HIV Integrase inhibitor
BI 224436
BI-224436 is a highly potent inhibitor of the HIV integrase. It has an excellent ADME profile and combines high solubility with very good cell permeability. in vivo, BI-224436 showed a good PK profile in all investigated species with oral bioavailability. Given the excellent antiviral potency demonstrated by this compound and its favorable properties, it was the first non-catalytic site integrase inhibitor (NCINI) to reach the clinic.
More information
Following the reverse transcription of viral RNA into cDNA, HIV-Integrase (IN) is responsible to integrate newly synthesized viral cDNA into the host cell genome. IN fulfils this function via a two-steps process: a 3’-dinucleotide processing reaction and a strand transfer reaction. In the first step, IN binds to viral DNA as part of the pre-integration complex (PIC) in the cytoplasm and excises a dinucleotide from each 3’-end. Thereafter, the PIC is transported into the nucleus where the 3’-ends of the viral DNA are covalently linked to the 5’-ends of the host cell DNA in a process known as strand transfer1.
Currently approved IN inhibitors (raltegravir, elvitegravir, dolutegravir) are IN strand transfer inhibitors (INSTIs) binding to the IN active site. In contrast, BI 224436 binds to a conserved allosteric pocket at the dimer interface of the catalytic core domain (CCD) of IN and acts through a distinct mechanism. The term non-catalytic-site integrase inhibitors (NCINIs) is used to differentiate both series of compounds having different MOA.
BI 224436 is the first NCINI validated in a phase-1a clinical trial and differs from INSTIs in many regards: 1) BI 224436 binds to an allosteric pocket, which functional effect is to inhibit the 3’-processing step of IN. Additionally, binding to this allosteric pocket also prevents protein-protein interaction with the host cell Lens Epithelial Derived Growth Factor (LEDGF) required for HIV-1 viral replication; 2) BI 224436 shows a distinct resistancy profile against virus mutations and maintains its antiviral activity against a panel of mutants that emerge during treatment failure with other inhibitor classes (INSTIs and NNRTIs)2.
HIV integrase in complex with an analog of BI 224436 (PDB code: 4NYF)
BI 224436 displays an IC50 = 15 nM in a LTR-cleavage assay measuring the 3’-processing hydrolysis reaction of a dinucleotide from the DNA 3’-end of each viral long terminal repeat. Moreover, BI 224436 exhibits excellent antiviral potency (EC50 11-27 nM) in a panel of wild-type and recombinant viruses with different aa124/aa125 variants of IN.
A hallmark of BI 224436 is the low influence of human serum on antiviral potency (ssEC50 2.1-fold) which is the assay used for human dose predictions for HIV clinical candidates.
| Probe name / negative control | BI 224436 | BI-0449 |
| MW [Da]a | 442.5 | 311.8 |
| LTR-cleavage assay (IC50) [nM]b | 15 | 6,840 |
| Luc-RGA (EC50) [nM]c | 11-27 | >40,000 |
| ssEC50 (50% HS), fold-changed | 2.1 | n.a. |
| MTT-C8166 (CC50) [nM]e | 110,000 | 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
bLong Terminal Repeat (LTR) DNA 3’-processing assay measures the enzymatic activity of HIV-1 integrase to perform the essential 3’-processing reaction. Integrase binds to the viral DNA LTR ends at the CAGT-3’ sequence and catalyzes the removal of the two terminal nucleotides. In this homogeneous assay, the HIV-1 LTR DNA substrate consists of two annealed oligonucleotides, a 31-mer modified at the 3’ end with a black hole quencher (BHQ) and a 31-mer modified at the 5’ end with rhodamine red-X N-hydroxysuccinimide (NHS) ester (5RhoR-XN). Enzymatic cleavage by integrase releases the terminal dinucleotides and black hole quencher, which allows the rhodamine fluorescence to be detected.
cLuciferase reporter gene assay (Luc-RGA): C8166 LTR-Luc cells infected with different HIV-1 viral strains was incubated at 37°C in presence of various concentration of inhibitors for 3 days. Viral strains used: HxB2 virus (A124/T125 IN variant); NL4.3 virus (T124/T125 IN variant); or recombinant NL4.3 virus (A124/T125, A124/A125, N124/T125, or N125/A125 IN variants)
dDetermined by measurement of EC50 +50% human serum. C8166 LTR-luciferase reporter cells were infected with HIV-1 NL4.3 virus in presence of 50% human serum. After 3-days of cell incubation at 37°C, Steady Glo was added and luminescence was monitored as a measurement of the HIV replication in the presence of various concentrations of inhibitor.
eCytotoxicity for C-8166 LTR-Luc cells was determined using the tetrazolium salt MTT metabolic assay after 3 days of incubation.
BI 224436 combines excellent solubility at all physiologically relevant pH values (Sol. pH 2.0-6.8 >0.84 mg/mL) with very good cell permeability as measured in the Caco-2 assay (A-B: 14 x10-6 cm/s) and minimal cytochrome inhibition (CYP2C9 IC50: 20 μM). BI 224436’s optimized quinoline C4-aryl substituent improved metabolic stability in human hepatocytes (13% QH).
| Probe name / negative control | BI 224436 | BI-0449 |
| LogD @ pH 11 | 0.3 | 0.1 |
| Solubility @ pH 2.0 / 6.8 [µg/mL] | >752 / >848 | n.a. |
| Caco-2 permeability AB @ pH 7.4 [*10-6 cm/s] | 14 | n.a. |
| Caco-2 efflux ratio | 0.3 | n.a. |
| Microsomal stability [% QH] (human/mouse/rat/monkey/dog) | 14 / 6.4 / 7 / 11 / 16 | <46 / >39 / <28/ n.a. / n.a. |
| Hepatocyte stability [% QH] (human/mouse/rat/monkey/dog) | 13 / 12 / 9 / 13 / 32 | n.a. |
| Plasma Protein Binding [%] (human/mouse/rat/monkey/dog) | 84.3 / 97.3 / 98.2 / 78.0 / 75.5 | n.a. |
| hERG [inh. % @ 100 µM] | 5.8 | n.a. |
| CYP 3A4 (IC50) [µM] | 23 | >50 |
| CYP 2C9 (IC50) [µM] | 20 | 19 |
| CYP 1A2 (IC50) [µM] | >30 | n.a. |
| CYP 2C19 (IC50) [µM] | >30 | >50 |
| CYP 2D6 (IC50) [µM] | >30 | >50 |
BI 224436 combines excellent aqueous solubility across pH values (2.0 – 6.8) and good cellular permeability. In addition, BI 224436 displays a good PK profile in all investigated animal species with oral bioavailability typically ranging from 54-100%. For rodents and dogs, in vivo clearance (CL, % QH) is lower than values predicted by in vitro hepatocyte stability whereas monkey species is showing an opposite trend (i.e., higher in vivo clearance than anticipated). For rats, low in vivo clearance might be attributed to biliary enterohepatic recirculation of the parent compound/acyl glucuronide3,6.
Given the overall favourable potency, in vitro ADME-CMC properties, in vivo PK profile, and clean animal toxicology studies, BI 224436 was advanced into phase-1a clinical development.
| BI 224436a | mouseb | ratb | monkeyc | dogc |
| Clearance [% QH] | 0.8 | 0.7 | 23 | 8 |
| Vss [L/kg] | 0.20 | 0.45 | 0.54 | 0.88 |
| t1/2 [h] | 2.6 | 8.8 | 1.4 | 5.9 |
| Cmax [μM] | 15 | 13 | 4.8 | 12 |
| AUC [μM*h] | 99 | 75 | 6.3 | 24 |
| F [%] | 100 | 54 | 82 | 81 |
a The oral formulation contained 1% MP, 0.3% Tween 80, 0.5% MC; the i.v. formulation contained 70% PEG, 30% water.
bi.v. dose: 0.2 mg/kg; p.o. dose: 0.4 mg/kg.
ci.v. dose: 1 mg/kg; p.o. dose: 2 mg/kg
No HIV-1 animal model routinely available for PD testing.
BI-0449 is a representative compound of the 3-acetic acid-4-aryl quinoline hit series discovered during HTS campaign. Compared to optimized BI 224436, it is ~450-fold less active in the biochemical LTR-cleavage assay. However BI-0449 is inactive in any HIV-1 replicon cellular assay up to the highest concentration tested (>40 μM).
-BI-0449.png)
BI-0449, which serves as a negative control.
| SELECTIVITY DATA AVILABLE | BI 224436 | BI-0449 |
SafetyScreen44™ with kind support of  | Yes | Yes |
| Invitrogen® | No | No |
| DiscoverX® | No | No |
| Dundee | No | No |
Download selectivity data:
BI%20224436_selectivityData.xlsx
BI-0449_selectivityData.xlsx
The X-ray co-crystal structure of target in complex with BI 224436 is not available. However, a closely related analog binding to a conserved allosteric pocket of the catalytic core domain of integrase has been reported (Figure 3, PDB code: 4NYF)1.
For a review on investigational HIV integrase inhibitors see reference 8.
BI 224436 was the first non-catalytic-site integrase inhibitor (NCINI) in the clinic. Its excellent potency and ADME profile make it a valuable in vivo tool compound.
Discovery of BI 224436, a Noncatalytic Site Integrase Inhibitor (NCINI) of HIV-1
Fader L. D., Malenfant E., Parisien M., Carson R., Bilodeau F., Landry S., Pesant M., Brochu C., Morin S., Chabot C., Halmos T., Bousquet Y., Bailey M. D., Kawai S. H., Coulombe R., LaPlante S., Jakalian A., Bhardwaj P. K., Wernic D., Schroeder P., Amad M., Edwards P., Garneau M., Duan J., Cordingley M., Bethell R., Mason S. W., Bös M., Bonneau P., Poupart M.-A., Faucher A.-M., Simoneau B., Fenwick C., Yoakim C., Tsantrizos Y.
Med. Chem. Lett. 2014, 5, 422-427.
Preclinical Profile of BI 224436, a Novel HIV-1 Non-Catalytic Site Integrase Inhibitor
Fenwick C., Amad M., Bailey M. D., Bethell R., Bös M., Bonneau P., Cordingley M., Coulombe R., Duan J., Edwards P., Fader L. D., Faucher A.-M., Garneau M., Jakalian A., Kawai S., Lamorte L., LaPlante S., Luo L., Mason S., Poupart M.-A., Rioux N., Schroeder P., Simoneau B., Tremblay S., Tsantrizos Y., Witvrouw M., Yoakim C.
Antimicrobial Agents and Chemotherapy 2014, 58, 3233-3244
Aligning Potency and Pharmacokinetic Properties for Pyridine-Based NCINIs
Fader L. D., Bailey M., Beaulieu E., Bilodeau F., Bonneau P., Bousquet Y., Carson R. J., Chabot C., Coulombe R., Duan J., Fenwick C., Garneau M., Halmos T., Jakalian A., James C., Kawai S. H., Landry S., LaPlante S. R., Mason S. W., Morin S., Rioux N., Simoneau B., Surprenant S., Thavonekham B., Thibeault C., Trinh T., Tsantrizos Y., Tsoung J., Yoakim C., Wernic D.
Med. Chem. Lett. 2016, 7, 797-801
Inhibitors of human immunodeficiency virus replication
Y. S. Tsantrizos et al.
2009, patent application WO 2009/062285.
Concise and Practical Asymmetric Synthesis of a Challenging Atropisomeric HIV Integrase Inhibitor
Fandrick K. R., Li W., Zhang Y., Tang W., Gao J., Rodriguez S., Patel N. D., Reeves D. C., Wu J. P., Sanyal S., Gonnella N., Qu B.,Dr. Haddad N., Lorenz J. C., Sidhu K., Wang J., Ma S., Grinberg N., Lee H., Tsantrizos Y., Poupart M.-A., Busacca C. A., Yee N. K., Lu B. Z., Senanayake C. H.
Angew. Chem. Int. Ed. 2015 54, 7144-7148
Minimizing the Contribution of Enterohepatic Recirculation to Clearance in Rat for the NCINI Class of Inhibitors of HIV
Fader L. D., Carson R., Morin S., Bilodeau F., Chabot C., Halmos T., Bailey M. D., Kawai S. H., Coulombe R., Laplante S., Mekhssian K., Jakalian A., Garneau M., Duan J., Mason S. W., Simoneau B., Fenwick C., Tsantrizos Y., Yoakim C.
Med. Chem. Lett. 2014, 5, 711-716
Aligning Potency and Pharmacokinetic Properties for Pyridine-Based NCINIs
Fader L. D., Bailey M., Beaulieu E., Bilodeau F., Bonneau P., Bousquet Y., Carson R. J., Chabot C., Coulombe R., Duan J., Fenwick C., Garneau M., Halmos T., Jakalian A., James C., Kawai S. H., Landry S., LaPlante S. R., Mason S. W., Morin S., Rioux N., Simoneau B., Surprenant S., Thavonekham B., Thibeault C., Trinh T., Tsantrizos Y., Tsoung J., Yoakim C., Wernic D.
Med. Chem. Lett. 2016, 7, 797-801
When you plan a publication, please use the following acknowledgement:
BI 224436 was kindly provided by Boehringer Ingelheim via its open innovation platform opnMe, available at https://www.opnme.com.