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1 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA
2 Sepracor Inc., Marlborough, Massachusetts 01752-7231, USA
Reprint requests to: Celia A. Schiffer, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA; e-mail: Celia.Schiffer{at}umassmed.edu; fax: (508) 856-2398.
(RECEIVED June 27, 2001; FINAL REVISION November 1, 2001; ACCEPTED November 6, 2001)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.25502.
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2.2 Åresolution. Two of the three analogs have only a single modification of indinavir, and their binding affinities to the variant HIV-1 protease are enhanced over that of indinavir. However, when both modifications were combined into a single compound, the binding affinity to the protease variant was reduced. On close examination, the structural rearrangements in the protease that occur in the tightest binding inhibitor complex are mutually exclusive with the structural rearrangements seen in the second tightest inhibitor complex. This occurs as adaptations in the S1 pocket of one monomer propagate through the dimer and affect the conformation of the S1 loop near P81 of the other monomer. Therefore, structural rearrangements that occur within the protease when it binds to an inhibitor with a single modification must be accounted for in the design of inhibitors with multiple modifications. This consideration is necessary to develop inhibitors that bind sufficiently tightly to drug-resistant variants of HIV-1 protease to potentially become the next generation of therapeutic agents. Keywords: HIV protease; drug resistance; drug design; protein crystallography
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Introduction |
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All of the HIV protease inhibitors currently prescribed for treatment of HIV-infected patients are competitive inhibitors that bind to the active site. Their design was based on both the three-dimensional structure of the active site of the protease and the primary sequences of its natural substrates. Unfortunately, exposure to protease inhibitors often leads to drug-resistant mutations in the protease gene. The resulting protease is resistant to the inhibitor, yet still maintains at least a portion of its function in cleaving its natural substrates. Patients receiving inhibitor therapy often have an initial reduction in viral load, followed by a rebound caused by the development of drug-resistant mutations. Both in vivo and in vitro studies involving HIV and currently available drugs have demonstrated the generation of mutations that give rise to drug resistance (Schinazi et al. 1997; Boden and Markowitz 1998; Molla et al. 1998). As the population of heterogeneous HIV-1 viruses increase within a patient, most likely a combination of protease inhibitors will be necessary for effective treatment.
More than 200 crystal structures of HIV protease have been solved over the past several years (http://www-fbsc.ncifcrf.gov/HIVdb). Crystal structures of specific drug-resistant protease inhibitor complexes have been solved recently (Baldwin et al. 1995; Chen et al. 1995; Hong et al. 1996,1997,1998; Kervinen et al. 1996; Silva et al. 1996; Ala et al. 1997,1998; Swairjo et al. 1998). Comparing these mutant and wild-type protease inhibitor complexes allows an analysis of the mechanisms by which these mutations reduce drug binding ability. Additionally, investigations of the kinetics for these drug-resistant variant proteases provide a quantitative comparison of the mutational effects on substrate cleavage and inhibitor binding affinities (Gulnik et al. 1995; Lin et al. 1995; Pazhanisamy et al. 1996; Schock et al. 1996; Ermolieff et al. 1997; Wilson et al. 1997,1998; Klabe et al. 1998).
The protease inhibitor known as indinavir (Crixivan) (Chen et al. 1994) (Fig. 1a
) is often one of the first lines of treatment for patients infected with HIV (J. Sullivan and K. Luzuriaga, pers. comm.). Indinavir is a peptidomimetic; a peptide analog in which the scissile amide bond is replaced by a reduced moiety. This protease inhibitor has several large, mainly hydrophobic groups that interact with the hydrophobic P2–P2' pockets in the active site of the protease (Wlodawer and Erickson 1993). Thus, nonpolar interactions contribute significantly to the inhibitory properties of indinavir to HIV protease, but these interactions are also often the sites at which drug-resistant mutations occur. Mutations at the active site such asV82T and I84V confer resistance to indinavir but several other mutations often occur simultaneously, combining a decrease in the affinity of the inhibitor to the protease and an increase in catalytic efficiency (Molla et al. 1998). The mutation L63P, located in "the hinge region" of the protease (Chen et al. 1995), frequently arises upon drug exposure and along with M46I has been shown to compensate for active site mutations, restoring the impaired enzyme's ability to catalyze the necessary cleavage reactions (Markowitz et al. 1995; Schock et al. 1996). In fact, in a comparison of catalytic efficiencies, the doubly substituted M46I/L63P demonstrated greater catalytic efficiency (110–360%) for each of the eight normal substrates than the wild-type enzyme (Schock et al. 1996). Thus, HIV-1 protease evolves to maintain activity while in the presence of inhibitors.
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) is the focus of this study. These mutations were chosen on the basis of their relative frequency of emergence reported in both in vivo and in vitro studies with various HIV protease inhibitors (Molla et al. 1998). Both structural investigations and activity analyses were used to characterize the differences in inhibitor binding and substrate cleavage between the drug-resistant and wild-type proteases. From the changes in structure, new inhibitors were designed. This work describes the X-ray crystal structure of this protease variant in complex with indinavir, as well as with a series of newly designed inhibitors. Analysis of the structures of these complexes, in combination with the relative binding activity of the inhibitors, elucidates why two independent modifications to indinavir are mutually exclusive and cannot be productively combined in a single compound. Understanding which functional groups can be productively combined to form a tight inhibitor is essential for designing the next generation of HIV-1 protease inhibitors.
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). The 3X-protease inhibitor complexes displayed well-ordered electron density for the protease and all four ligands. The electron density for the mutated side chains was unambiguous. These included residues 82 and 84, which are located in the active site, and residue 63, located at the hinge region away from the inhibitor-binding site (Fig. 2
). Because HIV protease is catalytically active as a homodimer, the mutations encoding two active site residues result in four amino acid changes in the active site where inhibitor/substrate bind. The change from a leucine to a proline at position 63 is not only frequently selected for during protease inhibitor drug therapy, but also commonly exists as a polymorphism found in viral isolates from untreated patients (Olsen et al. 1999). Thus, the change at position 63 is expected to have less impact on the inhibitor than those substitutions at positions 82 and 84.
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). However, in the wild-type HIV-2 protease indinavir complex the difference in B-factors was even larger, with the pyridine ring having an average of 54 Å2 relative to the average of 39 Å2 for the entire inhibitor (Chen et al. 1994). Although indinavir was modeled in a single conformation, additional difference density (Fo - Fc) was observed in proximity to the tertiary-butyl group. However, no sterically acceptable alternate conformations could be unambiguously modeled to account for this extra density. The average of the B-factors for the t-butyl moiety were 31.9 Å2, lower than the average for the entire inhibitor, thus providing support for the unique conformation modeled in that region of the molecule. Modeling of the nearby I47a (the monomers are distinguished from one another by referring to them as "a" and "b") in a second conformation (Fig. 3a) further ordered the electron density surrounding both I47a and the region around the t-butyl moiety. The added conformational variability at I47a may result from the cavity introduced into the region by the nearby protease mutation of I84aV.
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). Indinavir binds to the active site in the same conformation seen in the wild-type protease indinavir complex (Chen et al. 1994) with the functional groups occupying similar subsites within the protease. Indinavir's pyridyl and piperidine groups occupy the S3 and S1 subsites, whereas the benzyl moiety occupies the S1' subsite. The tertiary-butyl group and the indanol moiety occupy the S2 and S2' subsites, respectively (Fig. 1a). The catalytic aspartic acids of the enzyme, at the edge of the S1/S1' subsites, are within hydrogen bonding distance of indinavir's O2 of the hydroxyethylene group. The binding constant from isothermal titration calorimetry is 38.4 times worse for indinavir to 3X-protease compared with its binding to wild-type HIV-1 protease (Table 2). The loss in binding is entirely due to a loss in the enthalpy of binding of 2.48 kcal/mole, presumably mainly because of the loss of van der Waals contacts at residues 82 and 84. The IC50 for indinavir inhibiting this 3X-protease is 0.47 µM (Table 3, Fig. 4).
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). Thus, this modification to indinavir was not further pursued.
XN1336–51
An alternative attempt at producing an inhibitor that would fill the cavity produced by the substitution I84aV involved adding an s-methyl group in the benzylic position to produce the indinavir analog XN1336–51. Thermodynamically, although the enthalpy of binding is somewhat worse than indinavir, the entropy has improved, thus improving the overall Kd (Table 2). Inhibition studies measure the IC50 for XN1336–51 as 0.40 µM (Table 3, Fig. 4). The 3X-protease XN1336–51 crystal structure was solved to determine how the added methyl group contributed to the improvement in inhibition (Fig. 3b, arrow). The root mean square deviation (RMSD) between XN1336–51 and the indinavir 3X-protease complexes for all 
carbons was 0.15 Å, indicating relatively little change in the protease backbone as a result of the added methyl group on XN1336–51. The added group does induce a 0.20 Å shift that can be seen throughout the entire inhibitor molecule, away from V84a compared with the position of indinavir (Fig. 5a). In addition, a slight adjustment in the position of the side chain of V84a was observed, with a difference in 
1 of 12.7°. I47a of this inhibitor protease complex became more ordered as a result of the added methyl group and only one conformation of this side chain was necessary to fit the electron density. Thus, the methyl group added onto the inhibitor ordered the conformation of nearby groups in the active site.
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), although the thermodynamics of binding in the absence of substrate did not show improvement over indinavir (Table 2).
The RMSD for the superposition of the crystal structure of the 3X-protease 807–29–4 complex and the 3X-protease indinavir complex was 0.20 Å for all carbons, with the main deviations in the loop involving active site residues 80b-82b in the S1 subsite (Fig. 3c,e). This region of the S1 loop has moved away from the inhibitor an average of 0.56 Å relative to the corresponding region in the protease-3X indinavir complex. Similar variations in this region have been observed in other protease inhibitor structures (Baldwin et al. 1995; Ala et al. 1998). In this case, the adaptation in the protease as a result of replacing the pyridyl moiety paralleled the movement of the inhibitor. The modification in 807–29–4 resulted in a "tilt" in its position relative to the position of indinavir in their respective protease complexes (Fig. 5b). The inhibitor 807–29–4 in this complex has pushed away from the protease flaps with the modified region of the inhibitor closer to P81b. The indanol group at the opposite end of the inhibitor has also been pushed away from the S2' binding pocket. An additional adaptation by the protease in its complex with 807–29–4 was seen at L23b (Fig. 3c), the side chain of L23b has rotated away from the inhibitor piperidine ring, possibly because of the tilt in the ligand, which has shifted the piperidine ring towards L23b. A further adjustment in the protease occurred at the tip of the flap of monomer B. The side chain of I50b assumed two conformations, with the CD1 atom of the second conformation directed towards the space between the inhibitor's t-butyl group and protease residue V84a, as an attempt to fill the void created by the I84aV mutation (Fig. 3c). Although there was some deviation in the position of I50b in the other inhibitor protease complexes, the side chain rotamers were basically the same. Flexibility at I50 has also been seen in the quadruple-mutant protease (M46I,L63P,V82T,I84V) indinavir complex (Chen et al. 1995). In addition, in the 3X-protease 807–29–4 complex, the side chain of I50a has relocated to fill the pocket created by the I84bV mutation on the opposite side of the active site, near the piperidine ring of the inhibitor. Thus, the flexibility of both backbone and side chains in the flap region of the protease allows it to adapt in this complex as well as in related complexes. The precise nature of the adaptability depends upon the exact protease variant and the inhibitor bound.
XN1336–52
Because the two beneficial modifications made to indinavir occurred in different regions of the compound, both modifications were combined into a single compound, with the hope of obtaining a synergistic effect. However, both the inhibition (Table 3, Fig. 4) and the thermodynamic data (Table 2) for this new indinavir analog, XN1336–52, show it to be a less effective inhibitor than indinavir. Superposition of the crystal structure of the 3X-protease complex and the doubly modified XN1336–52 with the two singly modified inhibitor analog 3X-protease complexes can be seen in Fig. 5d. The RMSDs were 0.15 Å and 0.20 Å for the superposition of all carbon atoms of the 3X-protease XN1336–52 and 3X-protease XN1336–51 complexes and the 3X-protease XN1336–52 and 3X-protease 807–29–4 complexes, respectively. This observation indicates that the complex with the doubly modified analog is more similar to the complex containing the inhibitor analog with the smaller methyl group added (XN1336–51) than it is to the complex containing the larger modification (807–29–4). Thus, the s-methyl group addition in the benzylic position of the inhibitor appears to have more impact on structural adaptations in the protease than does the substitution of the methylenedioxyphenyl group for the inhibitor's pyridine ring (Fig. 3d,e).
Comparing complexes
Examination of the substituted residues in the active site of the 3X-protease indinavir complex reveals both similarities and differences with related structures. In two previously solved protease indinavir complexes, wild-type and quadruple-mutant (M46I,L63P,V82T,I84V) (Chen et al. 1994,1995), the conformations of residue 82a in the S1' loop were the same as those in the triple-mutant complexes. The CG1 atom of residue 82a makes van der Waals contacts with the benzyl ring of indinavir. In the quadruple-mutant protease indinavir complex and in the present 3X-protease indinavir complex, threonine has been substituted for the isosteric valine at that position. In these mutant protease structures, the side chain oxygen faces away from the active site, thus reducing its inhibitory effect. On the other hand, the corresponding side chain at position 82b in the S1 loop of the 3X-protease inhibitor complex is rotated 96° about the 1 bond relative to that in the wild-type structure. (The position of the side chain of T82b in the quadruple-mutant protease indinavir complex was similar to that seen for V82b in the wild-type complex.) Thus, in the present 3X-protease indinavir complex, the oxygen of the side chain of T82b (Fig. 3a) also points away from the inhibitor, unlike that seen in the related indinavir structures. The conformations of T82a and T82b in all of the complexes of the 3X-protease indinavir and indinavir analogs were basically the same (Fig. 3).
In contrast with V82T, the protease mutation I84V physically changes the volume of the S1 and S1' subsites within the active site. Each inhibitor occupies a slightly different position in the active site, causing slight shifts in the position of V84a in the S1 subsite, although the side chain rotamers remain the same. On the other hand, for each of the 3X-protease inhibitor complexes, V84b in the S1 subsite had two conformations differing by a rotation about 1 of 109°–122° (Fig. 3). Multiple conformations at I84 have previously been characterized for the wild-type protease inhibitor complex, presumably because of the relatively larger S1 pocket (Baldwin et al. 1995).
Thus far, all of the differences described here primarily involve local adaptations in the protease. However, these adaptations do not take into account any long-range adjustments (beyond the RMSDs) that the protease may make between the various inhibitor complexes. These differences can arise as a result of subtle changes in the positions of the inhibitor molecules bound in the active site. A set of double difference plots (Fig. 6) showing the differences in domain to domain distances for the various structures provides information that is otherwise not readily apparent. Figure 6a shows the carbon difference distance matrix for the comparison between the 3X-protease XN1336–51 complex and the 3X-protease indinavir complex. This plot shows little variation between the two complexes. In contrast, large structural differences can be seen in the double difference plot comparing the 3X-protease 807–29–4 complex and the indinavir complex (Fig. 6b). However, in the double difference plot between the XN1336–52 protease complex and the 3X-protease indinavir complex (Fig. 6c) little change is once again observed even though, as in 807–29–4, a methylenedioxyphenyl group is part of the inhibitor. In fact, as shown in Figure 6d, the 3X-protease XN1336–51 complex and the 3X-protease XN1336–52 complex are virtually identical. This indicates that the presence of an added methyl group in the S1' pocket of the active site has a stronger impact on the structure of the protease inhibitor complex than does the methylenedioxyphenyl moiety.
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The details of the IC50s and the thermodynamics of the four inhibitors vary upon comparison of their relative binding. This is not completely unexpected, as although the two methods are measuring similar properties, they are not identical. The IC50s (Table 3, Fig. 4) of these competitive inhibitors are determined as described in the methods by blocking the cleavage of a peptide AcSQNYPVV-NH2 by the 3X-protease. Thus, the inhibitors compete with the binding of substrates and with product release. This competition increases IC50s. The thermodynamic measurements (Table 2), on the other hand, are made in the absence of substrates and are measuring the direct binding of the inhibitors to the 3X-protease. As the absolute binding of these various inhibitors is fairly similar, subtle changes due to possible product inhibition could account for the change in the relative inhibition and binding between the two techniques. Nevertheless, both techniques verify the nonadditivity of the two functional groups when combined in the XN1336–52 compound.
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This study focused on a multiple drug-resistant variant of HIV protease having amino acid substitutions L63P, V82T, and I84V. Both in vitro and in vivo studies involving a variety of protease inhibitors have shown that these substitutions commonly emerge (Condra et al. 1995; Molla et al. 1998). Solving the crystal structure of this protease with the inhibitor indinavir led to the design and analysis of indinavir analogs. Modifications made to different regions of the indinavir molecule resulted in similar improvements in inhibition, despite very different structural adaptations in the protease. The addition of an s-methyl group at the benzylic position of indinavir in one analog, XN1336–51, led to an increase in van der Waals interactions between the inhibitor and nearby atoms in the active site of the protease. In another analog, 807–29–4, substitution of the pyridine ring of indinavir with a methylenedioxyphenyl group resulted in structural rearrangements that propagated throughout the protease dimer.
Combining these two modifications into a single indinavir analog, XN1336–52, however, resulted in a compound with decreased inhibition and binding affinity relative to indinavir. The protease was also unable to adapt and assume the structurally beneficial rearrangements seen in either of its complexes with inhibitors containing the individual modifications alone. The challenge remains to develop an inhibitor that optimally combines the advantageous aspects of both analogs. Perhaps such an inhibitor would be a non-indinavir type molecule, having a novel scaffold that both fills the active site pocket formed by the I84V mutation and allows the movements within the protease seen around residue P81 in the S1 loop of the 807–29–4 complex, hopefully preserving the higher affinity.
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Protease expression and purification
The gene encoding HIV protease was cloned into the plasmid pXC34 (ATCC), which contains a 
PL promoter (Cheng and Patterson 1992). The protease was expressed by heat induction in Escherichia coli TAP 106 cells using this plasmid. Cells from 12 L of fermentation were lysed and the protein was purified from inclusion bodies (Hui et al. 1993). The inclusion body centrifugation pellet was dissolved in 50% acetic acid followed by another round of centrifugation to remove impurities. Size exclusion chromatography was used to separate high molecular weight proteins from the desired protease. This was carried out on a 2.1-L Sephadex G-75 superfine (Sigma Chemical) column equilibrated with 50% acetic acid. The protein was refolded in 10 mM formic acid (Todd et al. 1998). A final purification was performed with a Pharmacia Superdex 75 FPLC column equilibrated with 0.05 M sodium acetate at pH 5.5, 5% ethylene glycol, 10% glycerol, and 5 mM DTT.
Synthesis of inhibitors
The synthesis and preparation of the protease inhibitors used in this study are shown in Figure 1b. The penultimate piperazines (compound I in Fig. 1b) were prepared according to the method published in US patent 5,436,067, assigned to Merck & Co., Inc. T-amylamine was substituted for t-butylamine in the preparation of the piperazine amide for synthesis of the precursor to XN1336–27. In the cases of the precursors to XN1336–51 and XN1336–52, (S)-3-phenylbutyric acid (purchased from Fluka) replaced hydrocinnamic acid for the preparation of compound I (Fig. 1b). Preparation of the final products was accomplished by reductive alkylation of compound I by either pyridine-3-carboxaldehyde (XN1336–27, XN1336–51) or piperonal (807–29–4, XN1336–52), in the presence of sodium triacetoxyborohydride, as described (Abdel-Magid et al. 1996). The final products were purified by preparative thin layer chromatography using silica plates and 10% methanol in methylene chloride as the eluent. Structures and purities were confirmed by proton and carbon NMR and mass spectroscopy, with no evidence of hydration.
Crystallization and data collection
Crystals were set up using a three- to fivefold molar excess of inhibitor to protease, which ensures ubiquitous binding. The final concentration of protease was approximately 2 mg/mL in 0.05 M sodium acetate at pH 5.5, 5% ethylene glycol, 10% glycerol, 5 mM DTT. Equal volumes of the inhibitor–protein mixture and the reservoir solution were combined to set up hanging drops of 5 µL. The reservoir solution consisted of 126 mM phosphate buffer at pH 6.2, 63 mM sodium citrate, and ammonium sulfate in a range of 27–33% (Silva et al. 1996). Crystals were grown at ambient temperature and were evident within 24–72 hours. The data collection took place at room temperature on an R-AXIS-IV imaging plate system. The data were reduced and scaled using the programs DENZO and SCALEPACK (Otwinowski 1993), respectively. Crystals of both the indinavir and the indinavir analog protease complexes were of the P212121 space group, with one dimer per asymmetric unit.
Refinement
The program CNS [Crystallography and NMR System (Brunger et al. 1998)] was used to refine the structures. The wild-type HIV-1 protease indinavir complex (1HSG) (Chen et al. 1994) served as a model for solving the 3X-protease indinavir structure by molecular replacement. The 3X-protease indinavir complex was, in turn, used as a model for refining the corresponding 3X-protease XN1336–51 structure. The model used for refinement of the subsequent indinavir analog 3X-protease 807–29–4 complex was a high-resolution, well-refined substrate–protease structure recently solved in our laboratory (M. Prabu-Jeyabalan, E. Nalivaika, and C.A. Schiffer, unpubl.), having the same P212121 space group. The structure for 3X-protease 807–29–4 complex was then used as the model to refine the structure of the 3X-protease XN1336–52 complex. For each structure, an initial rigid body refinement was performed at 4Å resolution with difference Fourier electron density maps (Fo - Fc and 2Fo - Fc) subsequently computed. The program CHAIN (Sack 1988) was used for model building. Positional and B-factor refinements were carried out, and the difference Fourier electron density map (Fo - Fc) unambiguously revealed the positions of the inhibitors. The resolution limits were gradually extended in equal steps to accomplish the highest possible resolution. Solvent molecules were added manually at positions indicated by the electron density. For cross-validation, R-free values were monitored and simulated annealed omit maps were used to decrease model bias. The stereochemical parameters of the final structures were analyzed using the programs PROCHECK (Laskowski et al. 1993) and WHATIF (Vriend 1990; Hooft et al. 1996). There were no outliers in the Ramachandran maps. The crystallographic and refinement statistics are shown in Table 1. Complexes were superimposed using the -carbon backbone of the relatively immobile (Rose et al. 1998) terminal domain of all these structures (residues 1–9 and 86–99) with the program MIDAS (Ferrin et al. 1988). The coordinates have been deposited in the protein databank, 1K6C, 1K6P, 1K6T, and 1K6V for the 3X-protease indinavir, 807–29–4, XN 1336–51 and XN1336–52 complexes, respectively.
Protease inhibition assays
Inhibition of the triple-mutant protease by the various inhibitors was determined. Activity assays were performed at 37°C with the substrate AcSQNYPVV-NH2 (Sigma). Constant concentrations of protease, 0.05 mg/mL, and substrate, 492 µM, were used for all experiments. Inhibitor concentrations were varied from 0.0–0.80 µM. For each concentration of inhibitor, aliquots of the reaction mixture were removed at 15-min intervals, quenched with equal volumes of cold trichloroacetic acid, and assayed by reverse phase HPLC (Moore et al. 1989). The percent of activity remaining for each inhibitor concentration was plotted and IC50s were obtained (Fig. 4).
Thermodynamic binding assays
An isothermal titration calorimeter, a VP-ITC (MicroCal Inc.), was used to measure binding energies of inhibitors. Twenty to twenty-four 10–12-µl injections of 0.2 mM inhibitor were made into 29 µM wild-type HIV-1 protease and 22.7 µM 3X-protease HIV-1 protease at 20°C. The buffer in which both proteins and the inhibitor were suspended was 10 mM sodium acetate, 2.0% DMSO, and 2mM TCEP at pH 5.0. Heats of dilution were subtracted from the corresponding heats of reaction to obtain the heat due solely to the binding of the ligand to the enzyme. Data was processed and analyzed using the MicroCal Origin software package.
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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