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. Author manuscript; available in PMC: 2020 Aug 15.
Published in final edited form as: Bioorg Med Chem Lett. 2019 Jun 26;29(16):2182–2188. doi: 10.1016/j.bmcl.2019.06.047

Molecular and Cellular Studies Evaluating a Potent 2-Cyanoindolizine Catechol Diether NNRTI Targeting Wildtype and Y181C Mutant HIV-1 Reverse Transcriptase

Tomoaki Sasaki a,*, Zira TK Gannam a,*, Shalley N Kudalkar a, Kathleen M Frey a, Won-Gil Lee b, Krasimir A Spasov a, William L Jorgensen b, Karen S Anderson a,c,†
PMCID: PMC6690785  NIHMSID: NIHMS1039549  PMID: 31281023

Abstract

The development of efficacious NNRTIs for HIV/AIDS therapy is commonly met with the emergence of drug resistant strains, including the Y181C variant. Using a computationally-guided approach, we synthesized the catechol diether series of NNRTIs, which display sub-nanomolar potency in cellular assays. Among the most potent were a series of 2-cyanoindolizine substituted catechol diethers, including Compound 1. We present here a thorough evaluation of this compound, including biochemical, cellular, and structural studies. The compound demonstrates low nanomolar potency against both WT and Y181C HIV-1 RT in in vitro and cellular assays. Our crystal structures of both the wildtype and mutant forms of RT in complex with Compound 1 allow the interrogation of this compound’s features that allow it to maintain strong efficacy against the drug resistant mutant. Among these are compensatory shifts in the NNRTI binding pocket, persistence of multiple hydrogen bonds, and van der Waals contacts throughout the binding site. Further, the fluorine at the C6 position of the indolizine moiety makes multiple favorable interactions with both RT forms. The present study highlights the indolizine-substituted catechol diether class of NNRTIs as promising therapeutic candidates possessing optimal pharmacological properties and significant potency against multiple RT variants.

HIV/AIDS is a widespread epidemic affecting 35 million people globally with approximately 2.3 million newly infected individuals in 2012.[1] Therefore, the development of efficacious and potent HIV therapeutics has become an increasingly important goal over the past few decades. Notably, targeting of the virally encoded HIV-1 reverse transcriptase (RT), the essential polymerase that converts single-stranded RNA to double-stranded DNA, has been an effective drug target for HIV therapy.[2, 3] Two classes of RT inhibitors are FDA approved: nucleoside reverse transcriptase inhibitors (NRTIs), nucleoside analogs that mimic endogenous nucleotides and prevent further extension of the resultant DNA product, and non-nucleoside reverse transcriptase inhibitors (NNRTIs), which bind to an allosteric pocket 10Å away from the active site and inhibit RT activity.[2, 4] Combined administration of NRTIs and NNRTIs, along with drugs targeting other stages of the HIV viral life cycle, is a hallmark of Highly Active Antiretroviral Therapy (HAART), the current gold standard treatment to improve the quality of life of millions of HIV-infected patients worldwide.[2, 3, 5]

Due to the error prone nature of HIV-1 RT,[6] a major challenge in designing NNRTIs is the emergence of drug resistant viral strains.[3, 7] A number of very common drug resistant mutations in HIV-1 RT greatly complicate antiretroviral treatment strategies utilizing RT inhibitors.[2, 3, 8] One specific RT mutation wherein a tyrosine residue in the NNRTI pocket is mutated to a cysteine (Y181C) is among the most prevalent drug resistant mutation, observed to rapidly emerge after administration of NNRTIs including nevirapine, delavirdine, and other first-generation NNRTIs.[7, 9–11] A newer generation of NNRTIs was developed in an effort to successfully target drug resistant forms, including Y181C, which evade inhibition by early generation NNRTIs.[7, 12] However, while later NNRTIs are capable of targeting some of these variant forms of RT, they suffer from poor solubility/bioavailability and have demonstrated issues of toxicity. For example, the second generation NNRTI rilpivirine is soluble only to 0.02 μg/mL and has been shown to target the hERG ion channel, contributing to cardiotoxicity.[13–15] As such, it is imperative to develop further improved inhibitors that evade these mechanisms of drug resistance and toxicity while maintaining potency and bioavailability.

In the pursuit to design effective inhibitors for resistant forms of RT with optimal pharmacological properties, we use a computational chemistry approach to design small molecule compounds that have optimized properties to better accommodate the NNRTI pocket of RT.[16–18] Using a lead optimization strategy that utilizes free energy perturbation (FEP) calculations, we have developed the catechol diether series of NNRTIs that display picomolar potency.[16] Previously, we reported four co-crystal structures of RT bound to these NNRTIs containing a catechol diether scaffold, and we analyzed the intermolecular interactions that help elucidate the potency observed in our cell-based infectivity assays.[19] Our most recent work highlights the adaptation of a heterobicyclic 2-cyanoindolizine group in place of a cyanovinyl group in the catechol diether scaffold to circumvent reactivity concerns.[20] Previously determined cell-based assays suggest low nanomolar` to sub-nanomolar potency for the indolizine-substituted class of catechol diethers against RT, comparable to that of rilpivirine (Figure 1B). The current work focuses on a detailed molecular and cellular evaluation of the difluoro-substituted 2-cyanoindolizine catechol diether, Compound 1, which has been optimized for RT resistant activity (Figure 1A). Our previous assays suggested very potent activity on WT strains of HIV-1 IIIB but less effective on a clinical isolate (HIV-1 N119) containing the nevirapine-resistant Y181C mutation.[20] The current study utilized molecular clones of HIV-1 pNL4–3 containing WT or Y181C mutation.[21]

Figure 1. Non-nucleoside reverse transcriptase inhibitors.

Figure 1.

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(A) Chemical structures of rilpivirine, nevirapine, and Compound 1. Note in Compound 1 the catechol diether scaffold with the heterobicyclic replacement along with the positioning of the two fluorine atoms present in the scaffold. (B) Respective EC50 (inhibitory activity for HIV-1) and CC50 (cytotoxicity) values in MT-2 cell-based assays. NA – Not active (i.e., EC50 > CC50).

* – Reference [20]

† – Reference [13]

In order to determine the relative potencies of Compound 1 against WT RT and Y181C RT, a biochemical inhibition assay was used that utilizes PicoGreen, a fluorescent dye that detects formation of dsDNA, dsRNA, and hybrid RNA-DNA strands, as reported previously (Figure 2A; experimental methods available in Supplementary Data).[22] In order to allow sufficient time for binding, RT was pre-incubated with the NNRTI compound. Reactions were initiated by addition of substrate and co-substrates and subsequently quenched with EDTA. The output expressed in units of RFU is a direct measure of dsDNA product formed and therefore RT activity.

Figure 2. Evaluation of RT inhibition in vitro.

Figure 2.

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(A) Schematic cartoon of the steady state RT kinetic assay utilizing PicoGreen dye. Increasing concentrations of Compound 1 were incubated with 10nM active site concentration of (B) WT RT and (C) Y181C RT, respectively. Each inhibitor concentration was performed in triplicate. IC50 values were determined by fitting to a quadratic inhibition curve

Optimal enzyme concentration and incubation times were empirically determined at 10 nM RT and 30-minute reaction time to yield acceptable signal-to-noise ratios (data not shown). Each inhibitor concentration was performed in triplicate, including a pre-quenched negative control. The RFU value for the negative control was subsequently subtracted from all data points as background signal prior to analysis. Titration of increasing concentrations of Compound 1 yielded lower RFUs, and upon plotting RFUs as a function of inhibitor concentration and fitting the data points to a quadratic function, IC50 values were obtained. To test this system using a well-known inhibitor, we conducted a pilot experiment involving the titration of rilpivirine, yielding an IC50 of 680 ± 240 pM, mirroring its high picomolar potency found in cell-based experiments (Supplemental Figure 1). Consistent with its high potency observed in the cell-based assays, Compound 1 showed a low nanomolar IC50 of 4.5 ± 1.2 nM (Figure 2B). Remarkably, Compound 1 maintains high potency against Y181C RT, with an IC50 value of 5.5 ± 1.1 nM (Figure 2C).

To further confirm the inhibitory activity detected in biochemical assays, we performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based cellular viability assays, a method commonly used to screen the efficacy of anti-HIV compounds (experimental methods available in Supplementary Data).[23] The effect of the anti-HIV compound is assayed in the context of HIV-1 infected MT-2 cells by examining cellular viability 5 days post-infection. Cell viability can be detected with spectrophotometry based on in situ reduction of the MTT dye, which produces a colorimetric output.

We observed a concentration-dependent inhibition of HIV-1 infection for both WT and Y181C mutant RT, and an EC50 of 3.2 nM for Y181C, similar to the WT EC50 of 4.7 nM, was determined (Figure 1B, Supplemental Figure 2). Importantly, the EC50 values from our viral infectivity assay for both WT and Y181C strains were comparable to the IC50 values observed in our in vitro enzymatic assay (Figure 2, Supplemental Figure 2). Effective inhibition of WT and Y181C HIV-infected cells suggests that Compound 1 has potent inhibitory activity against WT and Y181C strains of HIV-1.

In order to determine the structural basis of Compound 1 inhibition of both WT and Y181C RT, the crystal structure of each construct in complex with the inhibitor was solved (PDB: 6DTX and 6DTW, respectively; experimental methods available in Supplementary Data). As with previous co-crystal structures of RT-NNRTI complexes[24], our crystal structures show RT in an open-cleft conformation. Clear electron density was observed at the NNRTI binding pocket, allowing unambiguous determination of the orientation and positioning of the 2-cyanoindolizine catechol diether scaffold of Compound 1 for both the WT RT and Y181C structures (Figure 3). The thumb, palm, and finger subdomains of the p66 subunit were found in a similar conformation as in previously reported RT-NNRTI co-crystal structures. Importantly, superimposition of our two reported structures show that the Y181C mutation in the p66 subunit did not cause large scale conformational changes in the RT fold, and thus, analysis of intermolecular interactions between RT and our compound of interest at the NNRTI binding pocket can be accounted for the difference in potencies observed in our steady state kinetic assays. The root-mean-square deviation (RMSD) values after superimposition of the NNRTI binding pocket of WT RT:Compound 1 and Y181C RT:Compound 1 was 0.765 Å, suggesting that the fold of the three-dimensional structure around the NNRTI binding pocket are well conserved in both structures. Similar to previously determined crystal structures of the catechol diether series containing indolizine rings, the cyano group projects into the tunnel region of the NNRTI binding pocket proximal to F227.[20] In addition, the side chain of K103 extends into the entrance channel of the NNRTI binding pocket, a region also utilized by several other NNRTIs containing morpholinyl substituents.[25]

Figure 3. Electron density map of NNRTI binding site.

Figure 3.

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2mFo-Fc electron density contoured to 1.5 σ for the Y181C RT:Compound 1 complex (PDB: 6DTX). Note the presence of a clear electron density, allowing for unambiguous fitting of the NNRTI in the electron density.

Compound 1 was rationally designed to replace the chemically reactive cyanovinylphenyl group with a bicyclic indolizine ring.[20] This bicyclic system allows for enhanced stacking interactions with other aromatic residues located in the proximity of the NNRTI binding pocket. Specifically, in both structures, clear stacking interactions are observed between Y188 and the five-membered ring of the indolizine moiety (Figure 4). In addition, favorable van der Waals contacts made between the hydrophobic residues and the catechol ring were manifested in previously reported structures.[19, 20, 26] The ring system interacts with many residues in the binding pocket: P95, L100, V108, Y188, and W229. An additional van der Waals interaction with F227 was identified, presumably due to the replacement of the cyanovinylphenyl group with a bicyclic ring.

Figure 4. Overlay of NNRTI binding pockets from WT RT:Compound 1 and Y181C RT:Compound 1 crystal structures.

Figure 4.

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WT RT (cyan; PDB: 6DTW) in complex with Compound 1 (magenta) overlaid with Y181C RT (green; PDB: 6DTX); in complex with Compound 1 (orange). Important binding residues labeled in black.

Additionally, we observe that the two structures differ in the orientation of the ethoxy linker and the uracil ring, such that the uracil ring of the WT RT structure extends further into the groove region of the NNRTI binding pocket flanked by K102 and P236. However, this slight conformational change does not result in loss of hydrogen bonding between the uracil moiety and the NNRTI bonding pocket. Three backbone hydrogen bonds still exist between K103/P236 and the uracil ring for both structures, suggesting that the Y181C mutation does not cause significant loss of interactions between the NNRTI and RT (Figure 5).

Figure 5. Compound 1 maintains hydrogen bonding interactions with Y181C RT.

Figure 5.

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(A) WT RT:Compound 1 (PDB: 6DTW) and Y181C RT:Compound 1 (PDB: 6DTX) crystal structures zooming into the NNRTI binding pocket. Atoms colored as indicated. (B) Summary of hydrogen bonds forming between the uracil ring moiety of Compound 1 and K103/P236 in the NNRTI binding pocket.

Previous structures with WT RT and a catechol diether NNRTI containing a single ring cyanovinylphenyl group showed an important stacking interaction between the catechol diether ring and Y181. The Y181C mutation abrogates this stacking interaction by replacement of an aromatic side chain with a cysteine. Our WT RT:Compound 1 structure shows Y181 in the previously reported “down” conformation (Figure 4).[19, 26] This is in contrast to some other published structures in which Y181 crystallized in the “up” conformation.[24, 27, 28]

Upon examining the overlay of our two structures, we observe that the Y181C mutation slightly shifts the catechol ring closer to the side chain of V179 similar to previously reported crystal structures of catechol diether compounds. [29] The presence of the bulky aromatic residue in wildtype RT allows formation of optimal van der Waal interactions with the catechol and indolizine rings (Figure 6A). Substitution of this tyrosine to cysteine removes this bulky residue preventing packing interactions between this residue and the catechol diether rings. However, in place of the lost tyrosine, we see compensatory van der Waals interactions in our Y181C structure. Thus, the catechol ring moves closer to V179, where the cysteine side chain favorably packs against the valine side chain (Figure 6B). Such a compensatory interaction likely results in conservation of the energetic stability of Compound 1 binding in the NNRTI pocket, and this may explain the similar potencies of Compound 1 against WT and Y181C RT in our steady state RT assays.

Figure 6: Inhibitor packing against proximal residues at the NNRTI binding pocket.

Figure 6:

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Compound 1 and nearby residues in (A) WT RT (PDB: 6DTW) and (B) Y181C RT (PDB: 6DTX) are shown using van der Waals spherical representation. Atoms colored as above.

Comparison of van der Waals sphere visualization of the WT and Y181C structures shows that WT RT packs more tightly around Compound 1 compared to Y181C RT (Supplemental Figure 3). This difference likely permits Compound 1 to have a high degree of conformational freedom in the absence of Y181. Such structural flexibility may allow Compound 1 to adopt and sample a variety of conformations, allowing formation of multiple stable interactions within the NNRTI binding pocket. Further, this flexibility may explain why the Y181C mutation does not necessarily result in a loss of potency with the catechol diether series of NNRTIs. In previous structural studies of earlier generation NNRTIs, conformational flexibility of NNRTIs in the binding pocket has been explored as a means of targeting multiple drug resistant forms of RT. [24] In Das et al., the flexibility offered by TMC278, a diarylpyrimidine NNRTI, was presented as one mechanism by which TMC278 maintains its high potency against wildtype and mutant forms of RT.[24] In a similar sense, in our Y181C RT:Compound 1 crystal structure, the binding pocket offers a higher level of conformational freedom by the NNRTI, possibly explaining how the flexibility of the binding pocket allows improved binding of the NNRTI, leading to the maintenance of high potency in a drug resistant mutant of RT despite the loss of stacking interaction and van der Waals interaction upon mutation of Y181.

Previous efforts in our NNRTI drug design strategy involved the optimization of the halogen substituent on the C6 atom on the central six membered ring.[19] Upon substitution of the C6 substituent with various halogens, we observed dramatic effects on potency in cell-based assays where the addition of chlorine and fluorine atoms yielded the most potent compounds.[19] Favorable packing interactions between the halogen and the Cγ of P95 were directly correlated with the degree of hydrogen bonding between the ethoxy uracil group and RT, suggesting that the presence of halogens with suitable van der Waals radius at the C6 position correctly positions the rest of the NNRTI in the binding pocket.

In our present structures, we find that the 6-fluorine substituent on the indolizine ring makes close contact with sidechain carbons of P95 for both WT RT and Y181C structures. Consistent with previous findings,[19] for WT RT, the interatomic distance between the fluorine and the Cγ atoms is 3.4 Å, which is in the same range as the previously reported crystal structures of WT RT with a catechol diether NNRTI containing a cyanovinylphenyl ring (Figure 7A). Surprisingly, in the structure of Compound 1 in complex with Y181C RT, we find that the interatomic distance between the fluorine and the Cγ of P95 is a nearly identical 3.5 Å, indicating that the NNRTI maintains close contact with P95 despite the Y181C mutation (Figure 7B). Spherical representation of the fluorine and P95 allows visual identification of the close packing and the strong van der Waals interaction between the halogen substituent and the binding pocket. Despite the shifting of the ethoxy uracil ring in our Y181C RT:Compound 1 structure, we see that both WT and Y181C RT contain three hydrogen bonds between the uracil ring and the L103/P236 residues, further suggesting that the interaction involving the halogen helps position the ethoxy uracil ring to preserve favorable interaction with its neighboring residues. The maintenance of the interaction between this halogen moiety and P95 is reflected by the remarkably similar potencies in the steady state kinetic assays, strengthening the likelihood that efficacy of Compound 1 against both wildtype and drug resistant variants of RT is rooted in its ability to flexibly interact and maintain interactions within the NNRTI binding pocket.

Figure 7: Van der Waals interaction between Compound 1 and Pro95 in unaltered in Y181C RT.

Figure 7:

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The fluorine substituent of Compound 1 and the nearest carbon atom (Cγ) of Pro95 of (A) WT RT (PDB: 6DTW) and (B) Y181C RT (PDB: 6DTX). Optimal interaction distances can be visualized by the close distance between the two spheres representing the fluorine and Cγ atoms. Atoms colored as above.

The first generation NNRTIs such as nevirapine have been challenged with rapid emergence of drug resistant mutations that can prevent binding of the compound dissolving the efficacy of the NNRTI over time.[3] A large part of this dilemma is due to the conformational rigidity of these first generation NNRTIs, which rely heavily on specific interactions present in the wildtype NNRTI binding pocket. Thus, in light of drug resistant mutations such as Y181C, these NNRTIs can no longer bind as effectively. To address this challenge, second generation NNRTIs containing a more flexible scaffold such as rilpivirine and etravirine have been developed. Possessing intrinsic conformational flexibility, these compounds circumvent issues faced by the first generation NNRTIs by effectively inhibiting wildtype and multiple drug–resistant variants of viral reverse transcriptase.[2] However, these compounds possess less than optimal pharmacological properties including low solubility and poor bioavailability. For instance, rilpivirine has an extremely low solubility in water at 0.020 μg/ml at pH 7.0 [13] and etravirine is poorly soluble in water with less than 1 μg/ml solubility.[30] The demand for NNRTIs effective against drug resistant variants of RT while possessing optimal pharmacological properties is high.

For this reason, numerous efforts across multiple groups have been made to design new NNRTIs in order to address both concerns of drug resistance and solubility. The development of novel benzophenone NNRTIs by another group effectively addresses the drug resistance concern since the set of compounds make important interactions with conserved moieties other than commonly mutated residues resulting in tight binding across multiple drug-resistant variants of RT.[31] Additionally, attachment of hydrophilic substituents to the terminal ring structure is an effort to improve water solubility.[31] In the commercial realm, parallel efforts by Merck and Co. in optimizing doravirine, a novel NNRTI which acts in low nanomolar potency against commonly arising drug resistant forms of RT,[32] has been accompanied with a guided synthesis strategy to produce compounds that possess improved solubility profiles to enhance oral bioavailability.[33] Such studies are but a few examples of structure-based drug design of NNRTIs to produce potent, adaptable, and pharmacologically favorable compounds which can be administered for HIV therapy.

Using similar approaches, we designed Compound 1, which is able to adapt and maintain potency for wildtype and drug resistant variants of RT. The IC50 and EC50 values, in biochemical and cellular assays, of Compound 1 against WT and Y181C RT are remarkably similar, and structural data suggest that several critical NNRTI-RT interactions are maintained with the Y181C mutant to rationalize the in vitro and cellular potency. From the structural standpoint, Compound 1 is able to flexibly maintain a number of important interactions in the NNRTI binding pocket, allowing us to rationalize the observed conserved potency across drug-resistant variants of RT. In addition, the solubility of Compound 1 is 43.8 μg/ml, an increase of more than 2000-fold over rilpivirine, making it a more attractive therapeutic candidate (Figure 1).[13, 20] Optimal pharmacological properties of NNRTIs are beneficial for later stage pharmacokinetic studies such as development of efficacious inhibitors for high solubility, low cytotoxicity and favorable pharmacological features including maximum concentration, prolonged serum residence time, and heightened in vivo safety profiles. [17, 34] Such improved solubility profiles can open new doors for HIV therapy by circumventing concerns regarding the oral bioavailability of particular formulations. The combination of robustness to drug resistant variants and improved solubility profile make Compound 1 and other catechol diether NNRTIs attractive candidates that may improve HAART treatment from the current composition.

In conclusion, our iterative drug design strategy has identified a series of catechol diether based NNRTIs that have remarkably high potency in the low nanomolar range against RT that is also realized as antiviral potency in HIV-infected MT-2 cells. Further optimization to address reactivity concerns has involved replacement of the reactive Michael acceptor cyanovinylphenyl group to a bicyclic moiety, allowing conservation the overall three-dimensional shape of the NNRTI. Compound 1 contains two fluorines at the C4 position of the catechol ring and the C6 position of the indolizine ring. We determined two crystal structures where Compound 1 was crystallized with WT and Y181C mutant RT. Analysis of key interactions reveals the conservation of hydrogen bonding and van der Waals forces between the WT and Y181C structures. Specifically, van der Waals packing and aryl-aryl interactions stabilize the bicyclic ring system of Compound 1, while extensive hydrogen bonding takes place between the backbones of K102 and K103 and the ethoxy uracil group. Despite loss of an aromatic side chain through mutation, compensatory interactions involving V179 allows close packing of Compound 1 within the NNRTI binding pocket, even in the absence of the bulky tyrosine side chain. Finally, the fluorine substituent extending from the indolizine ring is properly positioned at an optimal interaction distance for both variants of RT, strongly suggesting the maintenance of such interactions from a structural standpoint is the basis of the high potencies observed in our steady-state kinetic assays. To address concerns regarding drug resistance, newer generation NNRTIs that could flexibly interact within the NNRTI binding pocket of multiple drug-resistant variants of RT while maintaining strong binding and high potency may improve clinical outcomes of HIV/AIDS therapy.

Supplementary Material

1

Acknowledgements

This material is based upon work supported by the NIH Grant R01GM049551 (KSA), NIH Grant R01AI044616 (WLJ), NSF Grant DGE-1122492 (TS), NIH F31 fellowship support CA203254 granted by the National Cancer Institute (TS), NIH Virology Training Grant T32AI055403 (ZTKG), and NIH fellowship Support F32AI104334 (KMF). Resources of the CCMI Macromolecular X-ray Crystallography Facility were used for crystallographic experiments. This research used resources of the National Synchrotron Light Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-AC02–98CH10886. The WT and Y181C HIV clones for preparing viral strains were kindly provided by the Wainberg lab at McGill University.

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1
NIHMS1039549-supplement-1.pdf (1.5MB, pdf)

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