CMV polymerase inhibitor | BI-9553
Highlights
BI-9553 is a potent and selective non-nucleoside CMV polymerase inhibitor (EC50 < 30 nM). This compound has been extensively characterized. It shows good cell permeability, reasonable hepatocyte stability across species, and good bioavailability in rat and mouse. Thus, BI-9553 is suitable for both in vitro and in vivo experiments.
Background information
Target Information
Human cytomegalovirus (HCMV) belongs to the beta-herpes virus family and is among the largest of the DNA viruses.1 HCMV can cause severe life-threatening infections especially in immunocompromised and immunonaïve patients. Congenital CMV infection is also a leading cause of birth defects, such as hearing loss. One essential enzyme for viral replication is the CMV DNA polymerase encoded by the UL54 gene.2 Inhibition of CMV polymerase enzymatic activity has been clinically validated, however current gold standard therapies face considerable challenges such as drug resistance and poor tolerability.3
Homology model of HCMV polymerase showing the binding mode of an inhibitor structurally related to BI-9553, bound to the active site. (Homology model based on the X-ray structure of Herpes simplex virus polymerase, PDB code: 2gv9
In vitro activity
BI-9553 shows a good potency with an EC50 < 30 nM in a qPCR cell-based assay. Cell activity could be dissociated from cytotoxicity depending on different cell lines.
| Probe name / negative control | BI-9553 | BI-0309 |
| MW [Da] | 510.9 | 492.5 |
| HCMV Pol. LAN (IC50) [nM]a | 46 | >5,000 |
| HCMV qPCR_AD169 (EC50) [nM]b | 28 | >5,800 |
| Syber Green II (IC50) [nM]c | >100,000 | n.a. |
| Cytotoxicity (CC50) [μM]d | 1-30 | n.a. |
a HCMV Polymerase LANCE TR-FRET Assay: purified recombinant HCMV polymerase (UL54) using a Digoxigenin-labeled oligonucleotide priming a heteropolymeric template. The enzymatic activity is measured by incorporating biotin-dUTP in the nascent complementary strand. The signal is generated by FRET from the donor (anti-Digoxigenin-Europium chelate binding with the primer) to the acceptor (Streptavidin-APC) binding to the biotin of the labelled nucleotides incorporated in proximity.
b qPCR cell-based assay: this assay evaluates the propensity of a compound to inhibit the replication of HCMV viral DNA during the first 72h. MRC-5 cells (5% FBS), HCMV virus strain is AD169, MOI= 0.05; in MRC-5 cells a SI with >1000 could be measured.
c DNA intercalation biochemical assay.
d Variable cytotoxicity observed in different cell lines.
In vitro DMPK and CMC parameters
BI-9553 has a good cell permeability and reasonable hepatocyte stability across species.
| Probe name / negative control | BI-9553 | BI-0309 |
| cLogP / LogD pH 7.4 / LogD pH11 | -/ 2.8 / 2.9 | -/-/ 1.09 |
| Solubility @ pH 7 [µg/ml] | 0.8 | < 1 |
| CACO permeability @ pH 7.4 [*10-6 cm/s] | 20.4 | 0.4 |
| CACO efflux ratio | n.a. | 37 |
| Microsomal stability [% QH] (human/mouse/rat) | 27 / <24 / <23 | 27 / <23 / <22 |
| Hepatocyte stability [% QH] (human/mouse/rat) | 38 / 24 / 8 | n.a. |
| Plasma protein binding [%] (human/mouse/rat) | 89.9 / 94.2 / 93.0 | n.a. |
| hERG [μM] | n.a. | n.a. |
| CYP 3A4 (IC50) [μM] | 12.4 | >50 |
| CYP 2C8 (IC50) [μM] | 18 | >50 |
| CYP 2C9 (IC50) [μM] | 13.5 | >50 |
| CYP 2C19 (IC50) [μM] | >30 | >50 |
| CYP 2D6 (IC50) [μM] | 21.3 | >50 |
In vivo DMPK parameters
BI-9553 shows a good clearance and MRT in rat and moderate ones in mouse. Bioavailability is good in both species.
| BI-9553 | Mousea | Ratb |
| Clearance [% QH] | 30.2 | 10.3 |
| Mean residence time (MRT) after iv dose [h] | 0.8 | 2.5 |
| tmax [h] | 0.5 | 3.2 |
| Cmax_DN [nM] | 1,509 | 1,158 |
| F [%] | 32 | 82 |
| Vss [l/kg] | 1.5 | 1.5 |
a Dose for mouse i.v. and oral: 2.0 mg/kg and 10 mg/kg
b Dose for rat i.v. and oral: 2.0 mg/kg and 5.0 mg/kg
Negative control
Despite having high structural similarity to BI-9553, the negative control BI-0309 is inactive in biochemical and cellular assays, due to a substitution of a non-polar para-chlorine by a polar para-phenol group.
BI-0309 which serves as a negative control
Selectivity
BI-9553 is not showing any effect on kinase activity (82 kinase panels tested @10 µM, all <36% inhibition) and did not show any activity in a panel of 44 receptors at 10µM (all <39% inhibition @10 µM). Negative control BI-0309 did not hit any receptor out of 44 targets @10µM.
| SELECTIVITY DATA AVILABLE | BI-9553 | BI-0309 |
SafetyScreen44™ with kind support of  | Yes | Yes |
| Invitrogen® | Yes | No |
| DiscoverX® | No | No |
| Dundee | No | No |
Download selectivity data:
BI-9553_selectivityData.xlsx
BI-0309_selectivityData.xlsx
Reference molecule(s)
Other CMV polymerase inhibitors are commercially available e.g. ganciclovir or valganciclovir.
Summary
BI-9553 is a selective, potent and well characterized non-nucleoside CMV polymerase inhibitor. BI-0309 is available as its negative control.
Supplementary data
2 D structure formats available
CMV polymerase inhibitor | BI-9553.png
CMV polymerase inhibitor | BI-9553.smiles
CMV polymerase inhibitor | BI-9553.sdf
Negative control | BI-0309.png
References
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BI-9553 was kindly provided by Boehringer Ingelheim via its open innovation platform opnMe, available at https://opnme.com.
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