In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 110, No. 10 ( 2013-03-05)
Abstract:
The identification of effective antirabies compounds that bind host targets supports our original hypothesis and the use of this approach for both the analysis of viral capsid formation and the discovery of antiviral drugs. Our work turned the conventional “identify the target first” approach to drug discovery on its head by first identifying an active compound through a whole-pathway screen. As conventional drug targets are exhausted, the development of techniques allowing the identification of unconventional targets grows increasingly important. Using this highly unconventional approach, we have revealed a class of next-generation drug targets that may prove valuable beyond their application in rabies therapeutics. To identify the target functionally, we adapted PAV-866 to ligand affinity chromatography. Our findings show that it binds to a host multiprotein complex containing ATP-binding cassette family E1, a host protein previously implicated in HIV capsid assembly. Once the target was isolated, we demonstrated that its presence was requisite for drug activity. CFPS reactions carried out in extracts depleted of the target lost all drug sensitivity, whereas reconstituted extracts demonstrated full restoration of the dose-dependent drug effect. We used a rabies CFPS system to establish a drug screen that assays for activity along the whole capsid-assembly pathway (see Fig. P1 ). We applied this screen to a portion of a compound library, successfully identified small molecules that interfere with the pathway by targeting host proteins, and improved them through optimization of the structure–activity relationship. The drug activity observed in the CFPS screen was corroborated by robust activity against infectious rabies in cell culture. In this paper, we describe the structure of PAV-866, an active anti-rabies compound. As often has been the case in the history of science ( 5 ), new views of natural phenomena lead to the development of innovative methods for solving problems. In our case, the view is that capsid formation is host-catalyzed rather than spontaneous, the problem addressed is the need for antiviral drugs, and the approach is to target host multiprotein complexes involved in capsid assembly. Our working hypothesis is that catalytic interactions between host proteins and viral capsid proteins are critical for the formation of functional viral capsids, and that subtle disruption of a subset of these early interactions will therefore block later events in the viral lifecycle. On that basis, we studied the putative rabies capsid-assembly pathway to identify small molecules active against rabies virus, perhaps the most lethal viral infection of humans and against which no small-molecule therapeutic currently exists. Viruses encase their genetic material in a protein shell known as the “capsid.” During infection, viral proteins are synthesized by host cellular machinery and have been thought to self-assemble by a spontaneous, thermodynamically driven process ( 1 ). Cell-free protein synthesis (CFPS) is a classical cell biological technique and a valuable tool for the dissection of biochemical pathways ( 2 ). This technique was used previously to reconstitute viral capsid assembly for several viral families, including HIV ( 3 , 4 ). These studies led to an entirely different view of viral capsid assembly: an intricate biochemical pathway involving discrete assembly intermediates and steps with catalytic roles for host proteins.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1210198110
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2013
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
Permalink
