Capsid Assembly Inhibitor
BI-3257
BI-3257 (or BD 1) is an inhibitor of HIV replication with nanomolar activity. It prevents the formation of the capsid core structure that encapsulates the viral genome and its associated enzymes, reverse transcriptase and integrase. It is equally potent against the wild-type and mutant viruses resistant to antiretroviral inhibitors. It is offered with a negative control, BI-3545.
More information
BI-3257 targets the HIV-1 capsid (CA) protein. CA is initially synthesized as the central region of the 55-kDa Gag polyprotein, which is the protein that mediates the assembly and budding of the immature virion. In this context, CA provides key protein-protein interactions required for immature virion assembly2,3. During viral maturation, proteolytic cleavage of Gag releases CA, allowing the protein to assemble into a cone-shaped central capsid that surrounds the viral RNA genome and its associated enzymes, reverse transcriptase and integrase4. The capsid structure is stabilized by multiple weak protein-protein interactions.
Complex of a compound highly related to BI-3257 with the N-terminal domain of the capsid monomer, as solved by X-ray crystallography (PDB code: 4E91)1.
BI-3257 displays an EC50 of 70 nM in a viral replication assay and induces disassembly of viral capsid structures with an IC50 of 160 nM. No cytotoxicity was found up to a concentration of 28 µM.
| PROBE NAME / NEGATIVE CONTROL | BI-3257 | BI-3545 |
| MW [Da, free base]a | 578.6 | 468.4 |
| Assay A (EC50) [nM] | 70 1,6 | n.d. |
| Assay B (IC50) [nM] | >413 | n.d. |
| Assay C (IC50) [nM] | 160 6 | >9,300 |
| Assay D (CC50) [nM] | >28,000 | n.d. |
a For the salt form you will get, please refer to the label on the vial and for the molecular weight of the salt, pleae refer to the FAQs
Assay A: Standard antiviral replication assay1; C8166-LTR-luciferase cells were infected with HIV-1 NL4-3 for 1.5 h and were seeded in assay wells already containing inhibitors. The EC50 corresponds to a 50% reduction of viral replication as observed via spectroscopic measurement of the luciferase expression levels.
Assay B: Capsid assembly assay7; An assay that targets the assembly of the capsid protein (CA) in vitro into capsid-like oligomers. The assay makes use of a recombinant CA protein fused to nucleocapsid (NC) as found in the immature Gag polyprotein. in vitro assembly of the CA-NC fusion protein into capsid-like oligomers can be stimulated by single-stranded DNA oligonucleotides of TG repeat sequence at low protein concentrations. They stimulate assembly by binding to NC and remain bound in the assembled capsid-like oligomers. The assay will measure the assembly of CA-NC oligomers in the presence of a mixture of two TG repeat oligonucleotides: one labeled with biotin, the other labeled with fluorescein. CA-NC oligomers will be captured on strepavidin-coated 384 well plates and washed and detected by measuring the level of fluorescence they contain as only capsid-like oligomers will be able to bind both kinds of oligonucleotides simultaneously.
Assay C: Capsid disassembly assay7; This assay adds a step to the HIV Capsid Assembly assay (Assay B). The whole assembly process is performed, and then inhibitors are incubated with assembled CA complexes, that will lead to disassembly. The level of disassembly is measured by the fluorescence captured on neutravidin-coated 384 well plates.
Assay D: See reference 5
BI-3257 shows a good cell permeability and a half-life of 86 and 67 min in human and rat microsomes, respectively. BI-3257 shows rather low solubility.
| PROBE NAME / NEGATIVE CONTROL | BI-3257 | BI-3545 |
| logD @ pH 2/11 | 4.5 / 4.4 | 4.1 / 0.9 |
| Solubility @ pH 2.0/6.8 [µg/mL] | 0.13 / <0.1 | 4.8 / 573 |
| Caco-2 permeability AB @ pH 7.4 [*10-6 cm/s] | 9.9 6 | n.a. |
| Microsomal stability (human/rat) [t1/2, min] | 86 / 67 6 | n.a. |
| CYP 3A4 (IC50) [µM] | 3.6 6 | n.a. |
| CYP 2D6 (IC50) [µM] | >30 6 | n.a. |
BI-3545 is structurally highly related to BI-3257 but is much more soluble due to the presence of a carboxylate group. It was found inactive in the capsid disassembly assay at a concentration of 9.3 µM.
BI-3545 which serves as a negative control
BI-3257 displays an EC50 of 70 nM in a viral replication assay and induces disassembly of viral capsid structures with an IC50 of 160 nM. In selectivity panels it showed no relevant off-target effects.
The negative control BI-3545 showed in 1 out of 44 targets (PDE4D2) inhibition with more than 50% @ 10 µM.
| SELECTIVITY DATA AVILABLE | BI-3257 | BI-3545 |
SafetyScreen44™ with kind support of  | Yes | Yes |
| Invitrogen® | No | No |
| DiscoverX® | No | No |
| Dundee | No | No |
Download selectivity data:
BI-3257_selectivityData.xlsx
BI-3545_selectivityData.xlsx
The X-ray crystal structure of target in complex with BI-3257 was not determined. However, the structure with a highly related compound is available (PDB code: 4E91)1.
We have disclosed a second chemotype binding to the same pocket of the CA N-terminal domain as BI-3257 (see compound BM11). Compounds of this type have a distinct binding mode and induce a somewhat different structural change in the CA protein. Also, BM1-type compounds likely primarily inhibit the maturation of the conical capsid structure, while BI-3257 acts at an earlier stage of the replication cycle: it already inhibits the assembly of the CA-containing Gag polyprotein, which mediates the assembly and budding of the immature virion. Another compound described earlier and binding to a similar surface location on the capsid protein is CAP-1. However, this compound binds less deep in the protein and has a binding affinity of only ~800 µM9. Other molecules binding to at least two additional locations on CA have been described (see 1 and references therein).
BI-3257 inhibits replication of HIV-1 with nanomolar potency by inhibiting the assembly of the capsid core of the virus. A related negative control is available (BI-3545).
Distinct effects of two HIV-1 capsid assembly inhibitor families that bind the same site within the N-terminal domain of the viral CA protein
Lemke C. T., Titolo S., Von Schwedler U., Goudreau N., Mercier J-F., Wardrop E., Faucher A-M., Coulombe R., Banik S. S R, Fader L., Gagnon A., Kawai S. H., Rancourt J., Tremblay M., Yoakim C., Simoneau B., Archambault J., Sundquist W. I., Mason S. W.
J Virol. 2012, 86:6643–6655.
Discovery of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly
Fader L. D., Bethell R., Bonneau P., Bös M., Bousquet Y., Cordingley M. G., Coulombe R., Deroy P., Faucher A-M., Gagnon A., Goudreau N. Grand-Maître C., Guse I., Hucke O., Kawai S. H., Lacoste J-E., Landry S., Lemke C. T., Malenfant E., Mason S., Morin S., O'Meara J., Simoneau B., Titolo S., Yoakim C.
Bioorg Med Chem Lett 2011, 21:398–404.
Optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of HIV capsid assembly inhibitors 2: structure-activity relationships (SAR) of the C3-phenyl moiety
Fader L. D., Landry S., Goulet S., Morin S., Kawai S. H., Bousquet Y., Dion I., Hucke O., Goudreau N., Lemke C. T., Rancourt J., Bonneau P., Titolo S., Amad M., Garneau M., Duan J., Mason S., Simoneau B.
Bioorg Med Chem Lett 2013 23:3401–3405.
1-Phenyl-1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione derivatives as HIV replication inhibitors and their preparation and use for the treatment of HIV infection
Simoneau B., Deroy P., Fader L., Faucher A. M., Gagnon A., Grand-Maitre C., Kawai S., Landry S., Mercier J. F., Rancourt J.
WO 2011/100838, 2011
Optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of HIV capsid assembly inhibitors 1: addressing configurational instability through scaffold modification
Fader L. D., Landry S., Morin S., Kawai S. H., Bousquet Y., Hucke O., Goudreau N., Lemke C. T., Bonneau P., Titolo S., Mason S., Simoneau B.
Bioorg Med Chem Lett 2013 23:3396–3400.
When you plan a publication, please use the following acknowledgement:
BI-3257 was kindly provided by Boehringer Ingelheim via its open innovation platform opnMe, available at https://www.opnme.com.