Structure-Based Design and Two Aspartic Proteases

Elizabeth A. Lunney
Parke-Davis Pharmaceutical Research
Division of Warner-Lambert Company
2800 Plymouth Rd.
Ann Arbor, Michigan 48105-2430, USA


Structure-based design has been an integral player in molecular design for over a decade [1-3]. In that time, the advances in computational methods, X-ray crystallography, biochemistry and molecular biology have raised the potential of the methodology to new levels. Inhibitor design for two enzymes in the aspartic protease family span the lifetime of this approach, while exemplifying the application of structure-based design under diverse conditions. In the early stages, structure-based design was applied to renin as a target for antihypertension. These studies laid the ground work for later applications with various proteins including the aspartic protease found in HIV-1 (Human Immunodeficiency Virus - Type 1), the retrovirus implicated as the pathogen in the AIDS epidemic. By comparing the structure-based design work carried out for the two enzymes, one can appreciate where we have evolved over the years, as well as acknowledge inherent limitations that challenged the earlier studies with renin.


Renin, as part of the angiotensin/renin cascade, cleaves angiotensinogen to produce angiotensin I, which is then converted to the vasoconstrictor, angiotensin II [4]. By blocking the mechanism of action for this enzyme, hypertension might be successfully treated.

When structure-based design was initiated with renin, no X-ray crystal structure of the enzyme was available for use in the studies. However, crystal structures of homologous enzymes were being determined [5-7]. From these coordinates, models of renin were constructed [8, 9]. The aspartic protease crystal structures bound with inhibitors provided key information regarding the details of the intermolecular interactions of the enzyme with the peptide-like inhibitors, which were generally transition state mimetics. The transition state isostere bound at the catalytic dyad formed by two aspartic acids. The backbone of the peptide-like inhibitors engaged in a series of hydrogen bonds including interactions with a flexible flap region that covered the bound ligand (Scheme 1) [10].  The amino acid side chains occupied subsites on alternate sides of the backbone and have been designated P1, P1'...S1, S1' [11]. These experimental observations guided the design of novel inhibitors in the renin models. Targeted analogs were evaluated for their polar contacts and steric compatibilities with the enzyme. However, the docking procedures remained manual and qualitative due to the lack of well established protocols or computational tools. Rational design concepts from peptidomimetic chemistry and transition state mimetics were simultaneously applied. With the aim of reducing the peptidic elements that hinder oral bioavailability [4, 12], unnatural amino acids, amide isosteres and constrained moieties were incorporated into the structure-based inhibitor design. Modifications of the inhibitors were often made in an incremental manner, focusing on individual subsites.  Representative renin inhibitors resulting from these design strategies are shown in Table 1, (1-4) [13-16]. Ultimately, the ability to remove the peptidic nature and reduce the molecular weight of the potent inhibitors, while retaining activity, was limited and often led to unexpected results. However, retrospective reassessments of these studies did spur the development and application of computational methods and moved structure-based design forward.

  The renin models were refined over the years as additional X-ray structural information became available. Purified human renin was extremely scarce and it was not until the later years in the renin studies that an X-ray crystal structure became available [17-20] (Figure 1). While an advanced model compared favorably with the experimentally determined structure, structural features of the enzyme were not predicted [21]. These included a helix shift which reduced the size of the S3 pocket and a polyproline loop segment containing cis amide bonds located near the S1' and S3' sites. Therefore, while supporting the premise that models can be used in structure-based design, these results pointed out the desirability of having the X-ray structure for a more accurate 3-dimensional representation.

Therefore, the structure-based design approach in the renin era was limited not only by the computational tools available but by drawbacks that were inherent with the protein: the requirements of size and peptide character for its ligands and the lack of available X-ray structures for more precise inhibitor design.

HIV-1 Protease

In the late 1980's, a new aspartic protease emerged on the scene as a target for structure-based inhibitor design: HIV-1 protease. This enzyme cleaves gag and gag-pol proteins to produce structural and functional proteins in the virus [22]. It has been shown that inhibition of this process produces non-infectious virions [23].

  Ironically, in the same year that a renin crystal structure was finally reported, the X-ray structure of the HIV-1 protease was also determined [24, 25] (Figure 2). For the latter enzyme, this occurred in the early stages of inhibitor design and represented a enormous advantage relative to the renin research. HIV-1 protease is a homodimer, with each monomer contributing an aspartic acid to the catalytic dyad. Two flexible flap regions cover the ligand upon binding. Various X-ray structures of HIV-1 protease bound with peptide-like inhibitors show that the ligands bind in an extended manner similar to that observed with the renin ligands: the transition state isosteres bind at the catalytic dyad and the residue side chains occupy pockets on alternate sides of the backbone  ( Figure 3) [26, 27]. The amide bonds engage in a series of hydrogen bonds with the enzyme, but unlike renin, also with a conserved water molecule (H2O 301) positioned between the flap regions and the ligand. This key binding mode difference, determined through structure elucidation, has provided a unique strategy for nonpeptide inhibitor design not possible with renin.

Major technological advances in software and hardware, X-ray crystallography and biochemistry have afforded for HIV-1 protease scientific achievements not feasible during the heart of the renin era. Through the use of various types of computational database searching, unique nonpeptide ligands have been discovered. Haloperidol, 5  (Table 2), has been identified as an HIV-1 protease inhibitor by evaluating the fit of compounds found in the Cambridge Crystallographic database [28] to the binding site of the enzyme using the program dock [29, 30]. Unique nonpeptide inhibitors, which displace H2O 301, e. g., 6, were designed following a search of a 3-dimensional database with a pharmacophore model of inhibitor binding at the active site [31]. De novo design employing the iterative process of X-ray crystallography, molecular modeling and synthesis afforded the potent inhibitors 7 and 8 [32, 33].

In our laboratories, mass screening of our extensive chemical database with the HIV-1 protease provided two nonpeptide 'hits', 9 and 10 [34]. These analogs were excellent starting points to begin structure-based design of potent, novel inhibitors. Prior to obtaining the X-ray structure of these nonpeptide 'hits', we were successful in predicting the bound conformation for 9 using computational docking simulations [35]. The following is a description of this study and illustrates in more detail a structure-based design application.

A number of questions arose as we initiated the docking experiments for 9 in HIV-1 protease. Although the peptide-like inhibitors are shown to bind to the conserved H2O(301), would these nonpeptide ligands do the same or would they displace the water? Secondly, in the X-ray structure of the native enzyme, density is observed for a water molecule bound at the catalytic site (H2O(CAT)). The transition state isosteres in the peptide-like inhibitors displace H2O(CAT). Would our nonpeptide 'hits' bind accordingly? To explore these possibilities, three sets of docking simulations with different solvation states were carried out: one in which both H2O(301) and H2O(CAT) would be retained in the binding site, a second in which only H2O(301) would be retained and a third in which no water molecules would be present. The program Autodock, which couples Monte Carlo methodology with simulated annealing, was used to carry out the simulations [36]. The most interesting result occurred for the simulation in which no water was retained in the active site. The result indicated that the coumarin ring could span the active site and interact simultaneously with the catalytic dyad through its hydroxyl group and with Ile50/150 in the flap regions through its lactone   (Figure 4). Thus, both waters could be displaced by the nonpeptide. The fused phenyl ring bound in the S1 pocket and the phenoxy side chain bound through the S1' to the S3' site. Subsequently, the X-ray structure of 9 was determined and, interestingly, indicated two modes of binding. In both cases the two water molecules, H2O(301) and H2O(CAT), were displaced by the ligand. One mode confirmed what had been predicted by the modeling, whereas the second showed the inhibitor shifted in the binding site such that the lactone only interacted with one of the Ile50/150 in the flap regions. The flexible side chain in 9 now folded to bind in the S2' region. The X- ray structure provided the experimental data that elucidated the binding mode for the small molecule and allowed our design efforts to move forward. Experimentally determined structures of the pyrone analogs, e. g., 11, co-crystallized with the protease revealed a similar mode of binding [37, 38]. These X-ray results clearly indicated that branching of the substituents on the coumarin and pyrone rings could afford access to multiple pockets and improve potency. As is seen with 12  (Table 2), this proved to be the case. However, the X-ray structure determined for the protease bound with this inhibitor showed a novel mode of binding  (Figure 5) [39]. The hydroxyl group bound to only one aspartic acid of the catalytic dyad, while a water molecule interacted with the second. The dual orientations observed for 9 and the novel mode of binding for 12, pointed to the complexity of binding for these small molecules and the need for additional X-ray structures as the inhibitor design evolved. Indeed, during the course of our project, multiple key X-ray structures bound with inhibitors enabled us to elaborate the initial micromolar 'hits' to ultimately subnanomolar inhibitors.

Thus, with HIV-1 protease, we had the advantage of chemical mass screening in identifying nonpeptide inhibitor leads for the project. Not only did we have an X-ray structure of the enzyme with which to initiate our design, within a relatively short amount of time we had available multiple complexes with our nonpeptide inhibitors. These structures helped confirm and refine our modeling efforts with the novel ligands. In addition, we were able to apply advanced computational methods in docking the mass screen 'hits' and predicting the mode of binding. As with other laboratories, the structure-based design efforts with HIV-1 protease have produced extremely potent, nonpeptide inhibitors.


The structure-based design studies carried out for the two aspartic proteases, renin and HIV-1 protease, represent essentially two extremes for the methodology. The limitations in the renin work imposed by the state of the technology at the time and inherent features of the enzyme resulted in slow progress in inhibitor design. Conversely, HIV-1 protease had the luxury of advances in many fields including computer software and hardware, and X-ray crystallography. This enzyme also presented a unique mode of binding that has been capitalized upon in nonpeptide design. However, the experience and knowledge acquired with the renin studies with regards to, e. g., docking and intermolecular interactions, were invaluable as the structure-based design work with HIV-1 protease was undertaken. This adds to the advantages which make HIV-1 protease an 'ideal' target for structure-based design. What we learn with this protease and other current structure-based design studies will, in turn, benefit future endeavors in the field. This ever-increasing knowledge along with the continual advances in technology paint an extremely bright picture for structure-based design methodology in the future.


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[Table 1]

Table 1
Renin Inhibitors

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[Table 2]

Table 2
HIV-1 Protease Inhibitors

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[Scheme 1]

Scheme 1
Hydrogen bonding scheme observed in five X-ray structures of endothiapepsin bound with renin inhibitors [10]
( * * * * Non-conserved hydrogen bond)
( - - - - Conserved hydrogen bonds)

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[Figure 1]

Figure 1
Stereoview of the X-ray crystal structure of human
renin bound with an inhibitor [17].
Click on Image for full size

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[Figure 2]

Figure 2
Stereoview of the X-ray crystal structure of HIV-1 protease (ribbon
representation) bound with an inhibitor (cyan) [25]. Asp25/125 and
Ile50/150 are shown in red; H2O(301) is shown in yellow.
Click on Image for full size

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[Figure 3]

Figure 3
Stereoview of the X-ray crystal structure of a peptidomimetic
inhibitor, A74704 (yellow), bound in the HIV-1 protease active
site (cyan) [27]. H20(301) is shown in green.
Click on Image for full size

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[Figure 4]

Figure 4
Stereoview of the docking result for 9 from the Autodock
simulation carried out without water molecules [35].
Asp25/125, Ile50/150 and Arg108 are shown in cyan.
Click on Image for full size

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[Figure 5]

Figure 5
Stereoview of the X-ray crystal structure of HIV-1
protease bound with the branched pyrone inhibitor, 12 [39].
Asp25/125 and Ile50/150 are shown in cyan; the bridging
water molecule is shown in red.
Click on Image for full size

Return to the main article.

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