Nb₁₉₀-Rev interaction model.

<p>(<b>A</b>) Cartoon representation of the Nb₁₉₀ homology model. The coloring corresponds to the conservation of the amino acids calculated with the Consurf server, ranging from highly variable residues (red), over intermediate conservation (green), to highly conserved residues (blue). (<b>B</b>) Electrostatic surface of Nb₁₉₀ and helix-tun-helix motif of Rev: blue (negative), red (positive) and white (neutral). The interaction between Nb₁₉₀ and Rev is a combination of electrostatic interactions between RevK20 and the negative pocket of Nb₁₉₀ and further hydrophobic stabilization by the surrounding residues. (<b>C</b>) Cartoon representation of Nb₁₉₀ (green) docked onto Rev (N-terminal helix-turn-helix motive) (light blue). Important residues for binding interaction according to the alanine scanning results are colored dark blue for Rev and yellow for Nb₁₉₀.). Aliphatic hydrogens and backbone atoms have been hidden for clarity. (<b>D</b>) Close-up of the Nb₁₉₀-Rev interaction pattern, with residue RevK20 forming an extensive hydrogen bonding network (depicted by blue lines) with neighboring residues T33 and D107 in Nb₁₉₀. In addition, RevY23 makes a π-π interaction with Nb₁₉₀F100. This latter residue is also further stabilized by hydrophobic contacts with RevK20 and RevH53. RevH53 and RevL60 interact with Nb₁₉₀L101, while RevV16 makes contact with Nb₁₉₀Y105. Nb₁₉₀D98 stabilizes the CDR3 loop by hydrogen bonds with Nb₁₉₀N96.</p

Mapping of the Rev epitope.

<p>(<b>A</b>) Relative affinity of wild-type Nb₁₉₀ for the K20A and Y23A Rev mutants by FRAP of Nb₁₉₀-mKO when bound to RevM10-GFP. Values are averages ± SEM (n ≥8). (<b>B</b>) Half-time values for the recovery times after photobleaching in panel A. (<b>C</b>) Relative affinity of Nb₁₉₀T33A for the V16A, H53A and L60A Rev mutants by FRAP of Nb₁₉₀-mKO when bound to RevM10-GFP. Values are averages ± SEM (n ≥6). (<b>D</b>) Half-time values for the recovery times after photobleaching in panel C. (<b>E</b>) Schematic overview of the alanine scan performed on the head multimerization surface of Rev. Residues that were mutated to alanine are shown in bold. Mutated positions that resulted in a decreased affinity for the Nb₁₉₀T33A mutant are shown in bold red. Mutated positions that resulted in a decreased affinity for the wild-type nanobody have a red circle.</p

Rev and Nb₁₉₀ protein organization and mutation scheme.

<p>(<b>A</b>) Schematic representation of the HIV-1 Rev functional domain organization and secondary structures. The N-terminal domain of Rev forms a helix-loop-helix while de C-terminal domain is intrinsically unfolded. Three functional domains are shown: the Nuclear Export Signal (NES), the Nucleolar Localization Signal (NoLS) that also serves as RNA Binding Domain (RBD) and the first and second multimerization domain (Multimer. 1 and Multimer. 2). (<b>B</b>) Mutation scheme of the Nb₁₉₀ alanine scan. The three hyper-variable CDR regions are underlined as defined by the IMGT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060259#pone.0060259-Lefranc1" target="_blank">[50]</a>. Residues that were mutated to alanine are shown in bold.</p

Discovery of Thiophene[3,2‑<i>d</i>]pyrimidine Derivatives as Potent HIV‑1 NNRTIs Targeting the Tolerant Region I of NNIBP

Our previous studies led us to conclude that thiophene­[3,2-<i>d</i>]­pyrimidine is a promising scaffold for diarylpyrimidine (DAPY)-type anti-HIV agents with potent activity against resistance-associated human immunodeficiency virus (HIV) variants (<i>J. Med. Chem</i>. <b>2016</b>, <i>59</i>, 7991–8007; <i>J. Med. Chem</i>. <b>2017</b>, <i>60</i>, 4424–4443). In the present study, we designed and synthesized a series of thiophenepyrimidine derivatives with various substituents in the right wing region of the structure with the aim of developing new interactions with the tolerant region I of the binding pocket of the HIV-1 non-nucleoside reverse transcriptase (NNRTI), and we evaluated their activity against a panel of mutant HIV-1 strains. All the derivatives exhibited moderate to excellent potency against wild-type (WT) HIV-1 in MT-4 cells. Among them, sulfonamide compounds <b>9b</b> and <b>9d</b> were single-figure-nanomolar inhibitors with EC₅₀ values of 9.2 and 7.1 nM, respectively. Indeed, <b>9a</b> and <b>9d</b> were effective against the whole viral panel except RES056. Notably, both compounds showed potent antiviral activity against K103N (EC₅₀ = 0.032 and 0.070 μM) and E138K (EC₅₀ = 0.035 and 0.045 μM, respectively). Furthermore, <b>9a</b> and <b>9d</b> exhibited high affinity for WT HIV-1 RT (IC₅₀ = 1.041 and 1.138 μM, respectively) and acted as classical NNRT inhibitors (NNRTIs). These results are expected to be helpful in the design of thiophenepyrimidine-based NNRTIs with more potent activity against HIV strains with RT mutations

Design, Synthesis, and Evaluation of Thiophene[3,2‑<i>d</i>]pyrimidine Derivatives as HIV‑1 Non-nucleoside Reverse Transcriptase Inhibitors with Significantly Improved Drug Resistance Profiles

We designed and synthesized a series of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase inhibitors (NNRTIs) with a piperidine-substituted thiophene­[3,2-<i>d</i>]­pyrimidine scaffold, employing a strategy of structure-based molecular hybridization and substituent decorating. Most of the synthesized compounds exhibited broad-spectrum activity with low (single-digit) nanomolar EC₅₀ values toward a panel of wild-type (WT), single-mutant, and double-mutant HIV-1 strains. Compound <b>27</b> was the most potent; compared with ETV, its antiviral efficacy was 3-fold greater against WT, 5–7-fold greater against Y181C, Y188L, E138K, and F227L+V106A, and nearly equipotent against L100I and K103N, though somewhat weaker against K103N+Y181C. Importantly, <b>27</b> has lower cytotoxicity (CC₅₀ > 227 μM) and a huge selectivity index (SI) value (ratio of CC₅₀/EC₅₀) of >159101. <b>27</b> also showed favorable, drug-like pharmacokinetic and safety properties in rats in vivo. Molecular docking studies and the structure–activity relationships provide important clues for further molecular elaboration

Discovery of Novel Diarylpyrimidine Derivatives as Potent HIV‑1 NNRTIs Targeting the “NNRTI Adjacent” Binding Site

A novel series of diarylpyrimidine derivatives, which could simultaneously occupy the classical NNRTIs binding pocket (NNIBP) and the newly reported “NNRTI Adjacent” binding site, were designed, synthesized, and evaluated for their antiviral activities in MT-4 cell cultures. The results demonstrated that six compounds (<b>20</b>, <b>27</b> and <b>31</b>–<b>34</b>) showed excellent activities against wild-type (WT) HIV-1 strain (EC₅₀ = 2.4–3.8 nM), which were more potent than that of ETV (EC₅₀ = 4.0 nM). Furthermore, <b>20</b>, <b>27</b>, <b>33</b>, and <b>34</b> showed more potent or equipotent activity against single mutant HIV-1 strains compared to that of ETV. Especially, <b>20</b> showed marked antiviral activity, which was 1.5-fold greater against WT and 1.5- to 3-fold greater against L100I, K103N, Y181C, Y188L, and E138K when compared with ETV. In addition, all compounds showed lower toxicity (CC₅₀ = 5.1–149.2 μM) than ETV (CC₅₀ = 2.2 μM). The HIV-1 RT inhibitory assay was further conducted to confirm their binding target. Preliminary structure–activity relationships (SARs), molecular modeling, and calculated physicochemical properties of selected compounds were also discussed comprehensively

Structure-Based Optimization of Thiophene[3,2‑<i>d</i>]pyrimidine Derivatives as Potent HIV‑1 Non-nucleoside Reverse Transcriptase Inhibitors with Improved Potency against Resistance-Associated Variants

This work follows on from our initial discovery of a series of piperidine-substituted thiophene­[3,2-<i>d</i>]­pyrimidine HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTI) (J. Med. Chem. 2016, 59, 7991−8007). In the present study, we designed, synthesized, and biologically tested several series of new derivatives in order to investigate previously unexplored chemical space. Some of the synthesized compounds displayed single-digit nanomolar anti-HIV potencies against wild-type (WT) virus and a panel of NNRTI-resistant mutant viruses in MT-4 cells. Compound <b>25a</b> was exceptionally potent against the whole viral panel, affording 3-4-fold enhancement of in vitro antiviral potency against WT, L100I, K103N, Y181C, Y188L, E138K, and K103N+Y181C and 10-fold enhancement against F227L+V106A relative to the reference drug etravirine (ETV) in the same cellular assay. The structure–activity relationships, pharmacokinetics, acute toxicity, and cardiotoxicity were also examined. Overall, the results indicate that <b>25a</b> is a promising new drug candidate for treatment of HIV-1 infection

Structure-Based Optimization of Thiophene[3,2‑<i>d</i>]pyrimidine Derivatives as Potent HIV‑1 Non-nucleoside Reverse Transcriptase Inhibitors with Improved Potency against Resistance-Associated Variants

This work follows on from our initial discovery of a series of piperidine-substituted thiophene­[3,2-<i>d</i>]­pyrimidine HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTI) (J. Med. Chem. 2016, 59, 7991−8007). In the present study, we designed, synthesized, and biologically tested several series of new derivatives in order to investigate previously unexplored chemical space. Some of the synthesized compounds displayed single-digit nanomolar anti-HIV potencies against wild-type (WT) virus and a panel of NNRTI-resistant mutant viruses in MT-4 cells. Compound <b>25a</b> was exceptionally potent against the whole viral panel, affording 3-4-fold enhancement of in vitro antiviral potency against WT, L100I, K103N, Y181C, Y188L, E138K, and K103N+Y181C and 10-fold enhancement against F227L+V106A relative to the reference drug etravirine (ETV) in the same cellular assay. The structure–activity relationships, pharmacokinetics, acute toxicity, and cardiotoxicity were also examined. Overall, the results indicate that <b>25a</b> is a promising new drug candidate for treatment of HIV-1 infection

Further Exploring Solvent-Exposed Tolerant Regions of Allosteric Binding Pocket for Novel HIV‑1 NNRTIs Discovery

Based on the detailed analysis of the binding mode of diarylpyrimidines (DAPYs) with HIV-1 RT, we designed several subseries of novel NNRTIs, with the aim to probe biologically relevant chemical space of solvent-exposed tolerant regions in NNRTIs binding pocket (NNIBP). The most potent compound <b>21a</b> exhibited significant activity against the whole viral panel, being about 1.5–2.6-fold (WT, EC₅₀ = 2.44 nM; L100I, EC₅₀ = 4.24 nM; Y181C, EC₅₀ = 4.80 nM; F227L + V106A, EC₅₀ = 17.8 nM) and 4–5-fold (K103N, EC₅₀ = 1.03 nM; Y188L, EC₅₀ = 7.16 nM; E138K, EC₅₀ = 3.95 nM) more potent than the reference drug ETV. Furthermore, molecular simulation was conducted to understand the binding mode of interactions of these novel NNRTIs and to provide insights for the next optimization studies