Dual-targeted anti-CMV/anti-HIV-1 heterodimers

Abstract

Despite the development of efficient anti-human immunodeficiency virus-1 (HIV-1) therapy, HIV-1 associated pathogens remain a major clinical problem. Human cytomegalovirus (CMV) is among the most common HIV-1 copathogens and one of the main causes of persistent immune activation associated with dysregulation of the immune system, cerebrovascular and cardiovascular pathologies, and premature aging. Here, we report on the development of dual-targeted drugs with activity against both HIV-1 and CMV. We synthesized seven compounds that constitute conjugates of molecules that suppress both pathogens. We showed that all seven compounds exhibit low cytotoxicity and efficiently inhibited both viruses in cell lines. Furthermore, we chose a representative compound and demonstrated that it efficiently suppressed replication of HIV-1 and CMV in human lymphoid tissue ex vivo coinfected with both viruses. Further development of such compounds may lead to the development of dual-targeted anti-CMV/HIV-1 drugs.

Keywords: CMV; HIV-1; Heterodimers; Human tissues; Viral diseases.

Figure 1.. Structure of the compounds used for the synthesis of heterodimer 3c or resulting from its hydrolysis

The anti-CMV compound 1a-1f were combined with the anti HIV-1 compound AZT to synthesize six AZT-based heterodimers 3a-3f. Upon hydrolysis, heterodimers 3a-3f generate AZT and the anti CMV compounds 2a-2f. The anti HIV-1 activity of all heterodimers and AZT are presented in Table 1. The anti CMV compounds 1a-1f had no anti HIV-1 activity, as previously reported [23]. The anti CMV compound 2c (chosen as a representative of the group of compounds 2a-2f) also did not have any anti HIV-1 activity (see Table 1). The anti CMV activities of the compounds 1a-1f are in the range 4–20 μM [23]. The anti-CMV activity of the heterodimers 3a-3f and compounds 2a-2f are presented in Table 2. AZT does not have any anti CMV activity [28].

Figure 2.. Inhibition by heterodimer 3c of HIV-1 replication in human tonsillar tissues

27 tonsillar blocks from each of 6 donors were infected with X4_LAI.04 and incubated with 3c at five concentrations ranging from 1 nM to 10 μM for 12 days or used as untreated controls. We evaluated HIV-1 replication from p24_gag core antigen present in tissue culture medium using a Luminex bead-based assay. The inhibition of HIV-1 was expressed as percentage of donor-matched untreated control for each concentration of 3c. Presented are means ± SEM of HIV-1 inhibition.

Figure 3.. Cell toxicity of heterodimer-inoculated ex vivo tissue cultures

At day 12 of culture, 27 human lymphoid tissue blocks treated or not with 3c were stained with anti-CD3QD655, anti-CD4QD605, anti-CD8QD800, anti-CD45RA-FITC, anti-CCR7-PECy7, anti-CXCR4-PE, and anti-CCR5-APC antibodies (stain 1) or stained with anti-CD3-QD655, anti-CD4-QD605, anti-CD8-QD800, anti-CD25-APC, anti-CD38-PE, anti-CD95-PE-Cy7, and anti-HLA-DR-BV570 antibodies (stain 2). Means ± SEM (error bars) of various cell subsets compared to matched untreated tissue performed with tissues from seven different donors are presented. Cell depletion for each cell subset was normalized by tissue-block weights.

Figure 4.. Suppression of HIV-1 and CMV replication in coinfected tonsillar tissues

(A) Blocks of human tissue were co-inoculated with HIV-1 (X4_LAI.04) and CMV (AD169) and cultured for 15 days at 37°C. Tissues were treated with 3c at 10 μM or left untreated (control). We evaluated HIV-1 replication by measuring p24gag in culture medium, using a Luminex bead-based assay. (B) The replication of CMV was measured with real-time PCR for viral DNA accumulated in culture medium. For each day, presented are means ± SEM of viral replication as percent of cumulative virus produced in untreated control condition in 6 tonsillar tissues from 6 different donors.

Figure 5.. Inhibition of HIV-1 and CMV replication by heterodimer 4c

(A) Structure of the compounds used in the synthesis of the 3TC-based heterodimer 4c or resulting from its hydrolysis. (B) Anti HIV-1 activity of heterodimer 4c in MT-4 cell cultures. Graph was obtained by fitting the data points to a sigmoidal dose-response curve using Prism software. EC₅₀ of 4c in MT-4 cell cultures was 2.0 μM (95% CI (1.4–2.8). (C) Inhibition of HIV replication in human tonsillar tissues. Human tonsillar tissues ex vivo were inoculated with X4_LAI.04 and incubated with 4c at five concentrations ranging from 1 nM to 10 μM for 12 days. HIV-1 inhibition was expressed as percentage of donor-matched untreated control for each concentration of 4c. Means ± SEM of HIV-1 inhibition in tonsillar tissues blocks from 3 donors, relative to results for matched untreated tissues are presented. (D & E) Suppression of HIV-1 and CMV replication in coinfected tonsillar tissues. As for heterodimer 3c, tonsils were coinfected with HIV-1 (X4_LAI.04) and CMV (AD169) and treated with 4c 10 μM or left untreated (control). (D) The replication of HIV-1 was assessed by measuring p24gag using a Luminex bead-based assay. (E) Replication of CMV was evaluated by real-time PCR by measuring viral DNA in culture medium. For each day, means ± SEM of viral replication as percent of total virus produced in untreated control condition in tonsillar tissues from 3 different donors are presented.

Figure 5.. Inhibition of HIV-1 and CMV replication by heterodimer 4c

(A) Structure of the compounds used in the synthesis of the 3TC-based heterodimer 4c or resulting from its hydrolysis. (B) Anti HIV-1 activity of heterodimer 4c in MT-4 cell cultures. Graph was obtained by fitting the data points to a sigmoidal dose-response curve using Prism software. EC₅₀ of 4c in MT-4 cell cultures was 2.0 μM (95% CI (1.4–2.8). (C) Inhibition of HIV replication in human tonsillar tissues. Human tonsillar tissues ex vivo were inoculated with X4_LAI.04 and incubated with 4c at five concentrations ranging from 1 nM to 10 μM for 12 days. HIV-1 inhibition was expressed as percentage of donor-matched untreated control for each concentration of 4c. Means ± SEM of HIV-1 inhibition in tonsillar tissues blocks from 3 donors, relative to results for matched untreated tissues are presented. (D & E) Suppression of HIV-1 and CMV replication in coinfected tonsillar tissues. As for heterodimer 3c, tonsils were coinfected with HIV-1 (X4_LAI.04) and CMV (AD169) and treated with 4c 10 μM or left untreated (control). (D) The replication of HIV-1 was assessed by measuring p24gag using a Luminex bead-based assay. (E) Replication of CMV was evaluated by real-time PCR by measuring viral DNA in culture medium. For each day, means ± SEM of viral replication as percent of total virus produced in untreated control condition in tonsillar tissues from 3 different donors are presented.

Scheme 1.

Synthesis of conjugates 3a-f. Reagents and conditions: (a) 1. BrCH₂COOEt, K₂CO₃, DMF; 2. LiOH, H₂O/EtOH; (b) AZT, DCC, Py.