Determinants of Active-Site Inhibitor Interaction with HIV-1 RNase H.

The ribonuclease H (RNH) activity of HIV-1 reverse transcriptase (RT) is essential for viral replication and can be a target for drug development. Yet, no RNH inhibitor to date has substantial antiviral activity to allow advancement into clinical development. Herein, we describe our characterization of the detailed binding mechanisms of RNH active-site inhibitors, YLC2-155 and ZW566, that bind to the RNH domain through divalent metal ions, using NMR, molecular docking, and quantum mechanical calculations. In the presence of Mg2+, NMR spectra of RNH exhibited split (two) resonances for some residues upon inhibitor binding, suggesting two binding modes, an observation consistent with the docking results. The relative populations of the two binding conformers were independent of inhibitor or Mg2+ concentration, with one conformation consistently more favored. In our docking study, one distinctive pose of ZW566 showed more interactions with surrounding residues of RNH compared to the analogous binding pose of YLC2-155. Inhibitor titration experiments revealed a lower dissociation constant for ZW566 compared to YLC2-155, in agreement with its higher inhibitory activity. Mg2+ titration data also indicated a stronger dependence on Mg2+ for the RNH interaction with ZW566 compared to YLC2-155. Combined docking and quantum mechanical calculation results suggest that stronger metal coordination as well as more protein-inhibitor interactions may account for the higher binding affinity of ZW566. These findings support the idea that strategies for the development of potent competitive active site RNH inhibitors should take into account not only metal-inhibitor coordination but also protein-inhibitor interaction and conformational selectivity.

UV resonance Raman investigation of the aqueous solvation dependence of primary amide vibrations.

We investigated the normal mode composition and the aqueous solvation dependence of the primary amide vibrations of propanamide. Infrared, normal Raman, and UV resonance Raman (UVRR) spectroscopy were applied in conjunction with density functional theory (DFT) to assign the vibrations of crystalline propanamide. We examined the aqueous solvation dependence of the primary amide UVRR bands by measuring spectra in different acetonitrile/water mixtures. As previously observed in the UVRR spectra of N-methylacetamide, all of the resonance enhanced primary amide bands, except for the Amide I (AmI), show increased UVRR cross sections as the solvent becomes water-rich. These spectral trends are rationalized by a model wherein the hydrogen bonding and the high dielectric constant of water stabilizes the ground state dipolar (-)O-C═NH2(+) resonance structure over the neutral O═C-NH2 resonance structure. Thus, vibrations with large C-N stretching show increased UVRR cross sections because the C-N displacement between the electronic ground and excited state increases along the C-N bond. In contrast, vibrations dominated by C═O stretching, such as the AmI, show a decreased displacement between the electronic ground and excited state, which result in a decreased UVRR cross section upon aqueous solvation. The UVRR primary amide vibrations can be used as sensitive spectroscopic markers to study the local dielectric constant and hydrogen bonding environments of the primary amide side chains of glutamine (Gln) and asparagine (Asn).

Structure of a dihydroxycoumarin active-site inhibitor in complex with the RNase H domain of HIV-1 reverse transcriptase and structure-activity analysis of inhibitor analogs.

Human immunodeficiency virus (HIV) encodes four essential enzymes: protease, integrase, reverse transcriptase (RT)-associated DNA polymerase, and RT-associated ribonuclease H (RNase H). Current clinically approved anti-AIDS drugs target all HIV enzymatic activities except RNase H, which has proven to be a very difficult target for HIV drug discovery. Our high-throughput screening activities identified the dihydroxycoumarin compound F3284-8495 as a specific inhibitor of RT RNase H, with low micromolar potency in vitro. Optimization of inhibitory potency can be facilitated by structural information about inhibitor-target binding. Here, we report the crystal structure of F3284-8495 bound to the active site of an isolated RNase H domain of HIV-1 RT at a resolution limit of 1.71Å. From predictions based on this structure, compounds were obtained that showed improved inhibitory activity. Computational analysis suggested structural alterations that could provide additional interactions with RT and thus improve inhibitory potency. These studies established proof of concept that F3284-8495 could be used as a favorable chemical scaffold for development of HIV RNase H inhibitors.

Clicking 3'-azidothymidine into novel potent inhibitors of human immunodeficiency virus.

3'-Azidothymidine (AZT) was the first approved antiviral for the treatment of human immunodeficiency virus (HIV). Reported efforts in clicking the 3'-azido group of AZT have not yielded 1,2,3-triazoles active against HIV or any other viruses. We report herein the first AZT-derived 1,2,3-triazoles with submicromolar potencies against HIV-1. The observed antiviral activities from the cytopathic effect (CPE) based assay were confirmed through a single replication cycle assay. Structure-activity-relationship (SAR) studies revealed two structural features key to antiviral activity: a bulky aromatic ring and the 1,5-substitution pattern on the triazole. Biochemical analysis of the corresponding triphosphates showed lower ATP-mediated nucleotide excision efficiency compared to AZT, which along with molecular modeling suggests a mechanism of preferred translocation of triazoles into the P-site of HIV reverse transcriptase (RT). This mechanism is corroborated with the observed reduction of fold resistance of the triazole analogue to an AZT-resistant HIV variant (9-fold compared to 56-fold with AZT).

Structural basis of the allosteric inhibitor interaction on the HIV-1 reverse transcriptase RNase H domain.

HIV-1 reverse transcriptase (RT) has been an attractive target for the development of antiretroviral agents. Although this enzyme is bi-functional, having both DNA polymerase and ribonuclease H (RNH) activities, there is no clinically approved inhibitor of the RNH activity. Here, we characterize the structural basis and molecular interaction of an allosteric site inhibitor, BHMP07, with the wild-type (WT) RNH fragment. Solution NMR experiments for inhibitor titration on WT RNH showed relatively wide chemical shift perturbations, suggesting a long-range conformational effect on the inhibitor interaction. Comparisons of the inhibitor-induced NMR chemical shift changes of RNH with those of RNH dimer, in the presence and absence of Mg(2+) , were performed to determine and verify the interaction site. The NMR results, with assistance of molecular docking, indicate that BHMP07 preferentially binds to a site that is located between the RNH active site and the region encompassing helices B and D (the 'substrate-handle region'). The interaction site is consistent with the previous proposed site, identified using a chimeric RNH (p15-EC) [Gong et al. (2011) Chem Biol Drug Des 77, 39-47], but with slight differences that reflect the characteristics of the amino acid sequences in p15-EC compared to the WT RNH.

Direct observations of conformational distributions of intrinsically disordered p53 peptides using UV Raman and explicit solvent simulations.

We report the first experimental measurements of Ramachandran Ψ-angle distributions for intrinsically disordered peptides: the N-terminal peptide fragment of tumor suppressor p53 and its P27S mutant form. To provide atomically detailed views of the conformational distributions, we performed classical, explicit-solvent molecular dynamics simulations on the microsecond time scale. Upon binding its partner protein, MDM2, wild-type p53 peptide adopts an α-helical conformation. Mutation of Pro27 to serine results in the highest affinity yet observed for MDM2-binding of the p53 peptide. Both UV resonance Raman spectroscopy (UVRR) and simulations reveal that the P27S mutation decreases the extent of PPII helical content and increases the probability for conformations that are similar to the α-helical MDM2-bound conformation. In addition, UVRR measurements were performed on peptides that were isotopically labeled at the Leu26 residue preceding the Pro27 in order to determine the conformational distributions of Leu26 in the wild-type and mutant peptides. The UVRR and simulation results are in quantitative agreement in terms of the change in the population of non-PPII conformations involving Leu26 upon mutation of Pro27 to serine. Finally, our simulations reveal that the MDM2-bound conformation of the peptide is significantly populated in both the wild-type and mutant isolated peptide ensembles in their unbound states, suggesting that MDM2 binding of the p53 peptides may involve conformational selection.

Dependence of the AmII'p proline Raman band on peptide conformation.

We utilized UV resonance Raman (UVRR) measurements and density functional theory (DFT) calculations to relate the AmII'p frequency to the psi angle. The AmII'p frequency shifts by approximately 25 cm(-1) as the psi angle is varied over allowed angles of the Pro peptide bond. The AmII'p frequency does not show any significant dependence on the phi dihedral angle. The conformation sensitivity of the AmII'p frequency derives from conformation-induced changes in the planarity of the Pro peptide bond; psi angle changes push the amide nitrogen out of the peptide bond plane. We use this AmII'p frequency dependence on the psi angle to track temperature-induced conformation changes in a polyproline peptide. The temperature-induced 7 cm(-1) downshift in the AmII'p frequency of the polyproline peptide results from an approximately 45 degrees rotation of the psi dihedral angle from psi = 145 degrees (ideal PPII conformation) to psi = 100 degrees (collapsed PPII conformation).

Dependence of amide vibrations on hydrogen bonding.

The effect of hydrogen bonding on the amide group vibrational spectra has traditionally been rationalized by invoking a resonance model where hydrogen bonding impacts the amide functional group by stabilizing its [(-)O-C=NH (+)] structure over the [O=C-NH] structure. However, Triggs and Valentini's UV-Raman study of solvation and hydrogen bonding effects on epsilon-caprolactum, N, N-dimethylacetamide (DMA), and N-methylacetamide (NMA) ( Triggs, N. E.; Valentini, J. J. J. Phys. Chem. 1992, 96, 6922-6931) casts doubt on the validity of this model by demonstrating that, contrary to the resonance model prediction, carbonyl hydrogen bonding does not impact the AmII' frequency of DMA. In this study, we utilize density functional theory (DFT) calculations to examine the impact of hydrogen bonding on the C=O and N-H functional groups of NMA, which is typically used as a simple model of the peptide bond. Our calculations indicate that, as expected, the hydrogen bonding frequency dependence of the AmI vibration predominantly derives from the C=O group, whereas the hydrogen bonding frequency dependence of the AmII vibration primarily derives from N-H hydrogen bonding. In contrast, the hydrogen bonding dependence of the conformation-sensitive AmIII band derives equally from both C=O and N-H groups and thus, is equally responsive to hydrogen bonding at the C=O or N-H site. Our work shows that a clear understanding of the normal mode composition of the amide vibrations is crucial for an accurate interpretation of the hydrogen bonding dependence of amide vibrational frequencies.

Dependence of glycine CH2 stretching frequencies on conformation, ionization state, and hydrogen bonding.

We experimentally and theoretically examined the conformation, pH, and temperature dependence of the CH2 stretching frequencies of glycine (gly) in solution and in the crystalline state. To separate the effects of the amine and carboxyl groups on the CH2 stretching frequencies we examined the Raman spectra of 2,2,2-d3-ethylamine (CD3-CH2-NH2) and 3,3,3-d3-propionic acid (CD3-CH2-COOH) in D2O. The symmetric (nusCH2) and asymmetric (nuasCH2) stretching frequencies show a significant dependence on gly conformation. We quantified the relation between the frequency splitting (Delta = nuasCH2-nusCH2) and the xi angle which determines the gly conformational geometry. This relation allows us to determine the conformation of gly directly from the Raman spectral frequencies. We observe a large dependence of the nusCH2 and nuasCH2 frequencies on the ionization state of the amine group, which we demonstrate theoretically results from a negative hyperconjugation between the nitrogen lone pair and the C-H antibonding orbitals. The magnitude of this effect is maximized for C-H bonds trans to the nitrogen lone pair. In contrast, a small dependence of the CH2 stretching frequencies on the carboxyl group ionization state arises from delocalization of electron density from carboxyl oxygen to C-H bonding orbitals. According to our experimental observations and theoretical calculations the temperature dependence of the nusCH2 and nuasCH2 of gly is due to the change in the hydrogen-bonding strength of the amine/carboxyl groups to water.

Peptide bond vibrational coupling.

Neutral trialanine (Ala3), which is geometrically constrained to have its peptide bond at Phi and Psi angles of alpha-helix and PPII-like conformers, are studied at the B3LYP/6-31+G(d,p) level of theory to examine vibrational interactions between adjacent peptide units. Delocalization of the amide I, amide II, and amide III3 vibrations are analyzed by calculating their potential energy distributions (PED). The vibrational coupling strengths are estimated from the frequency shifts between the amide vibrations of Ala3 and the local amide bond vibrations of isotopically substituted Ala3 derivatives. Our calculations show the absence of vibrational coupling of the amide I and amide II bands in the PPII conformations. In contrast, the alpha-helical conformation shows strong coupling between the amide I vibrations due to the favorable orientation of the C=O bonds and the strong transitional dipole coupling. The amide III3 vibration shows weak coupling in both the alpha-helix and PPII conformations; this band can be treated as a local independent vibration. Our calculated results in general agree with our previous experimental UV Raman studies of a 21-residue mainly alanine-based peptide (AP).

Peptide secondary structure folding reaction coordinate: correlation between uv raman amide III frequency, Psi Ramachandran angle, and hydrogen bonding.

We used UV resonance Raman (UVRR) spectroscopy to quantitatively correlate the peptide bond AmIII3 frequency to its Psi Ramachandran angle and to the number and types of amide hydrogen bonds at different temperatures. This information allows us to develop a family of relationships to directly estimate the Psi Ramachandran angle from measured UVRR AmIII3 frequencies for peptide bonds (PBs) with known hydrogen bonding (HB). These relationships ignore the more modest Phi Ramachandran angle dependence and allow determination of the Psi angle with a standard error of +/-8 degrees , if the HB state of a PB is known. This is normally the case if a known secondary structure motif is studied. Further, if the HB state of a PB in water is unknown, the extreme alterations in such a state could additionally bias the Psi angle by +/-6 degrees . The resulting ability to measure Psi spectroscopically will enable new incisive protein conformational studies, especially in the field of protein folding. This is because any attempt to understand reaction mechanisms requires elucidation of the relevant reaction coordinate(s). The Psi angle is precisely the reaction coordinate that determines secondary structure changes. As shown elsewhere (Mikhonin et al. J. Am. Chem. Soc. 2005, 127, 7712), this correlation can be used to determine portions of the energy landscape along the Psi reaction coordinate.

UV resonance Raman determination of polyproline II, extended 2.5(1)-helix, and beta-sheet Psi angle energy landscape in poly-L-lysine and poly-L-glutamic acid.

UV resonance Raman (UVR) spectroscopy was used to examine the solution conformation of poly-l-lysine (PLL) and poly-l-glutamic acid (PGA) in their non-alpha-helical states. UVR measurements indicate that PLL (at pH = 2) and PGA (at pH = 9) exist mainly in a mixture of polyproline II (PPII) and a novel left-handed 2.5(1)-helical conformation, which is an extended beta-strand-like conformation with Psi approximately +170 degrees and Phi approximately -130 degrees . Both of these conformations are highly exposed to water. The energies of these conformations are very similar. We see no evidence of any disordered "random coil" states. In addition, we find that a PLL and PGA mixture at neutral pH is approximately 60% beta-sheet and contains PPII and extended 2.5(1)-helix conformations. The beta-sheet conformation shows little evidence of amide backbone hydrogen bonding to water. We also developed a method to estimate the distribution of Psi Ramachandran angles for these conformations, which we used to estimate a Psi Ramachandran angle energy landscape. We believe that these are the first experimental studies to give direct information on protein and peptide energy landscapes.