molecules Brief Report
Combining New Non-Nucleoside Reverse Transcriptase Inhibitors (RTIs) with AZT Results in Strong Synergism against Multi-RTI-Resistant HIV-1 Strains Fei Yu 1,2,† , Wen Li 2,† , Lili Wang 3,† , Yu Dai 1 , Xin Lu 1 , Qian Wang 2 , Lan Xie 4 and Shibo Jiang 2,5, * 1 2
3 4 5
* †
College of Life Sciences, Hebei Agricultural University, Baoding 071001, China;
[email protected] (F.Y.);
[email protected] (Y.D.);
[email protected] (X.L.) Key Laboratory of Medical Molecular Virology of MOE/MOH, School of Basic Medical Sciences, Fudan University, 130 Dong An Rd., Xuhui District, Shanghai 200032, China;
[email protected](W.L.);
[email protected] (Q.W.) Research Center of Chinese Jujube, Hebei Agricultural University, Baoding 071001, China;
[email protected] Beijing Institute of Pharmacology & Toxicology, Beijing 100850, China;
[email protected] Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065, USA Correspondence:
[email protected]; Tel.: +86-21-54237673 These authors contributed equally to this work.
Received: 24 May 2018; Accepted: 28 June 2018; Published: 2 July 2018
Abstract: Reverse transcriptase inhibitors (RTIs), including nucleoside RTIs (NRTIs) and non-nucleoside RTIs (NNRTIs), are critical antiretroviral drugs for the treatment of human immunodeficiency virus (HIV) infection. Emergence of multi-RTI resistance calls for the development of more potent therapeutics or regimens against RTI-resistant strains. Here, we demonstrated that combining azidothymidine (AZT) with a new NNRTIs under development, diarylpyridine (DAPA)-2e, diarylanilin (DAAN)-14h, or DAAN-15h, resulted in strong synergism against infection by divergent HIV-1 strains, including those resistant to NRTIs and NNRTIs, suggesting the potential for developing these novel NNRTIs as salvage therapy for HIV/acquired immune deficiency syndrome (AIDS) patients. Keywords: HIV-1; HAART; NNRTIs; antiviral activity; combination; synergism
1. Introduction Reverse transcriptase (RT) is an important target for the development of anti-HIV-1 drugs (HIV: human immunodeficiency virus) because of its important role in the HIV-1 life cycle [1]. RT inhibitors (RTIs) include a variety of nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs) that inhibit the conversion of single-stranded viral RNA into double-stranded pro-viral DNA in the HIV-1 infection process [2]. These RTIs are key components of the highly active antiretroviral therapy (HAART) used in clinics [3,4]. However, the rapid emergence of multi-RTI resistance has led to the failure of patients to respond to the current HAART. Recently, Xie and colleagues have identified two classes of novel HIV-1 NNRTIs, diarylanilines (DAANs) and diarylpyridines (DAPAs) (see Figure 1), with extremely high anti-HIV efficacy and improved resistance profile [5–8]. As a further study, we combined new DAPA or DAAN-NNRTIs (i.e., DAPA-2e, DAAN-14h, and DAAN-15h) with azidothymidine (AZT) [9,10] to explore their potential synergistic antiviral effects against laboratory-adapted and primary as well as RTI-resistant HIV-1 strains. Meanwhile, NNRTI drugs nevirapine (NVP) [11] and etravirine (ETR or TMC125) [12] were used as controls because the synergy Molecules 2018, 23, 1599; doi:10.3390/molecules23071599
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(NVP) [11] and etravirine (ETR or TMC125) [12] were used as controls because the synergy between between AZT and NVP [13] or between AZT and ETR [14] have been previously reported. Herein, AZT and NVP [13] or between AZT and ETR [14] have been previously reported. Herein, we reported wetheir reported their synergistic results ofor new DAPA or DAAN-NNRTIs/AZT synergistic results of new DAPA DAAN-NNRTIs/AZT combinations. combinations.
Figure 1. 1. Chemical inhibitor(NRTI) (NRTI)azidothymidine azidothymidine Figure ChemicalStructure Structureofofthe thenucleoside nucleoside reverse reverse transcriptase transcriptase inhibitor (AZT) and five non-nucleoside reverse transcriptase inhibitors (NNRTIs), including Nevirapine (NVP), (AZT) and five non-nucleoside reverse transcriptase inhibitors (NNRTIs), including Nevirapine (NVP), Etravirine (TMC125), DAAN-14h,h, and diarylpyridines (DAPA)-2e. Etravirine (TMC125),diarylanilines diarylanilines (DAANs)-15 (DAANs)-15 h,h,DAAN-14 and diarylpyridines (DAPA)-2e.
2. Results and Discussion 2. Results and Discussion shown Table1,1,all allNNRTI/AZT NNRTI/AZT combinations against infection AsAs shown inin Table combinationsexhibited exhibitedsynergistic synergisticeffects effects against infection by the laboratory-adapted HIV-1 strains IIIB (subtype X4) and Bal (subtype R5), and primary HIV-1 by the laboratory-adapted HIV-1 strains IIIB (subtype X4) and Bal (subtype R5), and primary HIV-1 isolates 94US_33931N (subtype R5) and 93IN101 (subtype C, R5), with combination index (CI) in the isolates 94US_33931N (subtype R5) and 93IN101 (subtype C, R5), with combination index (CI) in range of 0.025 to 0.904. The DAAN-15h/AZT combination showed the strongest synergism against the range of 0.025 to 0.904. The DAAN-15h/AZT combination showed the strongest synergism HIV-1 IIIB infection with a CI of 0.071, and dose reduction of DAAN-15h was about 44-fold, while against HIV-1 IIIB infection with a CI of 0.071, and dose reduction of DAAN-15h was about 44-fold, that of AZT was about 21-fold. Combining AZT with the novel NNRTI DAPA-2e, DAAN-14h, or while that of AZT was about 21-fold. Combining AZT with the novel NNRTI DAPA-2e, DAAN-14h, DAAN-15h, all exhibited strong synergism, which is comparable to that of the combination of AZT or DAAN-15h, all exhibited strong synergism, which is comparable to that of the combination of AZT with the FDA-approved NNRTI drug TMC125 or NVP, suggesting that these new NNRTIs have the with the FDA-approved drug TMC125 or NVP, suggesting that these new who NNRTIs potential to be used for NNRTI HIV/acquired immune deficiency syndrome (AIDS) patients havehave failedthe potential to be used for HIV/acquired immune deficiency syndrome (AIDS) patients who have failed to respond to the currently used NNRTIs. to respond to the currently usedNNRTI/AZT NNRTIs. combinations against AZT-resistant strains 964 and 629. Subsequently, we tested Subsequently, we tested NNRTI/AZT combinations against AZT-resistant 964 41,109 and 629. When tested alone, the IC50 values of AZT against these two resistant strains were strains 15,178 and When tested alone, the IC values of AZT against these two resistant strains were 15,178 and nM, respectively, whereas50those of NNRTIs tested alone were in the range of 0.6 to 34 nM. In the 41,109 nM, respectively, of NNRTIs alone were in the range of range 0.6 to of 347nM. In the combinations, the IC50 whereas values ofthose AZT against thesetested two resistant strains were in the to 4797 combinations, ICof values of AZT against these two resistant strains were in the range of 7 to nM, whereas the those NNRTIs ranged from 0.02 to 18 nM, with CI < 0.3 (see Table 1). In general, AZT 50 combined with DAAN-14h, DAPA-2e, and from DAAN-15h stronger against the two 4797 nM, whereas those of NNRTIs ranged 0.02 toexhibited 18 nM, with CI < synergism 0.3 (see Table 1). In general, resistant strains than the combination of AZT TMC125exhibited or NVP (see Table synergism 1). For example, thethe AZT combined with DAAN-14h, DAPA-2e, andwith DAAN-15h stronger against CIresistant values for combinations AZT with new against AZT-resistant strains 9641). and 629 were two strains than theofcombination of NNRTIs AZT with TMC125 or NVP (see Table For example, the range for of 0.003–0.040 and of 0.109–0.237, while the CIAZT-resistant values for combinations AZT629 theinCI values combinations AZT withrespectively, new NNRTIs against strains 964ofand with the currently FDA-approved NNRTIs against AZT-resistant strains 629 and 629 ranged from were in the range of 0.003–0.040 and 0.109–0.237, respectively, while the CI values for combinations of 0.231 to the 0.262 and from 0.292 to 0.423, NNRTIs respectively (see AZT-resistant Table 1). AZT with currently FDA-approved against strains 629 and 629 ranged from Results suggest that these new NNRTIs may be used 0.231 to 0.262 and from 0.292 to 0.423, respectively (see Tableto1).treat patients against HIV-1 mutants resistant to the currently available RTIs. Results suggest that these new NNRTIs may be used to treat patients against HIV-1 mutants resistant to the currently available RTIs.
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Table 1. Combination index (CI) and dose reduction in inhibition of infection by the HIV-1 strains by combining NNRTIs and AZT. DAPA-2e HIV-1 Strains (Tropism) IIIB (X4) Bal (R5) 94US_33931N (R5) 93IN101 (C, R5) 964 (R5/X4) 629 (R5/X4) RTMDR1 (X4)
CI 0.134 0.364 0.652 0.089 0.003 0.156 0.169
IC50 (nM) Alone
in Mixture
99.21 70.50 11.51 34.24 3.35 34.49 24.46
3.05 8.42 4.23 0.29 0.01 2.37 1.61
AZT
Dose Reduction (Fold) 32.50 8.38 2.72 116.19 460.00 14.52 15.16
IC50 (nM) Alone
in Mixture
39.31 34.47 148.91 730.12 15,178.32 41,109.61 935.39
4.07 8.42 42.32 58.95 7.28 3562.15 96.82
DAAN-14h HIV-1 Strains (Tropism) IIIB (X4) Bal (R5) 94US_33931N (R5) 93IN101 (C, R5) 964 (R5/X4) 629 (R5/X4) RTMDR1 (X4)
CI 0.144 0.528 0.904 0.141 0.023 0.109 0.279
IC50 (nM) Alone
in Mixture
39.12 3.77 1.65 1.55 0.62 13.87 1.34
2.42 0.31 0.71 0.07 0.01 0.84 0.20
IIIB (X4) Bal (R5) 94US_33931N (R5) 93IN101 (C, R5) 964 (R5/X4) 629 (R5/X4) RTMDR1 (X4)
CI
Dose Reduction (Fold) 16.18 12.26 2.33 22.09 54.04 16.55 6.67
0.071 0.852 0.063 0.095 0.040 0.237 0.116
IC50 (nM) Alone
in Mixture
3.98 5.36 0.47 0.60 0.74 16.57 1.59
0.09 0.52 0.02 0.02 0.02 2.00 0.09
9.66 4.10 3.52 12.39 2083.61 11.54 9.66 AZT
IC50 (nM) Alone
in Mixture
39.31 34.47 148.91 730.12 15,178.32 41,109.61 935.39
3.22 15.39 70.74 70.25 69.23 2010.51 120.26
DAAN-15h HIV-1 Strains (Tropism)
Dose Reduction (Fold)
Dose Reduction (Fold) 12.20 2.24 2.11 10.39 219.26 20.45 7.78
AZT
Dose Reduction (Fold) 44.22 10.31 20.72 27.72 32.03 8.29 17.45
IC50 (nM) Alone
in Mixture
39.31 34.47 148.91 730.12 15,178.32 41,109.61 935.39
1.86 26.02 2.27 43.21 139.03 4797.90 54.48
Dose Reduction (Fold) 21.13 1.32 65.65 16.90 109.17 8.57 17.17
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Table 1. Cont. TMC125 HIV-1 Strains (Tropism) IIIB (X4) Bal (R5) 94US_33931N (R5) 93IN101 (C, R5) 964 (R5/X4) 629 (R5/X4) RTMDR1 (X4)
IC50 (nM)
CI 0.179 0.883 0.203 0.110 0.231 0.292 0.194
Alone
in Mixture
0.89 3.20 2.09 1.49 0.73 5.86 1.24
0.08 1.93 0.18 0.03 0.13 1.20 0.17
AZT
Dose Reduction (Fold) 10.66 1.66 11.86 46.05 5.58 4.89 7.21
IC50 (nM) Alone
in Mixture
39.31 34.47 148.91 730.12 15,178.32 41,109.61 935.39
3.35 9.65 17.62 64.76 789.89 3599.14 51.74
NVP HIV-1 Strains (Tropism) IIIB (X4) Bal (R5) 94US_33931N (R5) 93IN101 (C, R5) 964 (R5/X4) 629 (R5/X4) RTMDR1 (X4)
CI 0.199 0.892 0.316 0.025 0.265 0.429 0.132
IC50 (nM) Alone
in Mixture
11.74 307.91 24.15 33.64 1.28 29.68 255.16
1.47 50.25 2.91 0.10 0.27 6.82 18.55
Dose Reduction (Fold) 11.73 3.57 8.45 11.27 19.22 11.42 18.08
AZT
Dose Reduction (Fold) 8.01 6.13 8.28 343.92 4.73 4.35 13.75
IC50 (nM) Alone
in Mixture
39.31 34.47 148.91 730.12 15,178.32 41,109.61 935.39
2.93 25.12 29.15 16.30 809.01 8832.58 55.66
Dose Reduction (Fold) 13.41 1.37 5.11 44.78 18.76 5.02 16.81
Note: HIV = human immunodeficiency virus. IIIB and Bal are laboratory-adapted HIV-1 strains, 94US_33931N and 93IN101 are primary HIV-1 strains, 964 and 629 are AZT-resistant HIV-1 strains, and RTMDR1 is the multiple RTI-resistant HIV-1 strain. A CI of >1, 1, and
