Despite major advances in antiretroviral therapy (ART), people living with human immunodeficiency virus type 1 (HIV-1) (PLWH) may fail to achieve viral suppression due to acquired drug resistance and an increasing spread of drug-resistant HIV-1 (Andersson et al., 2021). While newer generations of anti-HIV-1 drugs have a higher genetic barrier to resistance, the resistance mutations against the highly effective integrase strand transfer inhibitors (INSTIs) can develop (Andersson et al., 2021; Beesham et al., 2022; Li et al., 2022). Also, a subset of PLWH with extensive ART exposure has limited or exhausted treatment options also due to drug toxicity, drug–drug interactions, and/or comorbidities. Therefore, there is a need to develop new antiretroviral agents that can overcome these hurdles.

The dipeptide WG-am emerged as notably elevated in elite controllers (ECs) in comparison to viral progressors (VPs) and healthy controls (HCs). We have demonstrated the low toxicity of WG-am both in isolation and in combination with other compounds (Sperk et al., 2021; Cena-Diez et al., 2022). Building on these findings, we have shown that it has anti-HIV-1 activity (Sperk et al., 2021; Cena-Diez et al., 2023a). WG-am binds to the CD4 binding pocket of HIV-1 gp120 blocking the interaction between the virus and the CD4 receptor and also inhibits HIV-1 reverse transcription (Cena-Diez et al., 2023a). This dual antiviral mechanism and the low toxicity of WG-am suggest that it has the potential to be used against drug-resistant HIV-1.

Previously, we described the low toxicity and synergistic profile of WG-am and another novel antiviral compound, ssON, against drug-resistant HIV-1 (Cena-Diez et al., 2022; Cena-Diez et al., 2023b). To further study the efficacy of WG-am in combination with approved antiretrovirals, we now investigated its anti-HIV-1 activity combined with four antiretroviral agents from four classes: the protease inhibitor (PI) darunavir (DRV), the INSTI raltegravir (RAL), the nucleotide reverse transcriptase inhibitor (NRTI) tenofovir disoproxil fumarate (TDF), and the non-nucleoside RTI (NNRTI) efavirenz (EFV). The synergistic antiviral activity was evaluated against 28 drug-resistant HIV-1 isolates.

Our results demonstrated that the combination of WG-am and each of the four antiretrovirals had a strong synergistic profile in the majority of HIV-1 isolates resistant to each of the four major drug classes. We propose WG-am to be a viable complement to existing HIV-1 therapies, particularly in the context of drug-resistant HIV strains.

The existence of highly treatment-experienced patients with few remaining therapeutic options and the spread of drug-resistant HIV-1 illustrate the need to develop new anti-HIV-1 drugs despite the great success of ART in most patients (Clutter et al., 2016; Biswas et al., 2019; Chen et al., 2020). Here, we describe that our novel compound WG-am has synergistic effects with four antiretrovirals—RAL, TDF, DRV, and EFV—using drug-sensitive and drug-resistant isolates. The synergistic effects were stronger with the INSTI RAL and the PI DRV, although synergy was seen also for TDF (NRTI) and EFV (NNRTI) (Table 1). Our data suggest the value of combining WG-am with drugs of any class.

Among the mix of factors that contribute to the development of an elite control profile, the dipeptide WG-am stands out for being capable of inhibiting HIV-1 infection on its own. The dual mechanism of WG-am makes it an interesting candidate for combination with antiretrovirals from different families, especially targeting multiresistant HIV-1. Thus, the mechanisms give it a higher threshold for development of mutations at which the virus can escape its inhibition. Furthermore, the fact that it functions as both an entry and RT inhibitor allows it to have a synergistic profile with all the antivirals analyzed regardless of their mode of action. The dual-action mechanism of WG-am manifests a synergistic effect when combined with the four tested compounds, notwithstanding their diverse mechanisms of action. WG-am’s first mechanism of action is entry inhibition, which is distinct from the mechanisms of the tested ARVs. We have earlier shown that WG-am does not act after the RT step. Therefore, the lack of antagonistic effect between WG-am and the INSTI raltegravir and the PI darunavir is not unexpected. However, such an effect could possibly be anticipated in our examination due to the interaction between WG-am and the RTIs TDF and EFV in the RT step. Despite this anticipation, our findings demonstrated a notable synergistic relationship between WG-am and these compounds. Previously, we showed a synergy of WG-am and TDF against NRTI-resistant isolates 7324–1 and 52534–2 using the CalcuSyn software (Cena-Diez et al., 2022; Cena-Diez et al., 2023b). However, in the current manuscript, we expanded this finding and now report a synergy extended to all isolates exhibiting resistance against NNRTIs and NRTIs. These results are in line with our earlier experimental data hypothesizing that WG-am might act on the early steps of RT with a mechanism different from the NRTI and NNRTI. This observation highlights the promising potential of WG-am in addressing resistance issues while amplifying the therapeutic efficacy of existing ARVs.

Newer INSTIs—dolutegravir (DTG), cabotegravir (CAB), and bictegravir (BIC)—are cornerstones in modern ART. However, resistance may still develop, and there is a cross-resistance to the first-generation INSTI [RAL, elvitegravir (EVG)] (Duarte et al., 2022). The high efficacy of WG-am:EFV, WG-am:TDF, and WG-am:DRV on isolates resistant to INSTIs could make such WG-am combinations attractive options in patients failing regimens with INSTI resistance if WG-am is further developed and shown to hold through in clinical trials. Also, resistance to CAB develops somewhat more often in HIV-1 subtype A6 strains (Hu et al., 2023), wherein resistance to the NNRTI rilpivirine is present. Therefore, the efficacy of WG-am on all subtypes, earlier reported by us (Cena-Diez et al., 2022), may be relevant in such patients.

All combinations showed a strong synergism against the two INSTI-resistant isolates 4736_2 and 4736_4, which have 13 common drug resistance mutations, e.g., including N155H which reduces RAL and EVG susceptibility approximately 10- and 30-fold, respectively, and contributes to reduced susceptibility to DTG and CAB (Rhee et al., 2019). The 8070–1 isolate had three major INSTI-resistant mutations (G140S, Y143H, Q148H) giving resistance to RAL as well as DTG and CAB. G140S in combination with Q148 confers >100-fold reduced susceptibility to RAL, 10-fold reduced susceptibility to CAB, and 2- to 5-fold reduced susceptibility to DTG (Yoshinaga et al., 2015; Zhang et al., 2018; Saladini et al., 2019). Nonetheless, all our drug combinations completely inhibited the infection with these INSTI-resistant isolates. It can be noted that the WG-am:RAL inhibited the infection by more than 95% in all isolates despite high resistance to RAL (>100-fold reduced activity).

In addition, all WG-am combinations had a strong synergy or at least an additive effect (ZIP SS), against all PI-, NRTI-, and NNRTI-resistant isolates, except WG-am:DRV against PI-resistant isolates CA96454 and CA96453 (major mutations 46I and 90M), respectively, which showed a weak additive using ZIP SS. However, in both cases, a strong synergism was found using Chou–Talalay’s method at all effective doses >50% (Supplementary Tables 5A, B). When we used post-analysis tools of these results using SynergyFinder 3.0, we showed that the absence of synergism was due to the lack of synergy at low concentrations despite the strong synergism found at high inhibitory concentrations.

One of the antiviral mechanisms reported for WG-am is inhibition of HIV-1 RT (Cena-Diez et al., 2023a). Our data support that WG-am can be combined with NRTI and NNRTI inhibitors, like EFV or TDF, against RTI-resistant isolates. Also, WG-am itself was able to completely inhibit NRTI- and NNRTI-resistant isolates.

The usage of two different software programs, CalcuSyn and SynergyFinder 3.0, to calculate the efficacy of the drug combinations increases the strength of our data. CalcuSyn utilizes Chou–Talalay’s method, providing ED50, ED75, and ED90 values, for different inhibitory areas thereby offering insights into drug combination efficacy at various concentrations. SynergyFinder 3.0 employs the ZIP synergy method and stands out for its intuitive visualization and post-analysis tools. None out of the 112 comparisons, commented above, were reported as synergistic by Chou–Talalay’s method and antagonistic by SynergyFinder 3.0. In addition, 85 out of 112 analyses were reported as additive and the rest as synergistic by ZIP SS. However, almost half of those (34/85) that were reported as additives had an MSA that indicated that they presented a synergistic profile at high inhibitory concentrations. On the other hand, Chou–Talalay’s algorithm divides the calculation into sections depending on the inhibition obtained (ED50, ED75, ED90). Since the intention is to inhibit the infection completely, we suggest that for the purpose of our study, the CI value ED90 provided by Chou–Talalay’s software could be more relevant.

The absence of synergy reported by the ZIP method at low concentrations could potentially be important. Ideally, it is desired to obtain synergy at low concentrations of the compounds in order to diminish toxicity problems. However, in the present analysis, also the higher concentrations evaluated are well below the maximum non-toxic concentrations (Supplementary Figure 1). Furthermore, the intention of combination therapy is to completely inhibit the replication; therefore, the presence of synergy in more than 70% of the combinations against the different isolates resistant to antiretrovirals at high inhibitory concentrations (>90%) is of great relevance.

The observed naturally enhanced presence of WG in EC, seemingly without associated toxicity or adverse effects, highlights a potential correlation between elevated WG-am levels and disease control. While our study does not conclusively attribute elite controller status to this dipeptide, it underscores the need for further investigation into their specific biological role, both in persons with or without HIV-1. Understanding the precise mechanism by which WG-am functions and its potential impact on overall human health is imperative. Our findings suggest a promising avenue for the inclusion of WG-am in future therapeutic strategies; however, rigorous preclinical and early phase I clinical studies are warranted to comprehensively evaluate its biocompatibility, safety profile, and potential integration into existing antiretroviral combination therapies.

In conclusion, our in-vitro results show that WG-am has the potential to become a novel antiretroviral agent that can overcome HIV-1 drug resistance against other classes of antiretrovirals, exhibiting synergistic activity with commonly used antiretroviral agents. This makes WG-am an attractive candidate for further development as a new anti-HIV-1 drug.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.

FG: Data curation, Investigation, Writing – review & editing. AS: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Visualization, Writing – review & editing. RC: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Software, Supervision, Validation, Writing – original draft, Writing – review & editing.