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Department of Biology and Biocalorimetry Center, Johns Hopkins University, Baltimore, Maryland 21218, USA
Reprint requests to: Dr. Ernesto Freire, Department of Biology and Biocalorimetry Center, Johns Hopkins University, Baltimore, MD 21218, USA; e-mail: ef{at}jhu.edu; fax: (410) 516-6469.
(RECEIVED February 27, 2002; FINAL REVISION May 15, 2002; ACCEPTED May 16, 2002)
1 Present address: 13944 Bromfield Road, Germantown, MD 20874, USA. 
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0206402.
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Keywords: HIV-1 protease; drug resistance; HIV-1 protease inhibitors; isothermal titration calorimetry; Amprenavir; TMC-126
Abbreviations: HIV, human immunodeficiency virus • IPTG, isopropyl-ß-D-thiogalactoside • MES, 2-(N-morpholino)ethanesulfonic acid • ACES, N-(2-acetamido)-2-aminoethanesulfonic acid • EDTA, (ethylenedinitrile)tetraacetic acid • DMSO, dimethyl sulfoxide • ME, 2-mercaptoethanol • Tris, tris(hydroxymethyl)aminoethane • Gly-Gly, glycylglycine • ITC, isothermal titration calorimetry
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Introduction |
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Previously, we reported the thermodynamic analysis of the binding of four protease inhibitors in clinical use (Indinavir, Saquinavir, Nelfinavir, and Ritonavir; Todd et al. 2000) and a second-generation inhibitor (KNI-764; Velazquez-Campoy et al. 2001a) to the wild-type and the drug-resistant double mutant V82F/I84V, known to affect all clinical inhibitors including Amprenavir (Markowitz et al. 1995; Ala et al. 1998; Klabe et al. 1998). The V82F/I84V mutation is located at the edges of the active site (Fig. 1
) in a well-structured and stable region of the protease (Todd et al. 1998); its main effect on the binding affinity of inhibitors appears to be related to a change in the three-dimensional geometry of the binding site because its polarity and charge remain unchanged (Todd et al. 2000). Unlike other clinical inhibitors, Amprenavir is severely affected by the single mutation I50V (Kim et al. 1995; Pazhanisamy et al. 1996; Markland et al. 2000). The I50V mutation is located at the tips of the flaps (Fig. 1), which are characterized by a low degree of structural stability (Todd et al. 1998). In this paper, we report the binding thermodynamics of Amprenavir, one of six HIV-1 protease inhibitors approved for clinical use, and a chemically related second-generation inhibitor presently under development, TMC-126 (Yoshimura et al. 2002). TMC-126 has been shown to exhibit IC50s on the order of 0.3–0.5 nM against a wide spectrum of HIV, including common resistant mutations that severely affect existing protease inhibitors (Yoshimura et al. 2002). The chemical structures of the two inhibitors are shown in Figure 2. With this paper, we have concluded the thermodynamic characterization of five of the six protease inhibitors presently in clinical use against the same wild-type and drug-resistant mutations. The same characterization has also been performed on two second-generation inhibitors (KNI-764 and TMC-126) that show low susceptibility to drug-resistant mutations. The thermodynamic studies presented here permit us to draw general conclusions about the behavior of inhibitors and their response to mutations.
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shows a typical calorimetric displacement titration for Amprenavir and wild-type HIV-1 protease. The left panel shows the direct titration of Amprenavir into the protease. This experiment indicates that Amprenavir binds to the wild-type HIV-1 protease with a favorable enthalpy change of -6.9 kcal/mole at 25°C. The experiment also indicates that the binding affinity is too high for accurate determination from a direct titration. Acetyl pepstatin was selected as the most appropriate weak inhibitor for displacement, because it shows a positive binding enthalpy that will amplify the magnitude of the signal in the displacement titration (Sigurskjold 2000; Velazquez-Campoy et al. 2001a). The center panel of Figure 3 shows the direct titration of acetyl pepstatin into the protease. The right panel shows the titration of Amprenavir into the HIV-1 protease prebound to acetyl pepstatin. From these experiments, we determined an association constant, Ka, for Amprenavir of 5.0 ± 0.3 x 109 M-1, corresponding to a Gibbs energy of binding of -13.2 kcal/mole. Accordingly, the binding of Amprenavir is almost equally favored by enthalpic (-6.9 kcal/mole) and entropic (-6.3 kcal/mole) contributions to binding. Additional experiments performed at different temperatures yield a heat capacity change of -440 ± 50 cal/K • mole, 
Cp = (
H/T)p, indicating that there is a significant hydrophobic desolvation effect upon binding. Other experiments performed in buffers with different enthalpies of ionization (acetate: Hion = 0.12 kcal/mole; MES: Hion = 3.72 kcal/mole; ACES: Hion = 7.51 kcal/mole; Fukada and Takahashi 1998) yielded similar binding enthalpies, indicating that under the conditions of the experiments (pH 5.0), there is no net proton transfer process coupled to inhibitor binding (Velazquez-Campoy et al. 2000). All the thermodynamic results of this paper are summarized in Table 1.
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H = 2.7 kcal/mole) against the mutant protease. The heat capacity change upon binding was estimated to be -400 ± 30 cal/K • mole, similar to that obtained with the wild-type protease, consistent with a similar degree of desolvation. Again, experiments using buffers with different ionization enthalpies indicated no proton transfer upon binding. Together, the thermodynamic data are consistent with a weaker inhibitor/protein interaction, reflected only in the binding enthalpy.
Binding energetics of Amprenavir to HIV-1 mutant protease V82F/I84V
The binding of Amprenavir to the double-mutant protease is also exothermic; however, the binding enthalpy to the V82F/I84V mutant is 
3 kcal/mole less favorable than to the wild-type protease. The double mutation reduces the binding affinity by a factor of 104, corresponding to a loss in Gibbs energy of binding of 2.7 kcal/mole, which can be almost completely accounted for by the loss of enthalpic interactions. The heat capacity change upon binding was estimated to be -370 ± 40 cal/K • mole, slightly smaller than that of the wild-type protease. As in the case of the I50V mutant, no net proton transfer process was observed upon binding.
Binding energetics of TMC-126 to HIV-1 wild-type protease
The binding energetics of the inhibitor TMC-126 to the HIV-1 protease was also measured by isothermal titration calorimetry (Fig. 4). TMC-126 binds very tightly to the protease with a very large favorable enthalpy change, -12.0 kcal/mole at 25°C (Fig. 4, left panel). From the displacement experiments an association constant, Ka, of 2.6 ± 0.1 x 1011 M-1 (3.9 ± 0.1 pM) was determined, corresponding to a Gibbs energy of binding of -15.6 kcal/mole. The calorimetric results indicate that for TMC-126 most of the binding energy is of enthalpic (-12.0 kcal/mole) rather than entropic (-3.6 kcal/mole) origin. For Amprenavir, on the other hand, the enthalpic and entropic contributions to the Gibbs energy of binding are similar. These results should be compared to those obtained for first-generation inhibitors (Indinavir, Nelfinavir, Saquinavir, Ritonavir), which showed entropically controlled binding affinities and unfavorable or only slightly favorable binding enthalpies (Todd et al. 2000). For these inhibitors, entropy contributions as large as -16 kcal/mole were required to compensate unfavorable binding enthalpies.
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Additional experiments performed at different temperatures yield a heat capacity change of -350 ± 70 cal/K • mole. Measurements performed in buffers with different enthalpies of ionization indicate that there is a net proton transfer (from the bulk solution to the protease–inhibitor complex) coupled to inhibitor binding. At pH 5.0, 0.39 ± 0.02 protons associate to the complex. Because TMC-126 does not have an ionizable group, the group responsible for the proton-transfer reaction must belong to the protease. Considering the structural similarity between Amprenavir and TMC-126, one could expect a similar net proton transfer for both inhibitors. However, the interactions of these two inhibitors with Asp 29 and Asp 30 are not the same. The bis-THF group in TMC-126 interacts very strongly with the backbone of the active-site amino acids Asp 29 and Asp 30 (Yoshimura et al. 2002). In particular, the two oxygens in the bis-THF group, which are absent in Amprenavir, are within hydrogen-bond distance of the main-chain amides of Asp 29 and Asp 30. This interaction may lead to a higher pKa increase in either of the carboxylate groups than the one observed with Amprenavir, and consequently a net proton absorption upon inhibitor binding.
Binding energetics of TMC-126 to HIV-1 mutant proteases
The binding energetics of TMC-126 to the resistant mutants I50V and V82F/I84V were also measured by isothermal titration calorimetry under identical conditions. The results are summarized in Table 1
. The binding of TMC-126 to both mutants is also exothermic; however, the magnitudes of the binding enthalpies are 2.9 and 2.4 kcal/mole less favorable than that to the wild-type protease. The binding constants of TMC-126 to the resistant mutants are 1.6 x 1010 M-1 for I50V and 2.3 x 1010 M-1 for V82F/I84V, respectively. The I50V and V82F/I84V mutants lower the binding affinity for TMC-126 by a factor of 16 and 11, respectively, corresponding to a loss of 1.7 and 1.5 kcal/mole in Gibbs energy of binding. Because the loss in Gibbs energy is smaller than the loss in binding enthalpy, the results indicate the presence of compensatory entropic effects (-1.2 and -0.9 kcal/mole, respectively). Previously, Xie et al. (2000) indirectly estimated binding affinities of TMC-126 from the shift in protease stability in urea denaturation experiments. Although the absolute values differ from binding affinities measured directly (perhaps owing to different experimental conditions or the assumptions involved in the indirect method), the differences in binding affinities between wild-type and drug-resistant mutant V82F/I84V proteases are similar (1.5 and 1.3 kcal/mole).
The heat capacity changes upon binding were estimated to be -390 ± 30 and -460 ± 80 cal/K • mole for the I50V and V82F/I84V mutants, respectively, which appear to be slightly larger than the value measured for the wild-type protease. This observation would be consistent with a slightly better burial of the inhibitor with the mutant proteins. If this is the case, the compensatory entropic effect would originate from a higher desolvation of the inhibitor/mutant complexes. Similarly, the proton intakes measured for the inhibitor complexes with the mutant proteases are slightly smaller than the one measured for the wild-type protease.
The I50V mutation affects Amprenavir in a rather unique and severe fashion. Additional calorimetric experiments performed with existing clinical inhibitors (data not shown) reveal that the binding affinities of Indinavir, Nelfinavir, Saquinavir, and Ritonavir are only affected by a factor of 25, 55, 59, and 69 by the I50V mutation, respectively, compared with a factor of 147 for Amprenavir. The origin of the large effect of I50V on Amprenavir has been attributed to the strong interactions made by this inhibitor with the tips of the flap. The larger volume of the mutant cavity diminishes the packing density of the inhibitor and consequently the strength of the binding energy. Despite its structural similarity to Amprenavir, TMC-126 is not affected as much by the I50V mutation because most of its binding interactions are directed toward the bottom of the binding site, especially against Asp 29 and Asp 30 (Yoshimura et al. 2002).
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Discussion |
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With the results presented in this paper, we have completed the thermodynamic characterization of five of the six protease inhibitors presently in clinical use and of two second-generation inhibitors. The experiments have been performed under identical conditions and against the same wild-type and drug-resistant mutations (Todd et al. 2000; Velazquez-Campoy et al. 2001a). Figure 5 summarizes the binding thermodynamics of all inhibitors measured so far. It is apparent from the data that second-generation inhibitors have significantly higher binding affinities than first-generation inhibitors, and that their binding affinity is determined by favorable enthalpy and entropy changes. The binding affinity of first-generation protease inhibitors is entropically driven and, except for Ritonavir, characterized by an unfavorable binding enthalpy.
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. The entropy gain is due to an increase in the number of degrees of freedom, either as a result of increased conformational dynamics within the complex or the release of water molecules associated with an increased desolvation. Either situation can be traced back to the ability of the inhibitor molecule to adapt to the binding-site distortions induced by the mutation. This is illustrated schematically in Figure 7, in which the binding-site cavities of the wild-type and V82F/I84V mutant protease are shown. In this figure the bulge and distortion created by the presence of the Phe ring are apparent. The total loss of two methyl groups in the I50V mutation (one per monomer) also changes the volume and geometry of the binding cavity without a change in polarity or hydrophobicity. These changes induce a loss of van der Waals interactions in all inhibitors that have been optimized against the wild type. This effect accounts for a similar decrease in the binding enthalpy for all inhibitors. However, conformationally constrained inhibitors respond differently than inhibitors showing a certain degree of flexibility or asymmetric functional groups in those regions facing the protein mutations. The loss of conformational constraints imposed by the elimination of certain interactions with the target allows a flexible inhibitor to search for a new energy minimum. An asymmetric functional group further permits an inhibitor to present different interaction surfaces against the mutated regions of the protein. A common situation will be one in which the inhibitor increases its burial from the solvent, thus increasing its desolvation and eliciting a more favorable binding entropy. A rigid or conformationally constrained inhibitor will not be able to trigger this effect. The experimental thermodynamic data support this hypothesis. It must be noted that an entropic compensation of this kind does not require an inhibitor to interact with a mutant as well as with the wild type. All that is required is that the lost interactions are compensated by an entropic gain.
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Protease purification
Plasmid-encoded HIV-1 protease was expressed as inclusion bodies in Escherichia coli 1458 (Todd et al. 1998, 2000; Todd and Freire 1999). Cells were suspended in extraction buffer (20 mM Tris, 1 mM EDTA, 10 mM 2-ME at pH 7.5) and broken with two passes through a French pressure cell (
16,000 p.s.i.). Cell debris and protease-containing inclusion bodies were collected by centrifugation (20,000g at 4°C for 20 min). Inclusion bodies were washed with three buffers. Each wash consisted of resuspension (glass homogenizer, sonication) and centrifugation (20,000g at 4°C for 20 min). In each step a different washing buffer was used: Buffer-1 (25 mM Tris, 2.5 mM EDTA, 0.5 M NaCl, 1 mM Gly-Gly, 50 mM 2-ME at pH 7.0), Buffer-2 (25 mM Tris, 2.5 mM EDTA, 0.5 M NaCl, 1 mM Gly-Gly, 50 mM 2-ME, 1 M Urea at pH 7.0), Buffer-3 (25 mM Tris, 1 mM EDTA, 1 mM Gly-Gly, 50 mM 2-ME at pH 7.0). Protease was solubilized in 25 mM Tris, 1 mM EDTA, 5 mM NaCl, 1 mM Gly-Gly, 50 mM 2-ME, 9 M urea, pH 9.0, clarified by centrifugation, and applied directly to an anion exchange Q-Sepharose column (Q-Sepharose HP, Pharmacia) previously equilibrated with the same buffer. The protease was passed through the column and then acidified by adding formic acid to 25 mM immediately upon elution from the column. Precipitation of a significant amount of contaminants occurred upon acidification. Protease-containing fractions were pooled, concentrated, and stored at 4°C at 5–10 mg/mL.
The HIV-1 protease was folded by 10-fold stepwise dilution into 10 mM formic acid at 0°C. The pH was gradually increased to 3.8, then the temperature was raised to 30°C. Sodium acetate at pH 5.0 was added up to 100 mM, and protein was concentrated. Folded protease was desalted into 1 mM sodium acetate at pH 5.0 using a gel filtration column (PD-10, Pharmacia) and stored at either 4°C or -20°C (2.5 mg/mL) without loss of activity in several weeks. After folding, the protease was estimated to be 99% pure.
Determination of kinetic parameters
The catalytic activities of the HIV-1 proteases were tested prior to thermodynamic analysis by following the hydrolysis of the chromogenic substrate Lys-Ala-Arg-Val-Nle-nPhe-Glu-Ala-Nle-NH2, where Nle stands for norleucine and nPhe stands for p-nitrophenylalanine (California Peptide Research, Inc.).
In the spectrophotometric assay, protease was added to a 120-µL microcuvette containing substrate at 25°C. Final concentrations in the standard assay were: 30 nM to 60 nM active protease, 0 µM to 170 µM substrate, 10 mM sodium acetate, and 1 M sodium chloride, pH 5.0. The absorbance was monitored at six wavelengths (296–304 nm) using an HP 8452 diode array spectrophotometer (Hewlett Packard) and corrected for spectrophotometer drift by subtracting the average absorbance at 446–454 nm. An extinction coefficient for the difference in absorbance upon hydrolysis (1800 M-1 cm-1 at 300 nm) was used to convert absorbance change to reaction rates. Hydrolysis rates were obtained from the initial portion of the data, where at least 80% of the substrate remains free. The concentration of active protease was determined by performing active-site titrations with KNI-272, a very potent inhibitor (at pH 5.0, Ki 16 pM), using protease concentrations much higher (2 µM) than the corresponding Ki. The wild-type protease used in this work was characterized by kcat = 8.1 ± 0.2 sec-1, Km = 14 ± 1 µM; the V82F/I84V mutant by kcat = 6.4 ± 0.1 sec-1, Km = 28 ± 1 µM; and the I50V mutant by kcat = 6.4 ± 0.2 sec-1 and Km = 83 ± 2 µM under the same conditions.
Isothermal titration calorimetry
Isothermal titration calorimetry experiments were performed using a high-precision VP-ITC titration calorimetric system (Microcal Inc.). The enzyme solution in the calorimetric cell was titrated with Amprenavir, TMC-126, or acetyl pepstatin (Bachem AG) dissolved in the same buffer. The inhibitor concentration was estimated by nitrogen content determination (acetyl pepstatin) or from stoichiometric determination with a standardized protease solution. The heat evolved after each inhibitor injection was obtained from the integral of the calorimetric signal. The heat from the binding reaction between the inhibitor and enzyme was obtained as the difference between the heat of reaction and the corresponding heat of dilution. The binding affinities of Amprenavir and TMC-126 were determined by using ITC displacement experiments. Acetyl pepstatin was selected as the weak inhibitor in the displacement titrations because this inhibitor is endothermic and amplifies the signal of a high-affinity exothermic inhibitor when displaced. The measured binding enthalpies and binding affinities of acetyl pepstatin for the different HIV-1 proteases used in this paper were: wild-type: H = 8.0 kcal/mole, Ka = 2.3 x 106 M-1; I50V mutant: H = 11.1 kcal/mole, Ka = 6.9 x 104 M-1; V82F/I84V mutant: H = 5.2 kcal/mole, Ka = 1.3 x 107 M-1. The extension and contribution of protonation/deprotonation processes to the binding was assessed by measuring the binding enthalpy in buffers with different enthalpies of ionization, that is, acetate, MES, and ACES as described before (Velazquez-Campoy et al. 2000). Analysis of the data was performed using software developed in this laboratory.
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Acknowledgments |
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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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