BRD9 inhibitor | BI-9564
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Highlights
BI-9564 was developed in collaboration with the Structural Genomics Consortium (SGC). The compound binds with high affinity to BRD9 KD(BRD9, ITC) = 14 nM and with lower affinity to closely related BRD7 KD(BRD7, ITC) = 239 nM. CECR2 was the only other identified off-target (KD(CECR2, ITC) = 258 nM), but with no effect in cells at 1 µM (FRAP assay). BI-9564 is completely negative on BET family members in the AlphaScreen (>100 µM). BI-9564 with its high potency, selectivity and good ADME parameters is a good probe to study BAF complex biology in vitro and in vivo.1 We also offer BI-6354 as a negative control for in vitro experiments.
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Target information
The mammalian switch/Sucrose Non-Fermentable (SWI/SNF) complex is one of four mammalian chromatin remodelling complexes. Recurrent inactivating mutations in certain subunits of this complex have been identified in different cancers. Despite its known roles in tumor suppression, the mammalian SWI/SNF complex has recently received attention as a potential target for therapeutic inhibition.2
The human bromodomain family encompasses 61 domains, found on 46 proteins and BRD9 and BRD7 proteins containing a single acetyl-lysine reader bromodomain and are components of the chromatin remodelling SWI/SNF BAF complex. A recent study highlighted a role of another SWI/SNF subunit, BRD9, in leukemia growth. The BRD9 bromodomain (BD) was shown to be required for the proliferation of acute myeloid leukemia (AML) cells.3
BRD9 with BI-7273, as observed by X-ray.1
In vitro Activity
| Probe name / Negative control | BI-9564 | BI-6354 |
| MW [Da] | 353.4 | 279.3 |
| ITC(BRD9) (KD) [nM]a | 14 | n.d. |
| ITC(BRD7) (KD) [nM]a | 239 | n.d. |
| AlphaScreen(BRD9) (IC50) [nM]a | 75 | 27192 |
| AlphaScreen(BRD7) (IC50) [nM]a | 3410 | 81896 |
| AlphaScreen(BRD4-BD1) (IC50) [nM]a | >100000 | >100000 |
a for detailed assay conditions see Ref. 1
In vitro DMPK and CMC parameters
BI-9564 has an attractive ADME/PK profile for in vivo proof-of-concept studies, namely, high solubility at pH 6.8, moderate to high in vitro metabolic stability, low plasma protein binding, and no cytochrome P450 inhibition.
| Probe name / negative control | BI-9564 | BI-6354 | ||||
| logP | 1.5 | n.d. | ||||
| Solubility @ pH 6.8 [µg/ml] | >90 | >59 | ||||
| CACO permeability @ pH 7.4 [*10-6 cm/s] | 11 | n.d. | ||||
| CACO efflux ratio | 4.5 | n.d. | ||||
| Microsomal stability (human/mouse/rat) [% QH] | <24 | 35 | <23 | <24 | n.d. | <23 |
| Hepatocyte stability (human/mouse/rat) [% QH] | 17 | 56 | 17 | n.d. | ||
| Plasma protein binding (human/mouse/rat) [%] | 42 | 35 | 23 | n.d. | ||
| CYP 3A4 (IC50)[µM] | >50 | n.d. | ||||
| CYP 2C8 (IC50)[µM] | >50 | n.d. | ||||
| CYP 2C9 (IC50)[µM] | >50 | n.d. | ||||
| CYP 2C19 (IC50)[µM] | >50 | n.d. | ||||
| CYP 2D6 (IC50)[µM] | 49 | n.d. | ||||
In vivo DMPK parameters
BI-9564 showed moderate to high absorptive permeability and moderate in vivo plasma clearances upon i.v. dosing. BI-9564 displayed high oral bioavailability.
| BI-9564 | MOUSE |
| Clearance [% QH]a | 59 |
| Mean residence time after iv dose [l/kg]a | 0.6 |
| tmax [h]b | 0.7 |
| Cmax [nM]b | 5400 |
| F [%]b | 88 |
| Vss [l/kg]a | 2.1 |
ai.v. 5 mg/kg
bp.o. 20 mg/kg
In vivo pharmacology
BI-9564 showed efficacy at oral doses of 180 mg/kg in a disseminated mouse model of AML with a median TGI value of 52% on day 18, which translated into an additional survival benefit compared to that of the control group.1
Negative control
BI-6354 is available as an in vitro negative control. It shows only very weak potency on BRD9 and BRD7 and no potency on BRD4. Also see the “In vitro activity” section.
BI-6354 which serves as an in vitro negative control
Selectivity
BI-9564 was screened on 48 bromodomains, 55 GPCRs and a large kinase panel (324 kinases). Beside BRD9 and BRD7, CECR2 was the only bromodomain off-target (258 nM, ITC), but with no cellular effect at 1 µM in FRAP assay. All GPCRs except M1(h) (75%) and M3(h) (86%) showed less than 40% crtl inhibition at 10 µM. From the 324 kinases only 3 kinases (ACVR1, TGFBR1, ACVR2B) showing an % ctrl inhibition of > 40%, for which the measured IC50 values were > 5 µM.
| BI-9564 | Selectivity data available |
| Cerep® | No |
| Panlabs® | No |
| Invitrogen® | Yes |
| DiscoverX® | Yes |
| Dundee | No |
Co-crystal structure of the BI probe compound and the target protein
The Xray crystal structure of target in complex with BI-9564 is available (PDB code: 5F1H).1
reference molecules
LP994, I-BRD95, BI-72731, “compound 28”6.7
Summary
BI-9564 binds with high affinity to BRD9 KD(BRD9, ITC) = 14 nM and with lower affinity to closely related BRD7 KD(BRD7, ITC) = 239 nM. CECR2 was the only other identified off-target (KD(CECR2, ITC) = 258 nM), but with no effect in cells at 1 µM (FRAP assay). BI-9564 is completely negative on BET family members in the AlphaScreen (>100 µM).
BI-9564 with its high potency, selectivity and good ADME parameters is a good probe to study BAF complex biology in vitro and in vivo.1,7 We also offer BI-6354 as a negative control for in vitro experiments.
Supplementary data
References
Structure-based design of an in vivo active selective BRD9 inhibitor
Martin L. J., Koegl M., Bader G., Cockcroft X.-L., Fedorov O., Fiegen D., Gerstberger T., Hofmann M. H., Hohmann A. F., Kessler D., Knapp S., Knesl P., Kornigg S., Müller S., Nar H., Rogers C., Rumpel K., Schaaf O., Steurer S., Tallant C., Vakoc C. R., Zeeb M., Zoephel A,, Pearson M., Boehmelt G., McConnell D.
J. Med. Chem. 2016, 59, 4462−4475.
Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance
Fedorov O., Castex J., Tallant C., Owen D. R., Martin S., Aldeghi M., Monteiro O., Filippakopoulos P., Picaud S., Trzupek J. D., Gerstenberger B. S., Bountra C., Willmann D., Wells C., Philpott M., Rogers C., Biggin P. C., Brennan P. E., Bunnage M. E., Schüle R., Günther T., Knapp S., Müller S.
Sci. Adv. 2015, 10, e1500723.
Sensitivity and engineered resistance of myeloid leukemia cells to BRD 9 inhibition
Hohmann A. F., Martin L. J., Minder J. L., Roe J. S., Shi J., Steurer S., Bader G., McConnell D., Pearson M., Gerstberger T., Gottschamel T., Thompson D., Suzuki Y., Koegl M., Vakoc C. R.
Nat. Chem. Biol. 2016, 12, 672–679.
LP99: Discovery and Synthesis of the First Selective BRD7/9 Bromodomain Inhibitor
Clark P. G. K., Vieira L. C. C., Tallant C., Fedorov O., Singleton D. C., Rogers C. M., Monteiro O. P., Bennett J. M., Baronio R., Müller S., Daniels D. L., Méndez J., Prof. Knapp S., Brennan P. E., Dixon D. J.
Angew. Chem. Int. Ed. Engl. 2015, 54, 6217-21.
Discovery of I-BRD9, a Selective Cell Active Chemical Probe for Bromodomain Containing Protein 9 Inhibition
Theodoulou N. H., Bamborough P., Bannister A. J., Becher I., Bit R. A., Che K. H., Chung C. W., Dittmann A., Drewes G., Drewry D. H., Gordon L., Grandi P., Leveridge M., Lindon M., Michon A.-M., Molnar J., Robson S. C., Tomkinson N. C. O., Kouzarides T., Prinjha R. K., Humphreys P. G.
J. Med. Chem. 2016, 59, 1425–1439.
Design and synthesis of potent and selective inhibitors of BRD7 and BRD9 bromodomains
Hay D. A., Rogers C. M., Fedorov O., Tallant C., Martin S., Monteiro O. P., Müller S., Knapp S., Schofielda C. J., Brennan P. E.
Med. Chem. Commun. 2016, 6, 1381-1386.
An Advanced Tool To Interrogate BRD9
Karim R. M., Schönbrunn E.
J. Med. Chem. 2016, 59, 4459−4461.
