme catalysts precisely to guarantee enzymatic efficiency by favoring the formation of an anhydrous environment. On the other hand, effective inhibitors attach to polar surfaces by wrapping the dehydrons, with a net effect of blocking the catalytic or mechanically active sites.
This perspective clearly leads to a strategy to identify hot spots for inhibitor binding based on the location of functional packing defects in protein enzymes. The results reveal how to improve the inhibitor binding specificity and affinity for the HIV-1 integrase by focusing on packing defects so far overlooked placed along the viral DNA track.
Acknowledgements
A. F. gratefully acknowledges financial support from INGEN, the Indiana Genomics Initiative, and thanks the Eli Lilly Corporation for an unrestricted grant.
Figure Captions
Figure 1a. Distribution of dehydrons in a monomeric HIV-1 protease unit. The backbone is represented as a light blue polygonal made up of virtual bonds joining a-carbons. Well wrapped backbone hydrogen bonds (Methods) are indicated as grey segments joining a-carbons, while dehydrons are marked in green. The catalytically active D25 is shown in black, and the most significant residues undergoing site mutation associated with drug resistance or substrate specificity are shown in red. The dehydrons, together with their functional roles, are: (G49, G52) (flap flexibility, dimerization inducer); (G78-T80) (induces dehydration of catalytic core); (A28, R87) (stickiness of substrate-harnessing region, dimerization inducer); (D29, N88) (stickiness of substrate-harnessing region); (T91, G94) (dimerization inducer).
Figure 1b. Homodimeric HIV-1 protease (pdb.1a30) adopting the same virtual-bond backbone representation as in Fig. 1a, except one monomer is shown in red and the other in blue. Only inter-molecular wrapping is shown. Thus, the line joining the a-carbon of a residue in a monomer and the center of a backbone hydrogen bond on the other monomer indicates penetration upon dimerization of at least one nonpolar group of the residue side chain into the desolvation domain of the hydrogen bond. Some dehydrons become well-wrapped hydrogen bonds upon dimerization. The remaining dehydrons dictate the pathway of the peptide-chain substrate through the HIV-1 protease. The substrate-anchoring residue D29 is marked in yellow, while the catalytic D25 is marked in black as above.
Figure 1c. Detail revealing the broken symmetry upon induced fit in HIV-1 dimerization. The flap plane is distorted in one of the monomers to better wrap (intermolecularly) the flap dehydron of the other.
Figure 1d. The tripeptide inhibitor EDL (red) wrapping the dehydrons (G49, G52), (A28, R87) and (D29, N88) in one monomer of HIV-1 protease within the dimer (pdb.1a30).
Figure 2a. Dehydron distribution in FIV protease, the protease from the feline immunodeficiency virus. The convention of Fig. 1 is followed for consistency. Mutation sites that affect substrate and inhibitor specificity (16) are marked in red if the substitution affects the wrapping of a dehydron, and in yellow otherwise.
Figure 2b. Dehydron distribution in the active dimeric FIV protease (pdb.3fiv). The intermolecular wrapping of dehydrons by L10 is highlighted.
Figure 3a. Dehydron distribution for HIV-1 integrase (pdb.b9f). The catalytic residues are marked in black and the other active residues are marked in yellow. Dehydron (E152, K156) constitutes a major epitope anchoring inhibitors drugs which dock along the major goove region parallel to the 146-164 helix.
Figure 3b. Ribbon rendering of the HIV-1 integrase as a visual aid.
Figure 3c. The HIV-1 integrase positioned as in Fig. 4, ref. 22, with the dehydrons (N117, N120) and (S119, T122) defining the anchoring track for the viral DNA, while the (E152, K156) defines the track for the host-cell DNA.
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