Structure-Based Discovery and Characterization of a Preclinical Drug Candidate for the Treatment of HIV-1 Infection

HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) area key component of the current HIV-1 combination drug regimens. Although they exhibit potent anti-HIV-1 activity and modest toxicity, the emergence of mutant strains limits their application in clinical. Our previous research efforts contributed to the identification of compound K-5a2, which exhibits nanomolar activity in HIV-1-infected MT-4 cells. In this study, K-5a2 was shown to have a high level of anti-HIV-1 activity against various lab-adapted strains and clinical isolate strains, being comparable to ETR. Moreover, we showed the feasibility of K-5a2 as a preclinical anti-HIV-1 candidate by establishing its synergistic or additive anti-HIV-1 activity in combination with other representative anti-HIV-1 drugs and candidates. In addition, K-5a2 exhibited no inhibitory activity to the primary CYP isoforms and favorable pharmacokinetics. Taken together, its robust anti-HIV-1 potency, synergistic or additive effects with other anti-HIV drugs, and favorable pharmacokinetic and safety profiles make K-5a2 a potent alternative drug for HIV/AIDS treatment.


Introduction
According to the Joint United Nations Program on HIV/AIDS (UNAIDS) report in 2021, more than 38.4 million people are currently infected with human immunodeficiency virus (HIV-1), including 1.5 million new infections in 2021. Although 75% of people living with HIV received highly effective antiretroviral therapy (HAART) in 2021, 650,000 people nevertheless died of HIV-related causes [1,2]. Reverse transcriptase (RT) plays an important role in the HIV-1 replication cycle, reversing viral single-stranded RNA into double-stranded DNA [3]. HIV-1 RT is a heterodimer comprised of p66 and p51 subunits. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) inhibit HIV-1 RT by binding to an allosteric hydrophobic pocket located about 10 Å from the DNA catalytic site of RT [4]. NNRTIs mainly interact with amino acid residues such as L100, K101, K103, K104, V106, V108, V179, Y181, Y188, G190, F227, W229, L234, P236, and Y318 of the NNRTI's binding pocket (NNIBP) [5]. NNRTIs are essential components in HARRT due to the effective antiviral activity, high selectivity, and modest toxicity [6]. To date, there are more than V108, V179, Y181, Y188, G190, F227, W229, L234, P236, and Y318 of the NNRTI's bindi pocket (NNIBP) [5]. NNRTIs are essential components in HARRT due to the effective tiviral activity, high selectivity, and modest toxicity [6]. To date, there are more than chemical types of NNRTIs, including dihydro-alkoxyl-benzyl-oxopyrimidine, dia ether, diarylpyrimidine (DAPY), among others [7,8]. Among them, six NNRTIs have be approved for AIDS therapy by the US Food and Drug Administration (FDA), includ the first-generation drugs nevirapine (NVP), delavirdine (DLV), and efavirenz (EFV), a the second-generation drugs etravirine (ETR), rilpivirine (RPV), and doravirine (DO (Figure 1) [9]. Moreover, ainuovirine (ANV) and elsulfavirine (ESV) have received a proval for treating HIV-1 infection in China and Russia, respectively [10,11]. However variety of drug-resistant mutants (such as L100I, K101P/E, K103N/S, Y181C/I Y188L/I/C/V, V106A/M, F227L, E138K/R, and G190A/S) have emerged clinically, due the high genetic diversity of HIV-1, and these mutations in RT can develop drug resistan to existing RT inhibitors [12]. Additionally, their potency is hampered by their poor ph macokinetic profiles, which result in an increased opportunity for mutations to devel Therefore, the continuous effort to develop novel HIV-1 NNRTIs is paramount for dev oping new therapeutic drugs with improved resistance profiles, low toxicity and pharm cokinetic profiles. Over the past 10 years, our team has focused on the development of novel anti-H 1 drug candidates, based on the computer-aided drug design, synthetic organic chemist activity evaluation and mechanism validation, structural biology, and druggability ev uation. Previous research efforts have contributed to the discovery of a number of pot DAPY NNRTIs, such as the thiophene [3,2-d]pyrimidine derivatives K-5a2, 25a, and 2 the dihydrofuro [3,4-d]pyrimidine derivatives 13c2 and 14b; and the 2,4,5-trisubstitu pyrimidine derivative 16c (Figure 2), which exhibit significantly improved drug resistan profiles compared to those of the approved second-generation NNRTIs ETR and RPV. particular, compound K-5a2 was demonstrated to have optimal pharmacological prop ties and safety profiles, and was selected as an anti-HIV-1 drug candidate for further p clinical evaluation [13]. In this study, we describe the pharmacodynamic and pharma kinetic milestones achieved in the preclinical research on K-5a2. Over the past 10 years, our team has focused on the development of novel anti-HIV-1 drug candidates, based on the computer-aided drug design, synthetic organic chemistry, activity evaluation and mechanism validation, structural biology, and druggability evaluation. Previous research efforts have contributed to the discovery of a number of potent DAPY NNRTIs, such as the thiophene[3,2-d]pyrimidine derivatives K-5a2, 25a, and 24b; the dihydrofuro [3,4-d]pyrimidine derivatives 13c2 and 14b; and the 2,4,5-trisubstituted pyrimidine derivative 16c (Figure 2), which exhibit significantly improved drug resistance profiles compared to those of the approved second-generation NNRTIs ETR and RPV. In particular, compound K-5a2 was demonstrated to have optimal pharmacological properties and safety profiles, and was selected as an anti-HIV-1 drug candidate for further preclinical evaluation [13]. In this study, we describe the pharmacodynamic and pharmacokinetic milestones achieved in the preclinical research on K-5a2.

Ethics Statement
Ethics approval for this study and consent processes were provided by the Ethics Committee of the Kunming Institute of Zoology, Chinese Academy of Sciences (Approval Number: KIZRKX-2021-013 and SWYX-2006011).

Compounds and Reagents
K-5a2 and ETR were synthesized in our lab. MVC and DC521022 were provided by Hong Liu, Shanghai Institute of Materia Medica, Chinese Academy of Sciences. AZT, 3TC, and DRV were purchased from Meilunbio ® (Dalian, China). FTC was purchased from Zhongshuo Pharmaceutical Technology Development Inc (Beijing, China). RAL was purchased from Merck Sharp & Dohme (New Jersey, USA). TDF was purchased from Huangshi Fuertai Pharmaceutical Technology Inc (Huangshi, China). DTG was purchased from Macklin. T-20 was provided by Chengdu Shengnuo Biopharmaceutical Co., Ltd. FTC was dissolved in 0.9% sodium chloride injection and stored at −20 °C. AZT and T-20 were dissolved in RPMI-1640 media and stored at −20 °C. Other compounds were dissolved in dimethyl sulfoxide (DMSO) and stored at 4 °C.
Thiazolyl blue tetrazolium bromide, sodium chloride, Triton X-100, penicillin, potassium chloride, disodium hydrogen phosphate, Tween 20, potassium dihydrogen phosphate, PHA-P, and anti-mouse IgG (Fc specific) antibodies produced in goats were purchased from Sigma (Missouri, USA). Horseradish peroxidase-conjugated affinity pure goat anti-rabbit IgG (H+L) was purchased from KPL. Sodium dodecyl sulfate was purchase from BioFR0XX. Dimethyl sulfoxide was purchased from VWR Life Science AM-RESCO. Streptomycin sulfate was purchase from Solarbio. N, N-Dimethylformamine was purchased from Chengdu Kelong Chemical Inc. IL-2 was purchased from Dalian mei-

Ethics Statement
Ethics approval for this study and consent processes were provided by the Ethics Committee of the Kunming Institute of Zoology, Chinese Academy of Sciences (Approval Number: KIZRKX-2021-013 and SWYX-2006011).

Compounds and Reagents
K-5a2 and ETR were synthesized in our lab. MVC and DC521022 were provided by Hong Liu, Shanghai Institute of Materia Medica, Chinese Academy of Sciences. AZT, 3TC, and DRV were purchased from Meilunbio ® (Dalian, China). FTC was purchased from Zhongshuo Pharmaceutical Technology Development Inc (Beijing, China). RAL was purchased from Merck Sharp & Dohme (Kenilworth, NJ, USA). TDF was purchased from Huangshi Fuertai Pharmaceutical Technology Inc (Huangshi, China). DTG was purchased from Macklin. T-20 was provided by Chengdu Shengnuo Biopharmaceutical Co., Ltd. FTC was dissolved in 0.9% sodium chloride injection and stored at −20 • C. AZT and T-20 were dissolved in RPMI-1640 media and stored at −20 • C. Other compounds were dissolved in dimethyl sulfoxide (DMSO) and stored at 4 • C.
Thiazolyl blue tetrazolium bromide, sodium chloride, Triton X-100, penicillin, potassium chloride, disodium hydrogen phosphate, Tween 20, potassium dihydrogen phosphate, PHA-P, and anti-mouse IgG (Fc specific) antibodies produced in goats were purchased from Sigma (St. Louis, MO, USA). Horseradish peroxidase-conjugated affinity pure goat anti-rabbit IgG (H+L) was purchased from KPL. Sodium dodecyl sulfate was purchase from BioFR0XX. Dimethyl sulfoxide was purchased from VWR Life Science AMRESCO. Streptomycin sulfate was purchase from Solarbio. N, N-Dimethylformamine was purchased from Chengdu Kelong Chemical Inc. IL-2 was purchased from Dalian meilunbio ® . Lymphocyte Separation Medium (Human) was purchased from Tianjin Haoyang Biotechnology Inc. RPMI-1640 medium, DMEM medium, new born calf serum (NBCS) and fetal bovine serum (FBS) were purchased from Gibco. Skimmed milk powder produced by Inner Mongolia Yili Industrial Group Inc. was used. Anti-p24 McAb P6F4 was prepared in our laboratory. A reverse transcriptase assay kit was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Anti-HIV-1 Activity Assay
The antiviral assay of compounds was performed based on the viral cytopathic effect (CPE) [15]. Briefly, HIV-1 IIIB C8166 and compounds were co-cultured for three days. At three days post-infection, the number of syncytia in each well of the 96-well plates was counted under an inverted microscope (100×) to determine CPE. The 50% effective concentration (EC 50 ) was calculated using Origin 2019b 32Bit (Northampton, MA, USA, 2019).
The anti-HIV-1 activity of the compounds was tested using an MTT assay, as mentioned early [16]. In brief, MT-4 cells (4 × 10 4 /well) were infected with HIV-1 IIIB at different serial concentrations with a TCID 50 of 1300. At three days post-treatment, 100 µL of drug medium was added to 96-well plates at 37 • C and 5% CO 2 for four days. Approximately 30 µL MTT was further added to each well and incubated for 4 h. The cultural supernatant was removed, and 150 µL 12% SDS and 50% DMF were added. Then, the plates were incubated overnight. The absorbance value of the samples was read using a Bio Tek 800TS at 570/630 nm. EC 50 was calculated using Origin 2019b 32Bit (Northampton, MA, USA, 2019).
The anti-HIV-1 laboratory-adapted strain activity was measured in the presence of human serum, as mentioned early [17]. Briefly, C8166 cells were infected with HIV-1 IIIB with a TCID 50 of 2000. After 4 h, cells were washed two times to discard free viruses and resuspended in RPMI-1640 with 10% FBS. C8166 cells (50 µL, 8 × 10 5 /mL) were added in 96-well plates with different concentrations of compounds in the presence of 20% or 40% human serum at 37 • C and 5% CO 2 . After incubation for three days, the percentage inhibition of p24 was measured by ELISA. EC 50 was calculated using Origin 2019b 32Bit (Northampton, MA, USA, 2019).

Reverse Transcriptase Inhibition Assays
The inhibitory effect of compounds on HIV-1 RT was detected according to the reverse transcriptase assay kit (Sigma-Aldrich) instructions. Briefly, 20 µL of recombinant HIV-1 RT (final concentration in reaction was 0.05 ng/µL) was added, 20 µL of RT inhibitor diluted in lysis buffer and reaction mixture was added per reaction tube, and the mixture was incubated for 1 h at 37 • C. The samples (60 µL) were transferred into MP module wells. The MP modules was covered with foil and incubated for 1 h at 37 • C. After rinsing with washing buffer 5 times, 200 µL of anti-DIG-POD working dilution (200 mU/mL) was added per well. The MP modules was covered with a cover foil and incubated for 1 h at 37 • C. After rinsing with washing buffer 5 times, 200 µL of ABTS substrate solution was added per well and incubated at room temperature until the color development was sufficient. The absorbance value of the samples was read using a Bio Tek 800TS reader at 405/490 nm; EC 50 was calculated using Origin 2019b 32Bit (Northampton, MA, USA, 2019).

Acute Toxicity Assays
Twenty male and 20 female BALB/c mice (18-22 g) were divided into two groups, respectively. Compound K-5a2 was suspended in PEG400/normal saline (125 mg/mL). The mice were given intragastric administration with a dosage of 5000 mg/kg after fasting for 12 h. The death and body weight of the mice were monitored.

Anti-HIV-1 Activity of K-5a2
HIV-1 can infect human T lymphocytes, monocyte-derived macrophages, dendritic cells, and other cells in vivo and in vitro [19,20]. The drugs show different anti-HIV-1 activities in different cell types. Therefore, cell lines from multiple sources are needed to study the effects of these drugs on different HIV-1 strains. To evaluate the antiviral activity of K-5a2, the laboratory-adaptive strain HIV-1 IIIB was used to infect C8166 cells, and ETR was used as a control. The results are represented by EC 50 (a mean 50% effective concentration), CC 50 (a mean 50% cytotoxic concentration), and SI (selectivity index, CC 50 /EC 50 ratio). As shown in Table 1, the results showed that K-5a2 had a potential inhibitory effect on HIV-1 IIIB -induced C8166 cytopathy, with a promising activity (EC 50 = 1.88 ± 0.87 nM), low cytotoxicity (CC 50 > 50.00 µM) and higher selectivity index (SI > 26595.74), being comparable to those of ETR (EC 50 = 1.74 ± 0.67 nM, CC 50 > 50.00 µM, SI > 28735.63) ( Table 1, Figures 3 and 4). In C8166 cells acutely infected with HIV-1 IIIB , K-5a2 exhibited an EC 50 value of 6.11 ± 0.57 nM, also comparable to that of ETR (EC 50 = 5.71 ± 1.31 nM) (Table 1, Figure 4). Meanwhile, both compounds showed no cytotoxicity at a concentration of 50.00 µM, which led to their higher SI values. These properties suggest that K-5a2 has the potential to be an anti-HIV-1 drug comparable to ETR.  Figures 3 and 4). In C8166 cells acutely infected with HIV-1IIIB, K-5a2 exhibited an EC50 value of 6.11 ± 0.57 nM, also comparable to that of ETR (EC50 = 5.71 ± 1.31 nM) (Table 1, Figure 4). Meanwhile, both compounds showed no cytotoxicity at a concentration of 50.00 µM, which led to their higher SI values. These properties suggest that K-5a2 has the potential to be an anti-HIV-1 drug comparable to ETR.     Figure 4). Meanwhile, K-5a2 showed lower cytotoxicity in human PBMCs, with a CC50 value of 39.74 ± 6.29 µM (Table 1, Figure 3). These results suggest that K-5a2 has potential antiviral activity against HIV-1 clinical isolate strains comparable to that of ETR. A previous study demonstrated that plasma proteins, especially α1 acid glycoprotein, markedly affected the in vitro antiviral activity of the drug [23]. Therefore, the anti-HIV activity of K-5a2 in the presence of human serum was further tested. In the presence

MT-4 cells are also HIV-1 sensitive T cells. Unlike C8166 cells, MT-4 cells will not
form syncytia after being infected with HIV-1 but will cause cell death within 6-7 days. When we evaluated K-5a2 for its inhibitory effect on HIV-1 IIIB replication in MT-4 cells, we found that K-5a2 has a good protective effect against HIV-1 IIIB infection-induced MT-4 cell death, with an EC 50 of 1.74 ± 0.11 nM, being about three-fold more potent than that of ETR (EC 50 = 6.15 ± 1.76 nM) (Table 1, Figure 4). The double-stranded DNA of HIV-1 in chronically infected H9 cells has been integrated into the genome of the host cell. Therefore, anti-HIV-1 drugs acting on the DNA replication, assembling, maturation, and release steps after the integration of the HIV-1 replication cycle undertake antiviral activity in chronically infected cells. However, the compounds acting on the steps of adsorption, fusion, reverse transcription, and nuclear import before the integration of the virus replication cycle did not affect the viral replication of H9 cells chronically infected with HIV-1I IIB . Next, we evaluated the inhibitory effect of K-5a2 on HIV-1 replication in HIV-1 IIIB chronically infected H9 cells. The results showed that K-5a2 and ETR could not inhibit the replication at a concentration of 50.00 µM ( Table 1), suggesting that K-5a2 plays a role in suppressing the virus in the pre-integration stage.
HIV-1 clinical isolates refer to virus strains isolated from the body fluids and tissues of HIV-1-infected patients. The genetic variability and biological characteristics of clinical isolates isolated at the early stage of HIV-1 infection may reflect the virus quasispecies in patients to a certain extent [21]. According to the research on HIV molecular epidemiology, the main circulating recombinant forms (CRFs) of HIV-1 are CRF07_BC, CRF01_AE, and CRF08_BC in China [22]. Therefore, we further evaluated the potency of K-5a2 against HIV-1 clinical isolate strains that are widely prevalent in China, including HIV-1 KIZ001 (CRF07_BC), HIV-1 TC-1 (CRF01_AE), and HIV-1 WAN (CRF01_AE). Notably, K-5a2 had a broad inhibitory effect on these clinical isolates in human peripheral blood mononuclear cells (PMBCs), with EC 50 values of 2.16 ± 0.24 nM, 2.25 ± 0.87 nM, and 5.56 ± 2.16 nM, respectively, which were equivalent to that of ETR (EC 50 = 3.58 ± 0.62 nM, 2.27 ± 0.95 nM, and 4.58 ± 0.89 nM, respectively) (Table 1, Figure 4). Meanwhile, K-5a2 showed lower cytotoxicity in human PBMCs, with a CC 50 value of 39.74 ± 6.29 µM (Table 1, Figure 3). These results suggest that K-5a2 has potential antiviral activity against HIV-1 clinical isolate strains comparable to that of ETR.
A previous study demonstrated that plasma proteins, especially α1 acid glycoprotein, markedly affected the in vitro antiviral activity of the drug [23]. Therefore, the anti-HIV activity of K-5a2 in the presence of human serum was further tested. In the presence of 20% and 40% human serum, K-5a2 could inhibit HIV-1 replication in C8166 cells acutely infected with HIV-1 IIIB , with EC 50 values of 7.83 ± 1.73 and 7.87 ± 6.01 nM, respectively, which is comparable to the performance of ETR (EC 50 value of 6.56 ± 4.89 and 2.09 ± 0.65 nM) ( Figure 5). The results demonstrated that 20% and 40% human serum did not shield the anti-HIV-1 activity of K-5a2.

Inhibitory Activity to HIV-1 RT
To validate the binding target of K-5a2, it was tested for its ability to inhibit reco binant HIV-1 RT enzymes, and ETR was selected as a control drug. The results show that K-5a2 exhibited potent inhibitory activities toward RT, with an EC50 of 1.11 ± 0.32 µ which is comparable to that of ETR (EC50 = 1.31 ± 0.32 µM) ( Table 2). These results dem strate that the target of K-5a2 is HIV-1 RT and it acts as a classical NNRTI. Table 2. Inhibitory activity of K-5a2 against HIV-1 RT.

Combination Antiviral Activity Assay
HAART, the standard HIV-treatment regimen in clinical, usually comprises a co bination of three or more anti-HIV drugs, so any late-stage preclinical candidate shou be evaluated for its combined antiviral activity with other approved drugs with differ targets or mechanisms. Ideally, there should be a synergistic effect, or at least it shou have an addition effect. In this experiment, K-5a2 is used in combination with curren recognized and marketed anti-HIV drugs. The anti-HIV-1 activity of K-5a2 was evalua in two drug combination studies with 11 drugs representing six categories of HAA drugs, namely, NNRTI-ETR, NRTIs-emtricitabine (FTC), zidovudine (AZT), lamivud (3TC), tenofovir (TDF), INSTIs-dolutegravir (DTG), raltegravir (RAL), PI-daruna (DRV), CCR5-receptor inhibitor-maraviroc (MVC), thioraviroc (DC521022), and FIfuvirtide (T-20). The experimental program and data processing were designed accord to Zhou's median effect principle [24]. A drug combination was defined based on the co bination index (CI) as follows: CI value < 0.9, synergistic effect; CI value 0.9 to 1.1, addit effect; CI value > 1.1, antagonism effect [25]. The results demonstrated that 10 of the ot drugs tested showed synergistic or additive effects with K-5a2 on HIV-1IIIB-infected C81

Inhibitory Activity to HIV-1 RT
To validate the binding target of K-5a2, it was tested for its ability to inhibit recombinant HIV-1 RT enzymes, and ETR was selected as a control drug. The results showed that K-5a2 exhibited potent inhibitory activities toward RT, with an EC 50 of 1.11 ± 0.32 µM, which is comparable to that of ETR (EC 50 = 1.31 ± 0.32 µM) ( Table 2). These results demonstrate that the target of K-5a2 is HIV-1 RT and it acts as a classical NNRTI. Table 2. Inhibitory activity of K-5a2 against HIV-1 RT.

Combination Antiviral Activity Assay
HAART, the standard HIV-treatment regimen in clinical, usually comprises a combination of three or more anti-HIV drugs, so any late-stage preclinical candidate should be evaluated for its combined antiviral activity with other approved drugs with different targets or mechanisms. Ideally, there should be a synergistic effect, or at least it should have an addition effect. In this experiment, K-5a2 is used in combination with currently recognized and marketed anti-HIV drugs. The anti-HIV-1 activity of K-5a2 was evaluated in two drug combination studies with 11 drugs representing six categories of HAART drugs, namely, NNRTI-ETR, NRTIs-emtricitabine (FTC), zidovudine (AZT), lamivudine (3TC), tenofovir (TDF), INSTIs-dolutegravir (DTG), raltegravir (RAL), PI-darunavir (DRV), CCR5-receptor inhibitor-maraviroc (MVC), thioraviroc (DC521022), and FI-enfuvirtide (T-20). The experimental program and data processing were designed according to Zhou's median effect principle [24]. A drug combination was defined based on the combination index (CI) as follows: CI value < 0.9, synergistic effect; CI value 0.9 to 1.1, additive effect; CI value > 1.1, antagonism effect [25]. The results demonstrated that 10 of the other drugs tested showed synergistic or additive effects with K-5a2 on HIV-1 IIIB -infected C8166 cells, with the exception being NNRTI-ETR (Table 3). To determine the dose reduction effect (DRI) of the drug combination, the DRI was calculated. The DRI value represents the degree of dose reduction produced by the drug combination compared with the dose of each drug. The results showed the DRI value of K-5a2 combined with other drugs was 1.20-25.50-fold in the two-drug combination. Among the combinations tested, the combination of K-5a2 and AZT had the highest DRI value, producing a 25.21-fold reduction compared to the use of K-5a2 alone ( Table 3). As mentioned above, a novel anti-HIV-1 drug may be used in combination with existing HIV-1 treatments. The first-line regimens of HAART in China usually include two NRTIs and one NNRTI. The experimental results in this study indicated that K-5a2 and NRTIs have a good synergistic effect and can be considered as an alternative drug combination for antiretroviral therapy. The combination of K-5a2 and other target drugs shows a synergistic or additive effect, indicating that K-5a2 has good combination prospects and strong inhibitory activity at low doses. Our research provides reference values for clinical trials.

In Vitro Effects of K-5a2 on CYP Enzymatic Inhibitory Activity
Cytochrome P450 (CYP450) is the most important enzyme in the microsome mixedfunction oxidase system, and more than 90% of drugs are metabolized by CYP450 in clinical [26]. Metabolic drug interactions induced and inhibited by CYP450 enzymes may alter the pharmacokinetics, efficacy, and toxicity of combined drugs [27]. ETR showed strong inhibitory activity against CYP2C9 and CYP2C19, with IC 50 values of 0.28 µM and 0.50 µM, respectively. As depicted in Table 4, K-5a2 exhibited an IC 50 value of 18.92 µM for CYP3A4M. In the case of CYP1A2, CYP2C9, CYP2C19, and CYP2D6, K-5a2 showed no inhibitory activity at a concentration of 50.00 µM. The results demonstrated that K-5a2 has no or weak inhibitory activity against the primary CYP isoforms, and has been associated with a reduced possibility of drug-drug interactions.

Pharmacokinetics of K-5a2 in Wistar Rats
A detailed pharmacokinetic study of K-5a2 was carried out to determine its druggability in BALB/c mice at three oral dosage levels (30, 60 and 120 mg/kg) and after one intravenous dose (2 mg/kg) ( Figure 6 and Table 5). After injection of 2 mg/kg K-5a2, the maximum concentration (Cmax) in male and female mice was 1078 µg/L and 1098 µg/L, and the half-time (T1/2) was 2.18 h and 2.35 h, respectively. After a single oral administration of K-5a2 at 30, 60, or 120 mg/kg, K-5a2 was rapidly absorbed and peaked within 0.25-0.5 h; the Cmax was linear with the dose. Moreover, oral bioavailability was 6.8 ± 1.3% and 8.5 ± 2.7% in male and female mice, respectively, which is sufficient for a drug candidate.

Pharmacokinetics of K-5a2 in Wistar Rats
A detailed pharmacokinetic study of K-5a2 was carried out to determine its drug bility in BALB/c mice at three oral dosage levels (30, 60 and 120 mg/kg) and after o intravenous dose (2 mg/kg) ( Figure 6 and Table 5). After injection of 2 mg/kg K-5a2, maximum concentration (Cmax) in male and female mice was 1078 μg/L and 1098 μg and the half-time (T1/2) was 2.18 h and 2.35 h, respectively. After a single oral administ tion of K-5a2 at 30, 60, or 120 mg/kg, K-5a2 was rapidly absorbed and peaked within 0.2 0.5 h; the Cmax was linear with the dose. Moreover, oral bioavailability was 6.8 ± 1. and 8.5 ± 2.7% in male and female mice, respectively, which is sufficient for a drug can date.

Assessment of Acute Toxicity
A single-dose toxicity evaluation of K-5a2 was conducted in male and fem BALB/C mice. When K-5a2 was gavaged at a dose of 5000 mg/kg, there were no obvio clinical symptoms and no obvious abnormalities in body weight (Figure 7), suggest that the maximum tolerated dose is greater than 5000 mg/kg.

Assessment of Acute Toxicity
A single-dose toxicity evaluation of K-5a2 was conducted in male and female BALB/C mice. When K-5a2 was gavaged at a dose of 5000 mg/kg, there were no obvious clinical symptoms and no obvious abnormalities in body weight (Figure 7), suggesting that the maximum tolerated dose is greater than 5000 mg/kg.

Conclusions
In general, the data presented in this study show that K-5a2, a new NNRTI, exhib different toxicity in different cells; the results are consistent with the positive control dr ETR. K-5a2 demonstrated high anti-HIV activity against various lab-adapted strains a clinical isolate strains, which was comparable to ETR. The potent antiviral activity of 5a2 against HIV-1 laboratory-adapted strains (HIV-1IIIB) and clinical isolate strains (H 1TC-1, HIV-1KIZ001, and HIV-1WAN) in different cell lines, including C8166, MT-4, and PBM was also shown. In particular, the activity of K-5a2 was approximately three times m potent than that of ETR in MT-4 cells. In the combination antiviral activity assay, Kdemonstrated a synergistic or additive effect when combined with other approved dru with different mechanisms, indicating that it can be used as a main component of HAA Moreover, K-5a2 exhibited no inhibitory activity against to the primary CYP isoforms, a showed favorable pharmacokinetic and safety properties. Taken together, the potent an HIV-1 activity, synergistic or additive effects with other anti-HIV-1 drugs, and favora druggability make K-5a2 a potent alternative drug for the further development HIV/AIDS treatment.  Informed Consent Statement: Not applicable; study did not involve humans.

Data Availability Statement:
The data presented in this study are available in the present articl

Conclusions
In general, the data presented in this study show that K-5a2, a new NNRTI, exhibits different toxicity in different cells; the results are consistent with the positive control drug ETR. K-5a2 demonstrated high anti-HIV activity against various lab-adapted strains and clinical isolate strains, which was comparable to ETR. The potent antiviral activity of K-5a2 against HIV-1 laboratory-adapted strains (HIV-1 IIIB ) and clinical isolate strains (HIV-1 TC-1 , HIV-1 KIZ00 1, and HIV-1 WAN ) in different cell lines, including C8166, MT-4, and PBMC, was also shown. In particular, the activity of K-5a2 was approximately three times more potent than that of ETR in MT-4 cells. In the combination antiviral activity assay, K-5a2 demonstrated a synergistic or additive effect when combined with other approved drugs with different mechanisms, indicating that it can be used as a main component of HAART. Moreover, K-5a2 exhibited no inhibitory activity against to the primary CYP isoforms, and showed favorable pharmacokinetic and safety properties. Taken together, the potent anti-HIV-1 activity, synergistic or additive effects with other anti-HIV-1 drugs, and favorable druggability make K-5a2 a potent alternative drug for the further development of HIV/AIDS treatment.