English

• Acquired immunodeficiency syndrome (AIDS) is a chronic infectious disease infected by human immunodeficiency virus (HIV) [1]. HIV is an RNA retrovirus divided into HIV-1 and HIV-2 subtypes [2], of which HIV-1 is the main epidemic pathogen; as of 2020, it is estimated that 37.7 million individuals live with HIV-1 [3]. HIV-2 is prevalent in West Africa, and it has spread to neighboring countries with socioeconomic links to the region [4]. Combinatorial antiretroviral therapy (cART) is an anti-HIV drug combination targeted at several stages of the virus life cycle and has shown to be effective in preventing HIV infection [5,6]. Nevertheless, the long-term use of cART has been hampered by the establishment of substantially cross-resistant HIV-1 strains and cumulative medication toxicities [7-9]. Additionally, it has been reported that drug resistance of HIV-2 may occur earlier than HIV-1 and selectively mutate at different sites [10]. Therefore, it is an urgent need for researchers to develop new classes of anti-AIDS agents with unique action modalities or targets, and reduced side effects.

  HIV-1 capsid protein (CA), a widely conserved protein required for HIV-1 replication, participating in multiple steps of the viral life cycle, has risen to prominence as an attractive target for therapeutic intervention [11-13]. Mature CA consists of about 1500 monomers, mainly arranged into hexameric lattices but also into 12 pentamers that induce curvatures at the core-periphery to assemble a closed conical ship [14,15]. CA monomer is made up of two alpha-helical domains, the N-terminal domain (NTD) and C-terminal domain (CTD), connecting by a short flexible linker [16]. A conserved interprotomer pocket (namely NTD-CTD interface) exists in the structure of assembled CA multimers, which has been proved to interact with host-cell proteins such as cleavage and polyadenylation specificity factor-6 (CPSF6) and nucleoporin 153 (NUP153) (Fig. 1) [17]. The CA-CPSF6 interaction promotes the pre-integration complexes (PICs) to enter the nucleus, modulates nuclear localization, and integrates viral reverse transcripted DNA into the transcriptional active areas of host genes [18,19]. NUP153, as a nucleoporin, is also critical for the nuclear transport of viral PICs [20,21]. Disruption of these interactions through mutagenesis can interfere with HIV-1 replication, suggesting that NTD-CTD interface is a major target for inhibitor design.

  Figure 1

  

  Figure 1.  Crystal structure of HIV-1 CA in complex with (A) CPSF6 peptide (PDB code: 4WYM) and (B) NUP153 peptide (PDB code: 6AYA). The figures are generated in PyMOL (www.pymol.org).

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  Consistent with this speculation, the NTD-CTD interface has been defined as the mature binding site of the CA-targeted small molecule inhibitor PF-3450074 (PF74, Fig. 2) [22,23]. In agreement with the binding multifunction of this pocket, PF74 exhibits a concentration-dependent bimodal mechanism for its antiviral activity: At lower concentrations, it competes with host factors CPSF6 and NUP153 to influence nuclear entry; and at higher concentrations, it interrupts uncoating and reverse transcription, probably by altering inter-hexamer interactions [24,25]. However, PF74 was unable to reach clinical development owing to its insufficient antiviral efficacy and metabolic instability. Additionally, the binding site of PF74 only partially overlaps with CPSF6 and NUP153, which provides sufficient space within the NTD-CTD interface for further modification of PF74.

  Figure 2

  

  Figure 2.  Design principle of novel small molecule inhibitors targeting HIV-1 CA NTD-CTD interface. The figures are generated in PyMOL (www.pymol.org).

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  An examination of the PF74-CA complex structure indicates that there are multiple polar amino acid residues (such as Tyr169, Arg173, Lys182) in the NTD-CTD interface which does not form significant interactions with PF74, indicating a new site for structural optimization to improve antiviral activity, anti-drug resistance, and druggability. In this paper, to further enhance the potency and drug-like features, we selected PF74 as the lead compound, designed and synthesized a series of new small molecule moderators of capsid assembly to target the hot spot residues in NTD-CTD interface based on a structure-guided design strategy and drug-like parameters (such as Fsp³)-inspired optimization (Fig. 2) [26]. It has been proven that introducing a methoxy at the para-position of the aniline and replacing phenylalanine with 3, 5-difluorophenylalanine is favorable for antiviral activity; hence, we kept the methoxy‑bearing aniline substituent and 3, 5-difluorophenylalanine on basic skeleton of PF74. Further, employing piperazinone rich in sp³ hybrid carbon as a linker, we replaced the indole moiety with different substituted benzenesulfonyl groups bearing multiple hydrogen bond donors or receptors, aiming to form additional interactions with surrounding key residues in the NTD-CTD interface to improve binding affinity and drug-like qualities. Herein, the newly synthesized compounds were screened for antiviral activity and conducted to structure-activity relationship (SAR) analysis. Furthermore, we used surface plasmon resonance (SPR), action stage determination studies, and molecular dynamics (MD) simulations to investigate the mechanism of action (MOA) of these compounds. Finally, the stability experiments of representative compounds 7b, 7m, 7n were also carried out in human liver microsome (HLM) and human plasma, respectively.

  The target compounds 7a-7n were prepared according to the general Scheme 1. Briefly, (tert‑butoxycarbonyl)-3, 5-difluoro-l-phenylalanine (1) was treated with 4‑methoxy-N-methylaniline and 2-(7-aza-1H-benzotriazole-1-yl)-1, 1, 3, 3-tetramethyluronium hexafluoro (HATU) in the presence of N, N-diisopropylethyl-amine (DIEA) and dichloromethane (DCM) to give 2, followed by the removal of tert‑butyloxycarbonyl (Boc) protection using trifluoroacetic acid (TFA) to yield the free amine 3. The acylation of 3 with bromoacetic acid in DCM resulted in the crucial intermediate 4. Further, 4 was reacted with 1-Boc-3-oxopiperazine via a nucleophilic substitution (SN₂) reaction to afford intermediate 5. Removing the Boc protection of 5 yielded the free amine 6, which was acylated with the matching substituted benzenesulfonyl chloride to generate the desired compounds 7a-7k. The amino analogues 7l-7n were created by hydrogenating the nitro group of 7i-7k.

  Scheme 1

  

  Scheme 1.  Reagents and conditions: (i) 4-Methoxy-N-methylaniline, HATU, DIEA, 0 ℃ to r.t.; (ii) TFA, DCM, r.t.; (iii) Bromoacetic acid, HATU, DIEA, 0 ℃ to r.t.; (iv) 1-Boc-3-oxopiperazine, Cs₂CO₃, DMF, 45 ℃; (v) TFA, DCM, r.t.; (vi) TEA, DCM, r.t.; (vii) H₂, 10%Pd·C, DCM, r.t.

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  Compounds in this program were evaluated for their antiviral activity in MT-4 cells based MTT assay containing wild-type (WT) HIV-1 III_B and HIV-2 (ROD). The selectivity index (SI value, CC₅₀/EC₅₀) of the test compounds was determined by assessing compound toxicity against MT-4 cells in parallel using an MTT assay. Table 1 displayed the findings of the analysis.

  Table 1

  

  Table 1.  Anti-HIV activity and cytotoxicity in MT-4 Cells infected with HIV-1 III_B virus and HIV-2 ROD virus.

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  The majority of the compounds in this series demonstrated remarkable anti-HIV-1 activities in submicromolar levels, of which seven compounds exceeded PF74 (EC₅₀ = 0.75 µmol/L). The unsubstituted benzene (7a, EC₅₀ = 0.20 µmol/L) and fluorine substitution at the meta- or ortho-position [7b (EC₅₀ = 0.18 µmol/L), 7c (EC₅₀ = 0.24 µmol/L)] led to a significant improvement in efficacy and SI values. However, the efficacy of these compounds with a halogen modification at the para-position on the benzene [7d (EC₅₀ = 0.79 µmol/L), 7e (EC₅₀ = 0.93 µmol/L), 7f (EC₅₀ = 0.86 µmol/L)] was modest decreased; these compounds also exhibited low SI values illustrating that the decrease in efficacy might be ascribed to a concurrent increase in toxicity. The methyl substitution at the para-position (7g, EC₅₀ = 0.91 µmol/L) also resulted in a modestly decrease in potency but with a marked increased SI value indicating a decrease in toxicity. An acetamido group at the para-position contributed to a 3.57-fold increase in potency and > 10 SI value (7h, EC₅₀ = 0.21 µmol/L). Compounds 7i (EC₅₀ = 2.84 µmol/L), 7j (EC₅0 = 1.49 µmol/L), 7k (EC₅₀ = 1.01 µmol/L) containing a nitro substitution had decreased anti-HIV-1 activity along with equaled or lower SI values. Notably, the amino substitution at the ortho-, meta- or para-position [7l (EC₅₀ = 0.52 µmol/L), 7m (EC₅₀ = 0.16 µmol/L), 7n (EC₅₀ = 0.12 µmol/L)] led to a rise in potency while maintaining low toxicity, resulting in an increase in the SI value. Interestingly, among the nitro/amino substitution, a clear SAR was observed that the hydrogen-bond donor (amino) has the same activity order as the hydrogen-bond receptor (nitro): para-position > meta-position > ortho-position. Finally, the identification of 7n represented a 6.25-fold improvement in potency over the lead compound PF74.

  Exceptionally, all of the compounds demonstrated improved anti-HIV-2 potency and higher SI values than PF74 (EC₅₀ = 4.16 µmol/L, SI = 43.03). Among them, 7h was shown to be the most potent of the series against HIV-2, with an EC₅₀ value of 0.03 µmol/L, which was 139-fold than PF74. Take the unsubstituted benzene compound 7a (EC₅₀ = 0.10 µmol/L) as a reference: (i) Among the ortho-substituted benzene derivatives, substitution with 2-fluorine (7b, EC₅₀ = 0.09 µmol/L) and 2-amino (7l, EC₅₀ = 0.14 µmol/L) equaled to 7a. However, the introduction of 2-nitro (7i, EC₅₀ = 1.19 µmol/L) decreased the antiviral activity. (ii) Among the meta-substituted benzene derivatives, substitution with 3-fluorine (7c, EC₅₀ = 0.12 µmol/L) and 3-amino (7m, EC₅₀ = 0.11 µmol/L) led to equaled efficacy compared with 7a, and 3-nitro (7j, EC₅₀ = 0.17 µmol/L) showed a modest decrease in potency; (iii) Among the para-substituted benzene derivatives, the electron-donating groups [4-methyl (7g, EC₅₀ = 0.12 µmol/L), 4-acetamido (7h, EC₅₀ = 0.03 µmol/L), 4-amino (7n, EC₅₀ = 0.05 µmol/L)] were advantageous for activity, while the electron-withdrawing groups such as 4-fluorine (7d, EC₅₀ = 0.36 µmol/L), 4-chlorine (7e, EC₅₀ = 0.44 µmol/L), 4-bromine (7f, EC₅₀ = 0.35 µmol/L) and 4-nitro (7k, EC₅₀ = 0.48 µmol/L) displayed decreased activity.

  Taken together, these newly synthesized compounds displayed excellent anti-HIV activities. Since HIV-1 is more prevalent and pathogenic than HIV-2, this study focused on the MOA of the representative compounds in the context of HIV-1.

  To determine the target specificity of the new compounds to HIV-1 CA, we, therefore, employed 7b, 7m, and 7n as the three most potent compounds to study a direct interaction with two distinct CA protein constructs: CA monomer and hexamer, using SPR based approach as previously described [27-29]. To assure the validity of experimental findings, PF74 was used as an in-line control in this assay.

  The resulting sensorgrams and the SPR isotherms were illustrated in Figs. S1 and S2 (Supporting information), respectively. Table 2 shows the corresponding kinetic parameter. The kinetic profile of the compounds 7b, 7m, 7n-CA monomer interaction was comparable to that of the lead compound PF74 with fast association and dissociation rates, and similar equilibrium dissociation constants as compared to PF74 (Fig. S1 in Supporting information and Table 2). SPR analysis exhibited that the three new compounds robustly interacted with the immobilized disulfide-stabilized CA hexamer and with a kinetic signature that was broadly similar to that of PF74. The on-rates of all four compounds were extremely fast, but the off-rates were slightly variable (Fig. S1), with PF74 having the slowest off-rate (Table 2). Alterations in off-rates for the hexameric CA, correlate with increased binding affinity (Table 2). Despite their varying off-rates, all compounds exhibited affinities with CA hexamer in the µmol/L range from 5- to 11-fold of PF74, with 7n having a marginally upgraded K_D over 7b and 7m (0.915, 1.724 and 1.754 µmol/L, respectively). Surprisingly, the interactions of 7b, 7m and 7n with monomer and hexamer followed a similar profile to PF74, with a preference for the hexamer. Conclusively, the SPR analysis results demonstrated that these inhibitors retained the target specificity towards HIV-1 capsid.

  Table 2

  

  Table 2.  SPR results of 7b, 7m, 7n, and PF74 binding to monomeric and hexameric CA constructs.

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  To establish whether or not our target compounds genuinely share the same binding pocket as PF74, we designed and implemented SPR-based competition assays using peptides generated from the proteins CPSF6 and NUP153, using 7n as a representative compound. The findings and analysis of these studies were shown in Fig. S3 (Supporting information), which revealed that our immobilized hexameric CA not only has surface activity but also has affinities that are consistent with previously published values [22-30]. Fig. S4 (Supporting information) displays that the competitive ability of 7n with CPSF6 and NUP153 peptides was comparable to that of PF74. Consistent with previously published results [24], PF74 inhibited the CA hexamer-CPSF6/NUP153 peptides interaction. Remarkably, our newly obtained compound 7n also inhibited the CA hexamer-CPSF6/NUP153 peptides interaction, suggesting that it was binding within the NTD-CTD interface as expected.

  Notably, the inhibition profile of 7n was opposed to that of PF74, with 7n competing more effectively with NUP153 and PF74 inhibiting CPSF6 better (Table S1 in Supporting information). To study the possible rationale for this reversal of the inhibitory profile between the two compounds, we used rigorous docking simulations for PF74 and 7n, and compared them to the crystal structures of the peptides bound to hexameric CA. Investigation of the docking poses revealed that 7n, also due to its increased size, occupies a larger space as compared to PF74 (Fig. 3). The orientation of 7n was comparable to that of the NUP153 peptide, suggesting that it might more effectively sterically block the binding site for NUP153.

  Figure 3

  

  Figure 3.  Docking analysis and structural overlay of PF74 (yellow, PDB code: 4XFZ) and 7n (purple) compared with CPSF6 (blue) and NUP153 (orange). The co-crystal structures of the CPSF6 (PDB code: 4WYM) peptide and the NUP153 (PDB code: 4U0C) peptide are highlighted in blue and orange, respectively.

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  Using direct engagement of HIV-1 CA monomers/hexamers and competition with CPSF6 and NUP153, we demonstrated low-micromolar affinity binding for 7b and 7m, while 7n engaged hexameric CA in the nanomolar range. The HIV-1 CA protein is essential for the early and late stages of the viral replication cycle. While hexameric/oligomeric CA functions predominantly during the early stages, CA is mainly monomeric during the late stage in the context of newly synthesized Gag polyprotein. We, therefore, sought to determine the stage of inhibition of our most potent compound (7n) as a representative for our compound series, using a modified single round infection (SRI) assay, efficiently separating the early and late stages of the HIV-1 life cycle [27].

  PF74 has been shown to have inhibitory characteristics in both the early and late-stages [29]; however, late-stage inhibition was observed only at higher concentrations of PF74 not used in our setup. As shown in Fig. S5 (Supporting information), 7n could exert its role in the early and late stages of the HIV-1 life cycle, while PF74 inhibits only the early stage in our experimental setup. Interestingly, 7n, although demonstrated as a similar affinity for the monomeric CA protein compared to PF74, displayed comparable inhibition in both stages, possibly suggesting a dual mechanism of action.

  Besides, we performed MD analysis on 7n-CA monomer complexes. Fig. S6 (Supporting information) showed the presence of different conformations of the protein and the compound, the whole trajectory was clustered to find the protein and the compound conformations. The clustering procedure produced 19 clusters, the two most populous of which were depicted in Fig. S7 (Supporting information). Site 1 (Fig. S7B) is located at the binding site indicated by the X-ray structure, displays the interactions between the most prominent conformer of 7n and the HIV-1 CA monomer. Where, Lys70, Met66, Gln63 and Leu56 are shared between the X-ray binding and site 1 of MD simulation binding. The methoxybenzene of 7n has hydrophobic interactions with Leu56, Met66, and Lys70, and the aminobenzene could have hydrophobic interaction with Lys70. These hydrophobic interactions give the compound tighter binding to this binding site. Also, the aminobenzene could have ion-induced dipole with the ammonium moiety of Lys70, giving further binding affinity for 7n. Additionally, hydrogen bond analysis showed that 7n forms hydrogen bonding with Asn57 for 7.0% of the MD simulation time. Fig. S7C shows the binding interactions of 7n in the second most populated cluster (site 2). During the MD simulation, these two conformations are mainly bound to the NTD region of CA monomer, and there are no critical interactions between the exposed bensulfamide piperazinone structure and this region. Therefore, it can be speculated that the high potency of this series of compounds may be caused by the key interactions between the bensulfamide piperazinone structure and the adjacent monomer.

  Since a significant pitfall of PF74 is its metabolic lability, one of our primary objectives in designing these chemicals was to increase their metabolic half-life. We, therefore, conducted metabolic stability assays of our most potent antiviral compounds (7b, 7m, and 7n) in both HLM and human plasma, along with PF74 as a control. PF74 was rapidly metabolized with a half-life (t_1/2) of 0.5 min, and improved stability was observed for 7m (t_1/2 = 7.7 min, 15.4-fold over PF74) and 7n (t_1/2 = 2.4 min, 4.8-fold over PF74). The CL_int(liver) of 7m and 7n was also decreased to 1/16 and 1/5 of that of PF74, with values of 161.8, 510.1, and 2576.2 mL/min/kg, respectively (Table S2 in Supporting information). 103.1% of 7b, 109.2% of 7m, and 100.4% of 7n maintained intact after incubation for 120 min at 37 ℃. On the contrary, PF74 was easily metabolized (maintaining amounts of 85.2% at 120 min) (Table S3 in Supporting information). Overall, the representative compounds were comparatively stable in HLM and human plasma, the improvement is most likely due to the introduction of two fluorine atoms on the benzene ring of phenylalanine and the replacement of the indole ring with more minor electron-rich moieties. Due to only modest improvements of the here presented compound series, future efforts will be directed towards further improvement of drug-like properties, including metabolic stability.

  In this paper, inspired by the structural and biochemical information of PF74, we used a structure-based drug design strategy to design moderators of the capsid NTD-CTD interface that would disrupt HIV-1 replication at multiple stages. This effort generated compound 7n, which was identified to be the most potent for inhibition of HIV-1 replication and gave a 6.25-fold increment in efficacy to its parent compound PF74. Moreover, these inhibitors exhibited a prominent antiviral activity profile against HIV-2 and take compound 7h as representation whose anti-HIV-2 activity was 139 times that of PF74, offering profitable lead compounds as encouraging HIV-2 inhibitor. In this paper, the MOA of representative compounds was discussed only on HIV-1. First, it was shown that 7n could directly bind to monomeric and hexameric HIV-1 CA with micromolar and nanomolar affinity, respectively. SPR-based competition experiments with CPSF6 and NUP153 peptides have proven that 7n binds within the NTD-CTD interface of hexameric CA, which is in agreement with our design efforts. SRI experiments displayed that 7n interferes with the HIV-1 life cycle in a dual-stage manner, influencing both early and late stages. Furthermore, employing MD simulation, we confirmed that 7n might bind to the same site as PF74 but with a different direction. Metabolic stability evaluation in HLM and human plasma indicated that 7m and 7n has moderately improved stability over PF74, providing the rationale for future improvement of the metabolic stability of this compound series.

  Taken together, this work culminated in the identification of phenylalanine-bearing HIV-1 capsid inhibitors with a novel chemotype and broad-spectrum anti-HIV activity, and highlights the potential of the NTD-CTD interface in the CA hexameric configuration as a promising target for the design of potent HIV inhibitors.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  Acknowledgments

  We gratefully acknowledge financial support from the National Natural Science Foundation of China (NSFC, Nos. 82173677, 81773574), the Key Project of NSFC for International Cooperation (No. 81420108027), the Shandong Provincial Key Research and Development Project (No. 2019JZZY021011), the Science Foundation for Outstanding Young Scholars of Shandong Province (No. ZR2020JQ31) and NIH/NIAID grant (No. R01AI150491, Cocklin, PI, Salvino, Co-I). We also would like to thank the OpenEye Scientific Software, Inc. for providing a free academic license.

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2022.06.034.

  
  
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• 

  Figure 1  Crystal structure of HIV-1 CA in complex with (A) CPSF6 peptide (PDB code: 4WYM) and (B) NUP153 peptide (PDB code: 6AYA). The figures are generated in PyMOL (www.pymol.org).

  下载: 全尺寸图片 幻灯片
  

  Figure 2  Design principle of novel small molecule inhibitors targeting HIV-1 CA NTD-CTD interface. The figures are generated in PyMOL (www.pymol.org).

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  Scheme 1  Reagents and conditions: (i) 4-Methoxy-N-methylaniline, HATU, DIEA, 0 ℃ to r.t.; (ii) TFA, DCM, r.t.; (iii) Bromoacetic acid, HATU, DIEA, 0 ℃ to r.t.; (iv) 1-Boc-3-oxopiperazine, Cs₂CO₃, DMF, 45 ℃; (v) TFA, DCM, r.t.; (vi) TEA, DCM, r.t.; (vii) H₂, 10%Pd·C, DCM, r.t.

  下载: 全尺寸图片 幻灯片
  

  Figure 3  Docking analysis and structural overlay of PF74 (yellow, PDB code: 4XFZ) and 7n (purple) compared with CPSF6 (blue) and NUP153 (orange). The co-crystal structures of the CPSF6 (PDB code: 4WYM) peptide and the NUP153 (PDB code: 4U0C) peptide are highlighted in blue and orange, respectively.

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  Table 1.  Anti-HIV activity and cytotoxicity in MT-4 Cells infected with HIV-1 III_B virus and HIV-2 ROD virus.

  下载: 导出CSV

  Table 2.  SPR results of 7b, 7m, 7n, and PF74 binding to monomeric and hexameric CA constructs.

  下载: 导出CSV
•