Antiviral Activity and Crystal Structures of HIV-1 gp120 Antagonists

As part of our effort to discover drugs that target HIV-1 entry, we report the antiviral activity and crystal structures of two novel inhibitors in a complex with a gp120 core. NBD-14204 showed similar antiviral activity against all the clinical isolates tested. The IC50 values were in the range of 0.24–0.9 µM with an overall mean of 0.47 ± 0.03 µM, showing slightly better activity against the clinical isolates than against the lab-adapted HIV-1HXB2 (IC50 = 0.96 ± 0.1 µM). Moreover, the antiviral activity of NBD-14208 was less consistent, showing a wider range of IC50 values (0.66–5.7 µM) with an overall mean of 3 ± 0.25 µM and better activity against subtypes B and D (Mean IC50 2.2–2.5 µM) than the A, C and Rec viruses (Mean IC50 2.9–3.9 µM). SI of NBD-14204 was about 10-fold higher than NBD-14208, making it a better lead compound for further optimization. In addition, we tested these compounds against S375Y and S375H mutants of gp120, which occurred in some clades and observed these to be sensitive to NBD-14204 and NBD-14208. These inhibitors also showed modest activity against HIV-1 reverse transcriptase. Furthermore, we determined the crystal structures of both inhibitors in complexes with gp120 cores. As expected, both NBD-14204 and NBD-14208 bind primarily within the Phe43 cavity. It is noteworthy that the electron density of the thiazole ring in both structures was poorly defined due to the flexibility of this scaffold, suggesting that these compounds maintain substantial entropy, even when bound to the Phe43 cavity.


Introduction
Advances in available therapeutics, particularly combination antiretroviral therapy, have significantly improved the treatment of HIV infections, facilitating the shift from a disease characterized by high morbidity and mortality to a manageable chronic disease. Despite this remarkable success, current treatments suffer from several limitations, including (1) reliance on daily adherence, (2) long-term use resulting in long-term toxicity, (3) limited treatment options due to the development of drug resistance, (4) high costs, and (5) the failure of current treatments to eradicate HIV. In addition, despite tremendous efforts and investment, no effective vaccine or microbicide has been developed, and significant hurdles must be overcome to achieve an effective cure. Thus, the continued development of small-molecule drugs with high potency against novel targets and minimal side effects remains imperative. The development of novel therapeutics will increase the number of available drugs, extend the scope of combination therapies, and present opportunities to formulate long-acting drugs.
Approximately 18 years after the first publication describing HIV-1 attachment inhibitors by Bristol Meyer & Squib (BMS) [1], the first drug targeting gp120, Fostemsavir [2] (Rukobia, Viiv Healthcare, Middlesex, TW8 9GS, UK), received FDA approval in 2021. This recognition by the FDA confirms the significance of our efforts to develop novel drugs targeting HIV-1 gp120, especially for the growing number of treatment-experienced patients with limited treatment options and for use in combination therapies for the millions of affected individuals worldwide.
Our group has made significant progress toward meeting this critical need by developing a new class of HIV-1 entry inhibitors (we termed NBDs) targeting the Phe43 cavity of HIV-1 gp120, a different binding site from that targeted by Fostemsavir. NBDs inhibit gp120 binding to the cellular receptor CD4 by direct binding to the Phe43 cavity of HIV-1 envelope glycoprotein gp120. On the contrary, Fostemsavir binds to an induced binding pocket under the β20-21 loop, distinct from the Ph43 cavity created upon CD4 binding. We reported the design, synthesis, antiviral activity, X-ray crystal structures, and preclinical assessments of many such NBDs [3][4][5][6][7][8][9][10][11][12][13][14][15][16]. Due to the critical role the structural information plays in designing inhibitors with improved binding and antiviral activity, we continue to strive to determine the crystal structures of NBDs to understand the binding interactions of this series of compounds in the Phe43 cavity.
We recently discovered that some of the most active gp120 antagonists also display antiviral activity against HIV-1 reverse transcriptase (RT) [15]. HIV uses RT to convert its RNA into viral DNA in a process called reverse transcription, a post-entry process in the HIV-1 life cycle. Nucleoside reverse transcriptase inhibitors (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) play a pivotal role in the highly active antiretroviral therapy (HAART) of AIDS patients.
This report showed detailed neutralizing activity of NBD-14204 ( Figure 1A) and NBD-14208 ( Figure 1B) against a selected set of HIV-1 pseudotyped viruses constructed with Env glycoproteins from a diverse class of clinical isolates. Earlier, we published the antiviral activity of these two NBDs against HIV-1 HXB2 [12,13]. In addition, we presented the crystal structures of these compounds in complexes with gp120 cores.  Our results have shown that compounds NBD-14204 and NBD-14208 have good antiviral activity against the laboratory-adapted Env-pseudotyped HIV-1 HXB-2 [12,13]. Therefore, we decided to evaluate their activity against a selection of 25 Env-pseudotyped HIV-1 clinical isolate clones of different subtypes, including the A, B, C, and D types and five recombinant (Rec) HIV-1 clones. All those clinical isolates use CCR5 coreceptor for entry apart from subtype B # 11563, dual tropic (CCR5/CXCR4). As shown in Table 1, NBD-14204 showed equal antiviral activity against all the clinical isolates tested, including the Rec clones. The IC 50 values were in the range of 0.24-0.9 µM with an overall mean of 0.47 ± 0.03 µM, showing slightly better activity against the clinical isolates than against the lab-adapted HIV-1 HXB2 (IC 50 = 0.96 ± 0.1 µM) . Representative dose-response curves are shown in Figure 1A. Moreover, the antiviral activity of NBD-14208 was less consistent, showing a wider range of IC 50 values (0.66-5.7 µM) with an overall mean of 3 ± 0.25 µM and better activity against the subtypes B and D (Mean IC 50 2.2-2.5 µM) than the A, C and Rec viruses (Mean IC 50 2.9-3.9 µM). Representative curves are shown in Figure 1B. Additionally, both compounds were inactive against the control pseudovirus VSV-G suggesting their activity is specific to HIV-1.

S375Y and S375H Mutants Are Not Resistant to NBD-14204 and NBD-14208
We previously reported that some amino acid substitutions located in the CD4-binding site of gp120 reduced the sensitivity of those mutant viruses to the NBD compounds, but the sensitivities of viruses carrying the S375H, S375W, and S375Y mutations were not reduced [17]. Therefore, in this study, we evaluated both NBD compounds against two mutant pseudovirus HIV-1 HXB2 having amino acid substitutions, S375Y and S375H, in the gp120 region. These mutants were sensitive to both NBD compounds, which showed similar activity against the mutants as was observed against HIV-1 HXB2 -WT (data not shown). S375 mutations, especially Y/N, are known to reduce sensitivity to HIV-1 attachment inhibitors [2,18]. In addition, a 48-week Phase 2b clinical trial reported several resistant mutants, including at S375, that reduced susceptibility to Fostemsavir [19].

NBD Compounds Showed Activity against HIV-1 Reverse Transcriptase (RT)
Some previously described NBD entry antagonists have shown activity against HIV-1 reverse transcriptase (RT) [11,15]. Therefore, we evaluated the RT inhibitory activity of NBD-14204 and NBD-14208 against that enzyme. We used Nevirapine and NBD-556 as controls. As expected, Nevirapine was highly potent against HIV-1 RT, with an IC 50 of 0.20 µM. However, 300 µM NBD-556 had no activity ( Table 2). Both compounds, NBD-14204 and NBD-14208, inhibited HIV-1 RT; their IC 50 s were 8.3 ± 1.2 µM and 5 ± 0.5 µM, respectively. Comparing those values with those obtained with the HIV-1 neutralization assays and calculating the ratios, we can hypothesize that the antiviral activity of NBD-14208 is primarily due to its activity against both gp120 and RT, while the primary activity of NBD-14204 is mostly against gp120 and its activity against RT may just play a secondary role. However, it was critically important to understand how some of the gp120 antagonists also show RT inhibitory activity. Since RTI and NNRTI drugs play a pivotal role in the highly active antiretroviral therapy (HAART) of AIDS patients, we wanted to learn more about the binding mode of these gp120 antagonists on HIV-1 RT. Therefore, we confirmed the binding of three such gp120 antagonists with HIV-1 RT by X-ray crystallography [15]. These compounds achieve dual-inhibitory activity by bridging dNTP-and NNRTI-binding sites to inhibit the polymerase activity of isolated RT in enzymatic assays. Similar binding was reported for compound G from Merck [20] (PDB: 5VZ6) and dihydroxybenzoyl naphthyl hydrazone [21] (DHBNH; PDB:2I5J). However, NBD binding extends into the NNRTIbinding pocket on one end and toward the polymerase active site and into the nucleotidebinding site on the other end, reaching further than either compound G or DHBNH1.

Crystal Structures of NBD-14204 and NBD-14208-Bound HIV-1 gp120 Core
To determine the crystal structures of HIV-1 gp120 core in complex with NBD-14204 or NBD-14208, we used a clade A/E 93TH057 HIV-1 gp120 core with His375 to Ser substitution [22,23], which enabled us to produce well-diffracting crystals consistently. The structures revealed that the compounds mimicked the binding of Phe43 of CD4 to gp120 and occupied the cavity in the CD4 binding site of gp120, as shown in previously reported structures of NBD compounds [4,6,9,24]. The trifluoro-methane group of NBD-14204 in region I (Figure 2A,B) contacted Thr257, Glu370, Ile371, and Trp427 with hydrophobic interaction. The nitrogen atom in the pyridine ring made a hydrogen bond to the carbonyl oxygen of Asn425. In addition, the pyrrole ring showed hydrophobic contact in region II with Trp427 ( Figure 2C). Similarly, the 2-fluoro,1-chloro-derivatized phenyl ring of NBD-14208 in the region I made hydrophobic contacts with Thr257, Glu370, and Trp427 in the cavity ( Figure 2C). Unlike NBD-14204, the nitrogen atom in the pyrrole ring of NBD-14208 made a hydrogen bond to the carbonyl oxygen of Asn425 ( Figure 2D). Both structures showed that their amine groups in region III made hydrogen bonds to Asp368 ( Figure 2C,D). However, the electron density of region III, particularly their thiazole groups, was poorly defined (Figure 2 E,F), even though the two structures were determined with relatively high resolutions of 1.9 Å and 2.1 Å for NBD-14204 and NBD-14208 ( Figure 3A and Table 3), respectively, indicating that the thiazole rings in the region III are flexible and need to be further optimized to enable the region to make favorable interactions with neighboring residues. Interestingly, an additional NBD-14208 compound was found outside the Phe43 cavity, under the bridging sheet where NBD-14208 and a HEPES molecule were nestled in the space formed by neighboring gp120 molecules in the crystal lattice ( Figure 3B).

Measurement of Antiviral Activity
NBD-14204 and NBD-14208 were evaluated by infecting TZM-bl cells with pseudovirus lab-adapted HIV-1 HXB-2 (CXCR4-tropic) in a single-cycle infection assay. Additionally, these compounds were evaluated against 25 HIV-1 pseudoviruses, pseudotyped with the Env from the clinical isolates panel. Briefly, TZM-bl cells were plated at 1 × 10 4 /well in a 96-well tissue culture plate and incubated overnight. The following day, aliquots of HIV-1 pseudovirus were pre-treated with the NBD compounds at graded concentrations for 30 min at room temperature and added to the cells. Following 3-day incubation, the cells were washed and lysed with 50 µL of lysis buffer (Promega, WI, USA). Twenty µL of the lysates were transferred to the wells of a white 96-well plate and mixed with the luciferase assay reagent (Promega, WI, USA). The luciferase activity was measured immediately with a Tecan Spark reader, and the percent of inhibition compared to the untreated positive control and the IC 50 (the half-maximal inhibitory concentration) values were calculated using the GraphPad Prism software. The neutralization activity of the compounds against the control pseudovirus VSV-G was evaluated in U87-CD4-CXCR4 cells as previously described [13].

Evaluation of Cytotoxicity
The cytotoxicity of NBD-14204 and NBD-14208 was evaluated in parallel with the neutralization assay in TZM-bl cells using the colorimetric CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (MTS) (Promega, WI, USA) following the manufacturer's instructions. Briefly, the cells were plated at 1 × 10 4 /well and cultured at 37 • C overnight. The following day, graded concentrations of the compounds were added to the cells and incubated for three days. Finally, the MTS reagent was added to the cells and incubated for 4 h at 37 • C. The absorbance was recorded at 490 nm. The percent of cytotoxicity and the CC 50 (the concentration for 50% cytotoxicity) values were calculated as above.

Reverse Transcriptase Inhibition Assay
The activity of the NBD-14204 and NBD-14208 was evaluated against the HIV-1 Reverse Transcriptase (RT) by using the Colorimetric Reverse Transcriptase Assay (Roche) and following the manufacturer's instructions. Nevirapine (NNRTI) and NBD-556 [36] were used as controls.

Crystallization, Data Collection, and Structural Refinement
Clade A/E 93TH057 gp120 core with H375S mutation was expressed in 293F cells, purified with a 17b-conjugated affinity column, and deglycosylated with Endoglycosidase H, as described previously [23]. The deglycosylated gp120 core was purified using HiLoad 16/600 Sperdex 200 pg size-exclusion chromatography column (GE Healthcare, NY, USA) with a running buffer containing 5 mM HEPES, pH 7.4, 150 mM NaCl. A molar excess of NBD-14204 or NBD-14208 in DMSO was added to the concentrated gp120 core to maintain the final concentrations of DMSO and gp120 of 5% and~10 mg/mL, respectively. The NBD-14204 or NBD-14208/gp120 complexes were then screened for crystallization conditions using Hampton Research, Wizard Screen, and Precipitation Synergy Screen. The diffracting quality crystals of the complexes grew at 12-14% PEG3350, 5% isopropanol, 0.1M HEPES, 7.4. Crystals were flash frozen in the presence of cryoprotectant containing 30% glycerol, 14% PEG3350, 5% isopropanol, and 0.1M HEPES, 7.4 for data collection at the synchrotron beamline, SER-CAT ID22, at Advanced Photon Source (Lemont, IL, USA). The diffraction data were collected and processed using HKL2000 [37]. The structures were solved by molecular replacement using Phaser [38] with PDB ID 3TGT as a search model, built with Coot [39], and refined using Phenix [40]. Figures were generated by using Ligplot [41], PyMOL [42], and Chimera [42].

Conclusions
This report presented the antiviral activity of two different series of NBD-type small molecules against a diverse set of HIV-1 Env-pseudotyped clinical isolates and their crystal structures. The antiviral data indicated that the pyridine-containing compound, NBD-14204, showed about five-fold better antiviral activity than the phenyl-containing compound, NBD-14208. Furthermore, NBD-14208 appeared to be more cytotoxic than NBD-14204. However, in crystal structures, both compounds have some common interactions. For example, the primary amine group in region III of both compounds forms an H-bond (saltbridge) with Asp368. Both the pyridine and phenyl rings also have similar hydrophobic interactions. However, one key difference is that the pyridine 'N' of NBD-14204 interacts with Asn425, whereas the pyrrole 'N' of NBD-14208 shows interaction with Asn425. These two inhibitors' structural information and antiviral activity are expected to provide critical information to further design this series of inhibitors with improved efficacy and selectivity index.