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Thermodynamic linkage between the binding of protons and inhibitors to HIV-1 protease.

ABSTRACT: The aspartyl dyad of free HIV-1 protease has apparent pK(a)s of approximately 3 and approximately 6, but recent NMR studies indicate that the aspartyl dyad is fixed in the doubly protonated form over a wide pH range when cyclic urea inhibitors are bound, and in the monoprotonated form when the inhibitor KNI-272 is bound. We present computations and measurements related to these changes in protonation and to the thermodynamic linkage between protonation and inhibition. The Poisson-Boltzmann model of electrostatics is used to compute the apparent pK(a)s of the aspartyl dyad in the free enzyme and in complexes with four different inhibitors. The calculations are done with two parameter sets. One assigns epsilon = 4 to the solute interior and uses a detailed model of ionization; the other uses epsilon = 20 for the solute interior and a simplified representation of ionization. For the free enzyme, both parameter sets agree well with previously measured apparent pK(a)s of approximately 3 and approximately 6. However, the calculations with an internal dielectric constant of 4 reproduce the large pKa shifts upon binding of inhibitors, but the calculations with an internal dielectric constant of 20 do not. This observation has implications for the accurate calculation of pK(a)s in complex protein environments. Because binding of a cyclic urea inhibitor shifts the pK(a)s of the aspartyl dyad, changing the pH is expected to change its apparent binding affinity. However, we find experimentally that the affinity is independent of pH from 5.5 to 7.0. Possible explanations for this discrepancy are discussed.

SUBMITTER: Trylska J

PROVIDER: S-EPMC2144115 | biostudies-other | 1999 Jan

REPOSITORIES: biostudies-other

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Thermodynamic linkage between the binding of protons and inhibitors to HIV-1 protease.

Trylska J J Antosiewicz J J Geller M M Hodge C N CN Klabe R M RM Head M S MS Gilson M K MK

Protein science : a publication of the Protein Society 19990101 1

The aspartyl dyad of free HIV-1 protease has apparent pK(a)s of approximately 3 and approximately 6, but recent NMR studies indicate that the aspartyl dyad is fixed in the doubly protonated form over a wide pH range when cyclic urea inhibitors are bound, and in the monoprotonated form when the inhibitor KNI-272 is bound. We present computations and measurements related to these changes in protonation and to the thermodynamic linkage between protonation and inhibition. The Poisson-Boltzmann model ...[more]

PMID: 10210196

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Project description:KNI-272 is a powerful HIV-1 protease inhibitor with a reported inhibition constant in the picomolar range. In this paper, a complete experimental dissection of the thermodynamic forces that define the binding affinity of this inhibitor to the wild-type and drug-resistant mutant V82F/184V is presented. Unlike other protease inhibitors, KNI-272 binds to the protease with a favorable binding enthalpy. The origin of the favorable binding enthalpy has been traced to the coupling of the binding reaction to the burial of six water molecules. These bound water molecules, previously identified by NMR studies, optimize the atomic packing at the inhibitor/protein interface enhancing van der Waals and other favorable interactions. These interactions offset the unfavorable enthalpy usually associated with the binding of hydrophobic molecules. The association constant to the drug resistant mutant is 100-500 times weaker. The decrease in binding affinity corresponds to an increase in the Gibbs energy of binding of 3-3.5 kcal/mol, which originates from less favorable enthalpy (1.7 kcal/mol more positive) and entropy changes. Calorimetric binding experiments performed as a function of pH and utilizing buffers with different ionization enthalpies have permitted the dissection of proton linkage effects. According to these experiments, the binding of the inhibitor is linked to the protonation/deprotonation of two groups. In the uncomplexed form these groups have pKs of 6.0 and 4.8, and become 6.6 and 2.9 in the complex. These groups have been identified as one of the aspartates in the catalytic aspartyl dyad in the protease and the isoquinoline nitrogen in the inhibitor molecule. The binding affinity is maximal between pH 5 and pH 6. At those pH values the affinity is close to 6 x 10(10) M(-1) (Kd = 16 pM). Global analysis of the data yield a buffer- and pH-independent binding enthalpy of -6.3 kcal/mol. Under conditions in which the exchange of protons is zero, the Gibbs energy of binding is -14.7 kcal/mol from which a binding entropy of 28 cal/K mol is obtained. Thus, the binding of KNI-272 is both enthalpically and entropically favorable. The structure-based thermodynamic analysis indicates that the allophenylnorstatine nucleus of KNI-272 provides an important scaffold for the design of inhibitors that are less susceptible to resistant mutations.

Project description:The use of molecular simulation to estimate the strength of macromolecular binding free energies is becoming increasingly widespread, with goals ranging from lead optimization and enrichment in drug discovery to personalizing or stratifying treatment regimes. In order to realize the potential of such approaches to predict new results, not merely to explain previous experimental findings, it is necessary that the methods used are reliable and accurate, and that their limitations are thoroughly understood. However, the computational cost of atomistic simulation techniques such as molecular dynamics (MD) has meant that until recently little work has focused on validating and verifying the available free energy methodologies, with the consequence that many of the results published in the literature are not reproducible. Here, we present a detailed analysis of two of the most popular approximate methods for calculating binding free energies from molecular simulations, molecular mechanics Poisson-Boltzmann surface area (MMPBSA) and molecular mechanics generalized Born surface area (MMGBSA), applied to the nine FDA-approved HIV-1 protease inhibitors. Our results show that the values obtained from replica simulations of the same protease-drug complex, differing only in initially assigned atom velocities, can vary by as much as 10 kcal mol(-1), which is greater than the difference between the best and worst binding inhibitors under investigation. Despite this, analysis of ensembles of simulations producing 50 trajectories of 4 ns duration leads to well converged free energy estimates. For seven inhibitors, we find that with correctly converged normal mode estimates of the configurational entropy, we can correctly distinguish inhibitors in agreement with experimental data for both the MMPBSA and MMGBSA methods and thus have the ability to rank the efficacy of binding of this selection of drugs to the protease (no account is made for free energy penalties associated with protein distortion leading to the over estimation of the binding strength of the two largest inhibitors ritonavir and atazanavir). We obtain improved rankings and estimates of the relative binding strengths of the drugs by using a novel combination of MMPBSA/MMGBSA with normal mode entropy estimates and the free energy of association calculated directly from simulation trajectories. Our work provides a thorough assessment of what is required to produce converged and hence reliable free energies for protein-ligand binding.

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Thermodynamic linkage between the binding of protons and inhibitors to HIV-1 protease.

Publications

Thermodynamic linkage between the binding of protons and inhibitors to HIV-1 protease.

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