Multidrug Resistance to HIV-1 Protease Inhibition Requires Cooperative Coupling between Distal Mutations †

ABSTRACT: The appearance of viral strains that are resistant to protease inhibitors is one of the most serious problems in the chemotherapy of HIV-1/AIDS. The most pervasive drug-resistant mutants are those that affect all inhibitors in clinical use. In this paper, we have characterized a multiple-drug-resistant mutant of the HIV-1 protease that affects indinavir, nelfinavir, saquinavir, ritonavir, amprenavir, and lopinavir. This mutant (MDR-HM) contains six amino acid mutations (L10I/M46I/I54V/V82A/I84V/ L90M) located within and outside the active site of the enzyme. Microcalorimetric and enzyme kinetic measurements indicate that this mutant lowers the affinity of all inhibitors by 2-3 orders of magnitude. By comparison, the multiiple-drug-resistant mutant only increased the Km of the substrate by a factor of 2, indicating that the substrate is able to adapt to the changes caused by the mutations and maintain its binding affinity. To understand the origin of resistance, three submutants containing mutations in specific regions were also studied, i.e., the active site (V82A/I84V), flap region (M46I/I54V), and dimerization region (L10I/L90M). None of these sets of mutations by themselves lowered the affinity of inhibitors by more than 1 order of magnitude, and additionally, the sum of the effects of each set of mutations did not add up to the overall effect, indicating the presence of cooperative effects. A mutant containing only the four active site mutations (V82A/I84V/M46I/I54V) only showed a small cooperative effect, suggestin

Diffusion-Facilitated Direct Determination of Intrinsic Parameters for Rapid Photoinduced Bimolecular Electron-Transfer Reactions in Nonpolar Solvents

Bimolecular fluorescence-quenching reactions involving electron-transfer between electronically excited 5,10,15,20-tetraphenyl-21<i>H</i>,23<i>H</i>-porphine (TPP*) and 1,4-benzoquinone (BQ) or 1,4-naphthoquinone (NQ) were investigated using a set of alkane solvents that enabled the rapid reaction kinetics to be probed over a wide viscosity range, while minimizing changes in other relevant solvent parameters. Relative diffusion coefficients and reaction distances were recovered directly from analysis of fluorescence decay curves measured on a nanosecond time scale. The electron transfer from TPP* to BQ requires reactant contact, consistent with tightly associated exciplex formation in these nonpolar solvents. In contrast, electron transfer from TPP* to NQ displays a clear distance dependence, indicative of reaction via a much looser noncontact exciplex. This difference is attributed to the greater steric hindrance associated with contact between the TPP*/NQ pair. The diffusion coefficients recovered from fluorescence decay curve analysis are markedly smaller than the corresponding measured bulk relative diffusion coefficients. Classical hydrodynamics theory was found to provide a satisfactory resolution of this apparent discrepancy. The calculated hydrodynamic radii of TPP and NQ correlate very well with the van der Waals values. The hydrodynamic radius obtained for BQ is a factor of 6 times smaller than the van der Waals value, indicative of a possible tight cofacial geometry in the (TPP⁺/BQ^–)* exciplex. The present work demonstrates the utility of a straightforward methodology, based on widely available instrumentation and data analysis, that is broadly applicable for direct determination of kinetic parameter values for a wide variety of rapid bimolecular fluorescence quenching reactions in fluid solution

Effect of Vesicle Size on the Cytolysis of Cell-Penetrating Peptides (CPPs)

A specific series of peptides, called a cell-penetrating peptide (CPP), is known to be free to directly permeate through cell membranes into the cytosol (cytolysis); hence, this CPP would be a potent carrier for a drug delivery system (DDS). Previously, we proposed the mechanism of cytolysis as a temporal and local phase transfer of membrane lipid caused by positive membrane curvature generation. Moreover, we showed how to control the CPP cytolysis. Here, we investigate the phospholipid vesicle&rsquo;s size effect on CPP cytolysis because this is the most straightforward way to control membrane curvature. Contrary to our expectation, we found that the smaller the vesicle diameter (meaning a higher membrane curvature), the more cytolysis was suppressed. Such controversial findings led us to seek the reason for the unexpected results, and we ended up finding out that the mobility of membrane lipids as a liquid crystal is the key to cytolysis. As a result, we could explain the cause of cytolysis suppression by reducing the vesicle size (because of the restriction of lipid mobility); osmotic pressure reduction to enhance positive curvature generation works as long as the membrane is mobile enough to modulate the local structure. Taking all the revealed vital factors and their effects as a tool, we will further explore how to control CPP cytolysis for developing a DDS system combined with appropriate cargo selection to be tagged with CPPs