*2.2. NMR Experiments*

NMR experiments were performed on Bruker Avance Neo 600 MHz NMR spectrometer equipped with TXI triple resonance probe at 298 K. Two dimensional 15NHSQC spectra were recorded with 16 scans and 256 data points in the indirect dimension. Topspin 4.0.5 software was used to acquire and process the NMR data [19].

#### *2.3. HIV Inhibition Assay*

HIV-1 entry inhibition by LUMS1 was studied by using pseud-typed virus-based single-round infectivity assay, according to a previously reported method [20]. In this regard, LUMS1 at varying concentration was mixed with HXB2 strain of HIV-1 pseudo-typed viral particles at 37 ◦C followed by the addition of TZM-bl cells (NIH AIDS reagent program) at a concentration of 1 <sup>×</sup> 10<sup>4</sup> cells/100 <sup>μ</sup>L. After 48 h, cells were lysed and percent infection was measured through luciferase activity (BrightGlo, Promega, Maddison, WI, USA) for each dilution of inhibitor with respect to control containing no inhibitor. Similarly, the activity of LUMS1 against vesicular stomatitis virus (VSV) was also tested using virus pseudo-typed with VSV envelope and HIV-1 backbone.

### *2.4. HCV Infection Assay*

The anti-HCV activity of LUMS1 was evaluated using cell culture-derived infectious HCV (HCVcc) expressing an NS5A-GFP fusion protein in the presence of inhibitors as previously described [21]. Briefly, Huh-7.5 cells were seeded in 384-well plates (2.5 <sup>×</sup> <sup>10</sup><sup>3</sup> cells/well). LUMS1 were serially diluted in complete DMEM, added to each well of the plates, inoculated with HCVcc and incubated at 37 ◦C for 3 days. On day 3 post-infection (p.i.), cultured cells were fixed with 2% paraformaldehyde in PBS containing 10 μg/mL Hoechst 33,342 (Life Technologies, Waltham, MA, USA) for 30 min. HCV replication was analyzed by determining the number of GFP-positive cells using automated confocal microscopy (Opera, PerkinElmer, Waltham, MA, USA). Cytotoxicity was assessed by counting cell nuclei stained with Hoechst 33342 and normalized to untreated control cells.

#### *2.5. HCV Replication Assay*

HCV subgenomic replicon cells were treated with inhibitors as described previously [21]. Briefly, replicon cells were seeded in 384-well plates and treated with inhibitors for 72 h. The inhibitory effect of the protein on HCV RNA replication was monitored together with RNA polymerase inhibitor (sofosbuvir) as a reference compound. Viral replication and cytotoxicity was assessed as described above.

#### *2.6. HCV Pseudoparticle Assay*

Viral entry was assessed using HCV pseudoparticle (HCVpp) expressing a luciferase reporter gene as described before [22]. In brief, HEK293 T cells seeded for 1 day in T-75 flasks were co-transfected with 5 μg of an HCV E1/E2 envelope protein expression vector (genotype 1a) and 15 μg of pNL4.3.Luc.R– E–, HIV Gag-Pol expression packaging vector containing luciferase reporter gene using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). Supernatants containing the pseudoparticles were harvested at 72 h and used as HCVpp in the entry inhibition assay using Huh7.5 cells as described above in Section 2.3. EI-1, a known potent inhibitor of HCV entry [23] was used as a positive control at a single concentration of 10 μM.
