A mechanistic and structural investigation of modified derivatives of the diaryltriazine class of NNRTIs targeting HIV-1 reverse transcriptase

https://doi.org/10.1016/j.bbagen.2014.04.001Get rights and content

Highlights

  • Novel DATA NNRTI with improved solubility is a potent inhibitor of HIV RT.

  • The novel morpholinopropoxy substituent extends into the RT/solvent interface.

  • Derivatives adopt differential binding modes in the NNBP to inhibit polymerization.

  • Implications of the altered binding modes observed for novel DATAs are described.

Abstract

Background

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are vital in treating HIV-1 infection by inhibiting reverse transcriptase (RT). Drug toxicity and resistance drive the need for effective new inhibitors with improved physiochemical properties and potent antiviral activity. Computer-aided and structure-based drug design have guided the addition of solubilizing substituents to the diaryltriazine scaffold. These derivatives have markedly improved solubility and maintain low nanomolar antiviral activity against RT. The molecular and structural basis of inhibition for this series was determined to facilitate future inhibitor development with improved pharmacological profiles.

Methods

The molecular mechanism of inhibition was investigated using transient-state kinetic analysis. Crystal structures of RT in complex with each inhibitor were obtained to investigate the structural basis of inhibition.

Results

The diaryltriazine and its morpholine derivative have RT inhibition constants of 9 ± 2 nM and 14 ± 4 nM, respectively. They adopt differential binding modes within the non-nucleoside inhibitor binding pocket to distort the catalytic site geometry and primer grip regions. The novel morpholinopropoxy substituent extends into the RT/solvent interface of the NNIBP.

Conclusions

Kinetic and structural analyses show that these inhibitors behave as conventional NNRTIs and inhibit the polymerization step. This study confirms that appending solubilizing substituents on the azine ring of diaryltriazine class of NNRTIs that extend into the RT/solvent interface effectively maintains low nanomolar potency and improves physiochemical properties.

General significance

The modification of NNRTI scaffolds with solubilizing substituents, which extend into the RT/solvent interface, yields potent antivirals and is an effective strategy for developing novel inhibitors with improved pharmacological properties.

Introduction

An estimated 35 million people worldwide are infected with HIV-1. The administration of highly active antiretroviral therapy (HAART), a combination of inhibitors targeting essential viral enzymes of the HIV-1 life cycle, has improved patients' quality of life. Key among the enzymes inhibited by HAART is HIV-1 reverse transcriptase (RT), a viral polymerase that transforms single-stranded RNA into double-stranded DNA that is subsequently integrated into the host genome [1]. The error prone nature of RT [2] along with inhibitor side effects and dosing regimens stemming from poor physiochemical properties [3], [4] necessitate the development of novel antiretroviral inhibitors with improved resistance and pharmacological profiles to combat this disease.

To this end, our previous efforts utilized computer-aided and structure-based rational drug design to develop non-nucleoside reverse transcriptase inhibitors (NNRTIs) with both potent antiviral activity and improved physiochemical properties [5], [6]. A key physiochemical property for an effective orally administered drug is aqueous solubility. The second-generation FDA-approved diarylpyrimidine (DAPY) NNRTIs etravirine and rilpivirine, with reported aqueous solubility of ≪ 1 [7] and 0.02–0.24 μg/ml [8], [9], respectively, have poor solubility, limiting the ease of formulation and bioavailability [7]. Given the poor solubility of approved DAPY NNRTIs, our drug design efforts focused on the addition of solubilizing groups to a structurally related class of NNRTIs, the diaryltriazines (DATAs). The impact of structural modifications of DATAs on antiviral activity and physiological properties has been previously examined but a detailed molecular and structural investigation is lacking [10]. Our previous work showed the addition of a solubilizing morpholinopropoxy substituent yielded a novel, structurally diverse DATA compound with 63- to 700-fold greater aqueous solubility than the parent DATA inhibitor and both FDA-approved NNRTIs etravirine and rilpivirine [6] (Fig. 1).

To understand how the morpholinopropoxy substituent significantly improved solubility while maintaining nanomolar levels of potency against wild-type (WT) HIV-1 [6], we sought to understand the molecular mechanism and structural basis of inhibition of RT by compound 1 and its morpholinopropoxy derivative, compound 2. We evaluated the mechanism of inhibition by transient kinetic analysis using pre-steady state burst experiments to examine single nucleotide incorporation. These studies illustrate the value of this approach for measuring the inhibition constant (Ki) of low nanomolar small molecule inhibitors of RT, a significant challenge arising from limits in assay sensitivity and stoichiometric binding. Transient kinetic analysis indicates that compounds 1 and 2 bind RT with high affinity and behave as conventional NNRTIs by inhibiting the polymerization reaction. Comparative structural analysis reveals that compounds 1 and 2 have different binding modes, with both inhibiting RT by preventing formation of a catalytically competent complex. Extension of the structural analysis provides possible implications on how resistance mutations within the non-nucleoside inhibitor binding pocket (NNIBP) may impact the antiviral activity of these inhibitors.

Section snippets

Purification of HIV-1 RT for transient-state analysis

Recombinant HIV-1 RT (p66/p51 heterodimer), a clone kindly provided by Stephen Hughes, Paul Boyer, and Andrea Ferris (Frederick Cancer Research and Development Center, MD), was expressed in E. coli BL21(DE3) pLysS cells and purified as described previously [11]. RT concentration was estimated by UV absorbance at 280 nm using an extinction coefficient of 260,450 M 1 cm 1 as previously described [12]. RT purity as judged by SDS-PAGE analysis with Coomassie staining was > 90%. RT active site

Affinity and molecular mechanism of inhibition of 1 and 2 for HIV-1 RT

The mechanism by which compound 1 and compound 2 inhibit HIV-1 RT was investigated using pre-steady-state kinetic analysis and the inhibition constant (Ki) of each inhibitor was determined. Under burst conditions we examined the effect of each inhibitor on the burst amplitude observed from single nucleotide incorporation reactions catalyzed by RT. RT and the 5′-32P-labeled-D21/D36 were pre-incubated with increasing concentrations of inhibitor and then rapidly mixed with MgCl2 and the next

Conclusions

Current antiviral therapies targeting HIV-1 RT are limited by poor physiochemical properties that limit dosing regimens and bioavailability. Thus there is a need to develop inhibitors with improved physiochemical properties that maintain potent antiviral activity. To this end, we previously reported significant enhancements in aqueous solubility for the DATA class of NNRTIs by appending solubilizing substituents to the azine scaffold. Importantly, this modification did not impact the antiviral

Acknowledgements

Gratitude is expressed to the National Institutes of Health (GM49551, AI44616, GM32136) for research support, (GM007324) for training support of ACM, and (AI104334) for fellowship support of KMF. We also thank the National Synchrotron Light Source at Brookhaven National Laboratory for beam time on X29A and training in their RapiData 2013 course.

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