HIV NNRTI
BI-2540
BI-2540 is a potent inhibitor of the HIV non-nucleoside reverse transcriptase (NNRT). This compound is also cross-reactive against clinically relevant mutants of reverse transcriptase. BI-2540 is suitable for both in vitro and in vivo experiments. In rats, it has shown a low clearance and good bioavailability upon oral administration.
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Description of the protein: The human immunodeficiency virus (HIV-1) reverse transcriptase (RT) enzyme performs the retrotranscription of the viral single-stranded RNA into double-stranded linear DNA. The viral genomic retrotranscription arises from the cooperative effect of two enzymatic functions of RT: a DNA polymerase activity (copy of the RNA/DNA template) and ribonuclease H activity (cleavage of RNA when part of a RNA/DNA duplex).
Protein domain structure: RT of HIV-1 is a heterodimer composed of subunit p66 (560 AA) and p51 (440 AA). The larger subunit p66 contains both active sites responsible for RT enzymatic functions (DNA polymerase and RNase H activities)1.
Molecular mechanism: there are two different classes for RT inhibitors: nucleoside RT inhibitors (NRTI) and non-nucleoside RT inhibitors (NNRTI). NNRTI bind to an allosteric site adjacent to the polymerase active site (~10 Å). Binding of NNRTI interferes with the chemical step of DNA synthesis by affecting the alignment of the primer terminus with the polymerase active site. Thus, NNRTIs efficacy stems from the structural rearrangement in the p66 subunit which precludes viral DNA synthesis.
Biology: HIV-1 largely infects CD4-positive T lymphocytes and macrophage cells, thus destroying the immune system. Lymphoid organs are a major reservoir of ongoing HIV-1 replication. HIV life cycle consists of seven steps: 1) binding, 2) fusion, 3) reverse transcription, 4) integration, 5) replication, 6) assembly, and 7) budding.
Disease link: HIV-1 infects >30 million people worldwide. Infected patients cannot be cured; therefore a “triple drug cocktail” of antiretroviral therapy (ART) must be continuously administered. Combination drug therapies suppress viral load by blocking viral replication and improving efficacy to overcome resistant variants1,2.
HIV NNRTI in complex with related structure GW564511 (PDB code: 3DLG)
BI-2540 is a potent HIV non-nucleoside reverse transcriptase (NNRT) inhibitor. For cellular efficacy against RT mutants please refer to the selectivity section.
| Probe name / negative control | BI-2540 | BI-2439 |
| MW [Da]a | 527.8 | 475.9 |
| HIV1-RT Pico, WT (IC50)b [nM] | 12 | 5053 |
| HIV replicon ELISA, WT (EC50)c [nM] | 0.76 | n.a. |
| HIV replicon LUC, WT (EC50)d [nM] | 2.6 | n.a. |
aFor the salt form you will get, please refer to the label on the vial and for the molecular weight of the salt, please refer to the FAQs
bEnzymatic assay: HIV-1 reverse transcriptase (Picogreen Fluorescence Assay); 15 min pre-incubation before adding substrate solution, 50 min incubation at 37°C, PicoGreen as a fluorescent intercalator.
cHIV viral replication assay: C8166 cells, 72h incubation at 37°C, readout: ELISA quantification of p24
dHIV-1 luciferase assay: C8166 cells, 72h incubation at 37°C, readout: RLU of luciferase, Steady Glo.
| Probe name / negative control | BI-2540 | BI-2439 |
| LogD @ pH 11 | 2.7 | 1.3 |
| Solubility @ pH 7.0 [µg/mL] | >52 | n.a. |
| Caco-2 permeability AB @pH 4 / 7.4 [*10-6 cm/s] | 23 | 12 |
| Caco-2 efflux ratio | 0.1 | 1.4 |
| Microsomal stability [% QH] (human) | <24 | n.a. |
| Plasma Protein Binding [%] (human) | 99.5 | 99.8 |
| CYP 3A4 (IC50) [µM] | 18 | >50 |
| CYP 2C9 (IC50) [µM] | >30 | 8.5 |
| CYP 1A2 (IC50) [µM] | >30 | >50 |
| CYP 2C19 (IC50) [µM] | >30 | >50 |
| CYP 2D6 (IC50) [µM] | >30 | >50 |
BI-2540 shows a low clearance and good bioavailability upon p.o. dosing in rats.
| BI-2540 | Rat |
| Clearance [% QH]a | 2.9 |
| Vss [l/kg]a | 2.3 |
| Mean residence time after i.v. dose [h]a | 12.8 |
| tmax [h]b | 1.0 |
| Cmax [μM]b | 11860 |
| AUC [μM*h]b | 125620 |
| F [%]b | 53 |
ai.v. dose: 1 mg/kg
bp.o. dose: 2.6 mg/kg
BI-2439 is a relatively close analog of BI-2540 with significantly lower activity (420-fold) in the HIV-RT Picogreen Fluorescence assay (IC50 = 5 µM) and is therefore offered as an in vitro negative control.
BI-2439 which serves as a negative control
Cellular efficacy against RT mutants: HIV-1 luciferase assays: C8166 cells, 72h incubation at 37°C; readout: RLU of luciferase, Steady Glo
| Mutations | BI-2549 | X-fold vs. wt |
| Wild type (WT) EC50 [nM] | 2.58 | -- |
| A98G EC50 [nM] | 3.94 | 1.5 |
| K103N EC50 [nM] | 2.36 | 0.9 |
| V106A EC50 [nM] | 12.8 | 5.0 |
| V106I EC50 [nM] | 4.19 | 1.6 |
| E138K EC50 [nM] | 15.0 | 5.8 |
| Y181C EC50 [nM] | 2.44 | 0.9 |
| Y188C EC50 [nM] | 0.38 | 0.1 |
| Y188L EC50 [nM] | 39.1 | 15 |
| G190A EC50 [nM] | 3.10 | 1.2 |
| P236L EC50 [nM] | 7.53 | 2.9 |
| L100I/K103N EC50 [nM] | 3.18 | 1.2 |
| K103N/G190A EC50 [nM] | 8.16 | 3.2 |
| K103N/V108I EC50 [nM] | 4.82 | 1.9 |
| K103N/Y181C EC50 [nM] | 6.54 | 2.5 |
| K103N/P225H EC50 [nM] | 4.77 | 1.8 |
| V106A/E138K EC50 [nM] | 119 | 46 |
| V106A/P236L EC50 [nM] | 199 | 77 |
| V106I/E138K EC50 [nM] | 38.1 | 15 |
| V106I/P236L EC50 [nM] | 36.1 | 14 |
| E138K/P236L EC50 [nM] | 60.3 | 23 |
| SELECTIVITY DATA AVILABLE | BI-2540 | BI-2439 |
SafetyScreen44™ with kind support of  | Yes | Yes |
| Invitrogen® | No | No |
| DiscoverX® | No | No |
| Dundee | No | No |
Download selectivity data:
BI-2540_selectivityData.xlsx
BI-2439_selectivityData.xlsx
No X-ray available.
Commercially available NNRTIs: Nevirapine, Delavirdine, Efavirenz, Dapivirine, Etravirine, Rilpivirine2.
BI-2540 is a potent inhibitor of HIV non-nucleoside reverse transcriptase (NNRT) and cross-reactive against clinically relevant mutants of reverse transcriptase.
Practical Synthesis of A Benzophenone-Based NNRT Inhibitor of HIV-1
Wang X.-J., Zhang L., Sun X., Lee H., Krishnamurthy D., O’Meara J. A., Landry S., Yoakim C., Simoneau B., Yee N. K., Senanayake C. H.
Org. Process Res. Dev. 2012, 16, 561-566.
When you plan a publication, please use the following acknowledgement:
BI-2540 was kindly provided by Boehringer Ingelheim via its open innovation platform opnMe, available at https://www.opnme.com.