1,5-Benzodiazepines, a Novel Class of Hepatitis C Virus Polymerase Nonnucleoside Inhibitors

[Guest guest]

http://aac.asm.org/cgi/content/abstract/52/12/4420

Antimicrobial Agents and Chemotherapy, December 2008, p. 4420-4431, Vol. 52, No.

12

0066-4804/08/$08.00+0 doi:10.1128/AAC.00669-08

1,5-Benzodiazepines, a Novel Class of Hepatitis C Virus Polymerase Nonnucleoside

Inhibitors

,

Origène Nyanguile,1* Frederik Pauwels,1 Walter Van den Broeck,1 Carlo W.

Boutton,1, Ludo Quirynen,1 Tania Ivens,1 Liesbet van der Helm,1 Geneviève

Vandercruyssen,1 Mostmans,1 Frédéric Delouvroy,1 Pascale Dehertogh,1

Maxwell D. Cummings,1 Jean-Francois Bonfanti,2 A. Simmen,1 and Pierre

Raboisson1

HCV Research, Tibotec, Mechelen, Belgium,1 and Pharmaceutical

and Research Development, Val de Reuil, France2

Received 21 May 2008/ Returned for modification 18 July 2008/ Accepted 26

September 2008

The exogenous control of hepatitis C virus (HCV) replication can be mediated

through the inhibition of the RNA-dependent RNA polymerase (RdRp) activity of

NS5B. Small-molecule inhibitors of NS5B include nucleoside and nonnucleoside

analogs. Here, we report the discovery of a novel class of HCV polymerase

nonnucleoside inhibitors, 1,5-benzodiazepines (1,5-BZDs), identified by

high-throughput screening of a library of small molecules. A

fluorescence-quenching assay and X-ray crystallography revealed that 1,5-BZD 4a

bound stereospecifically to NS5B next to the catalytic site. When introduced

into replicons, mutations known to confer resistance against chemotypes that

bind at this site were detrimental to inhibition by 1,5-BZD 7a. Using a panel of

enzyme isolates that covered genotypes 1 to 6, we showed that compound 4a

inhibited genotype 1 only. In mechanistic studies, 4a was found to inhibit the

RdRp activity of NS5B noncompetitively with GTP and to inhibit the formation of

the first phosphodiester bond during the polymerization cycle. The specificity

for the HCV target was evaluated by profiling the 1,5-BZDs against other viral

and human polymerases, as well as BZD receptors.

--------------------------------------------------------------------------------

* Corresponding author. Mailing address: Tibotec BVBA, Generaal de Wittelaan

L11B 3, 2800 Mechelen, Belgium. Phone: 32 15 461 296. Fax: 32 15 461 951.

E-mail: onyangui@...

Published ahead of print on 13 October 2008.

Supplemental material for this article may be found at http://aac.asm.org/.

Present address: Ablynx nv, Ghent, Belgium.

[Guest guest]

http://aac.asm.org/cgi/content/abstract/52/12/4420

Antimicrobial Agents and Chemotherapy, December 2008, p. 4420-4431, Vol. 52, No.

12

0066-4804/08/$08.00+0 doi:10.1128/AAC.00669-08

1,5-Benzodiazepines, a Novel Class of Hepatitis C Virus Polymerase Nonnucleoside

Inhibitors

,

Origène Nyanguile,1* Frederik Pauwels,1 Walter Van den Broeck,1 Carlo W.

Boutton,1, Ludo Quirynen,1 Tania Ivens,1 Liesbet van der Helm,1 Geneviève

Vandercruyssen,1 Mostmans,1 Frédéric Delouvroy,1 Pascale Dehertogh,1

Maxwell D. Cummings,1 Jean-Francois Bonfanti,2 A. Simmen,1 and Pierre

Raboisson1

HCV Research, Tibotec, Mechelen, Belgium,1 and Pharmaceutical

and Research Development, Val de Reuil, France2

Received 21 May 2008/ Returned for modification 18 July 2008/ Accepted 26

September 2008

The exogenous control of hepatitis C virus (HCV) replication can be mediated

through the inhibition of the RNA-dependent RNA polymerase (RdRp) activity of

NS5B. Small-molecule inhibitors of NS5B include nucleoside and nonnucleoside

analogs. Here, we report the discovery of a novel class of HCV polymerase

nonnucleoside inhibitors, 1,5-benzodiazepines (1,5-BZDs), identified by

high-throughput screening of a library of small molecules. A

fluorescence-quenching assay and X-ray crystallography revealed that 1,5-BZD 4a

bound stereospecifically to NS5B next to the catalytic site. When introduced

into replicons, mutations known to confer resistance against chemotypes that

bind at this site were detrimental to inhibition by 1,5-BZD 7a. Using a panel of

enzyme isolates that covered genotypes 1 to 6, we showed that compound 4a

inhibited genotype 1 only. In mechanistic studies, 4a was found to inhibit the

RdRp activity of NS5B noncompetitively with GTP and to inhibit the formation of

the first phosphodiester bond during the polymerization cycle. The specificity

for the HCV target was evaluated by profiling the 1,5-BZDs against other viral

and human polymerases, as well as BZD receptors.

--------------------------------------------------------------------------------

* Corresponding author. Mailing address: Tibotec BVBA, Generaal de Wittelaan

L11B 3, 2800 Mechelen, Belgium. Phone: 32 15 461 296. Fax: 32 15 461 951.

E-mail: onyangui@...

Published ahead of print on 13 October 2008.

Supplemental material for this article may be found at http://aac.asm.org/.

Present address: Ablynx nv, Ghent, Belgium.

[Guest guest]

http://aac.asm.org/cgi/content/abstract/52/12/4420

Antimicrobial Agents and Chemotherapy, December 2008, p. 4420-4431, Vol. 52, No.

12

0066-4804/08/$08.00+0 doi:10.1128/AAC.00669-08

1,5-Benzodiazepines, a Novel Class of Hepatitis C Virus Polymerase Nonnucleoside

Inhibitors

,

Origène Nyanguile,1* Frederik Pauwels,1 Walter Van den Broeck,1 Carlo W.

Boutton,1, Ludo Quirynen,1 Tania Ivens,1 Liesbet van der Helm,1 Geneviève

Vandercruyssen,1 Mostmans,1 Frédéric Delouvroy,1 Pascale Dehertogh,1

Maxwell D. Cummings,1 Jean-Francois Bonfanti,2 A. Simmen,1 and Pierre

Raboisson1

HCV Research, Tibotec, Mechelen, Belgium,1 and Pharmaceutical

and Research Development, Val de Reuil, France2

Received 21 May 2008/ Returned for modification 18 July 2008/ Accepted 26

September 2008

The exogenous control of hepatitis C virus (HCV) replication can be mediated

through the inhibition of the RNA-dependent RNA polymerase (RdRp) activity of

NS5B. Small-molecule inhibitors of NS5B include nucleoside and nonnucleoside

analogs. Here, we report the discovery of a novel class of HCV polymerase

nonnucleoside inhibitors, 1,5-benzodiazepines (1,5-BZDs), identified by

high-throughput screening of a library of small molecules. A

fluorescence-quenching assay and X-ray crystallography revealed that 1,5-BZD 4a

bound stereospecifically to NS5B next to the catalytic site. When introduced

into replicons, mutations known to confer resistance against chemotypes that

bind at this site were detrimental to inhibition by 1,5-BZD 7a. Using a panel of

enzyme isolates that covered genotypes 1 to 6, we showed that compound 4a

inhibited genotype 1 only. In mechanistic studies, 4a was found to inhibit the

RdRp activity of NS5B noncompetitively with GTP and to inhibit the formation of

the first phosphodiester bond during the polymerization cycle. The specificity

for the HCV target was evaluated by profiling the 1,5-BZDs against other viral

and human polymerases, as well as BZD receptors.

--------------------------------------------------------------------------------

* Corresponding author. Mailing address: Tibotec BVBA, Generaal de Wittelaan

L11B 3, 2800 Mechelen, Belgium. Phone: 32 15 461 296. Fax: 32 15 461 951.

E-mail: onyangui@...

Published ahead of print on 13 October 2008.

Supplemental material for this article may be found at http://aac.asm.org/.

Present address: Ablynx nv, Ghent, Belgium.

[Guest guest]

http://aac.asm.org/cgi/content/abstract/52/12/4420

Antimicrobial Agents and Chemotherapy, December 2008, p. 4420-4431, Vol. 52, No.

12

0066-4804/08/$08.00+0 doi:10.1128/AAC.00669-08

1,5-Benzodiazepines, a Novel Class of Hepatitis C Virus Polymerase Nonnucleoside

Inhibitors

,

Origène Nyanguile,1* Frederik Pauwels,1 Walter Van den Broeck,1 Carlo W.

Boutton,1, Ludo Quirynen,1 Tania Ivens,1 Liesbet van der Helm,1 Geneviève

Vandercruyssen,1 Mostmans,1 Frédéric Delouvroy,1 Pascale Dehertogh,1

Maxwell D. Cummings,1 Jean-Francois Bonfanti,2 A. Simmen,1 and Pierre

Raboisson1

HCV Research, Tibotec, Mechelen, Belgium,1 and Pharmaceutical

and Research Development, Val de Reuil, France2

Received 21 May 2008/ Returned for modification 18 July 2008/ Accepted 26

September 2008

The exogenous control of hepatitis C virus (HCV) replication can be mediated

through the inhibition of the RNA-dependent RNA polymerase (RdRp) activity of

NS5B. Small-molecule inhibitors of NS5B include nucleoside and nonnucleoside

analogs. Here, we report the discovery of a novel class of HCV polymerase

nonnucleoside inhibitors, 1,5-benzodiazepines (1,5-BZDs), identified by

high-throughput screening of a library of small molecules. A

fluorescence-quenching assay and X-ray crystallography revealed that 1,5-BZD 4a

bound stereospecifically to NS5B next to the catalytic site. When introduced

into replicons, mutations known to confer resistance against chemotypes that

bind at this site were detrimental to inhibition by 1,5-BZD 7a. Using a panel of

enzyme isolates that covered genotypes 1 to 6, we showed that compound 4a

inhibited genotype 1 only. In mechanistic studies, 4a was found to inhibit the

RdRp activity of NS5B noncompetitively with GTP and to inhibit the formation of

the first phosphodiester bond during the polymerization cycle. The specificity

for the HCV target was evaluated by profiling the 1,5-BZDs against other viral

and human polymerases, as well as BZD receptors.

--------------------------------------------------------------------------------

* Corresponding author. Mailing address: Tibotec BVBA, Generaal de Wittelaan

L11B 3, 2800 Mechelen, Belgium. Phone: 32 15 461 296. Fax: 32 15 461 951.

E-mail: onyangui@...

Published ahead of print on 13 October 2008.

Supplemental material for this article may be found at http://aac.asm.org/.

Present address: Ablynx nv, Ghent, Belgium.