Inhibitors of Signal Peptide Peptidase for Anti-Viral Therapy

Lead Research Organisation: MRC Virology Unit

Abstract

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Technical Summary

The objectives of this application are to perform mechanistic studies on proteolysis by signal
peptide peptidase (SPP) and to develop assays for identifying inhibitors of the protease.
Inhibitors of SPP activity could have potential as novel drug candidates for treatment of
hepatitis C virus (HCV) and for targeting immunoevasins encoded by viruses, such as US2,
which is encoded by human cytomegalovirus (HCMV).
HCV
Globally, approximately 130 million individuals have been infected with HCV. In the UK, HCV
infection has been diagnosed in 90,000 individuals but the true figure is closer to 270,000.
Chronic infection is a leading cause of liver disease, accounting for a high proportion of
cirrhosis and hepatocellular carcinoma. Predictions indicate that there will be about a 3-fold
rise in such HCV-associated diseases over the next decade. Current therapies to treat
infection have a 50% success rate, but a large cohort of patients fails to respond. Hence,
improved treatments are needed to reduce the future disease burden. In a landmark study,
we established that SPP, a cellular intramembrane-cleaving protease (I-CLiP), is essential for
maturation of HCV core (capsid) protein. Based on these data, a patent application claiming
SPP as a target for HCV was filed by MRCT, since SPP inhibitors would offer a novel approach
for intervention in HCV infection. Pharmaceutical companies have expressed interest in SPP,
but none has committed to this area, primarily due to 1) the absence of a robust assay and
2) the lack of proof of concept studies as HCV could not be cultivated under laboratory
conditions. From unpublished studies, we have now demonstrated that SPP cleavage of core
is needed for production of infectious HCV. Having established proof of principle, the
considered view is that the combination of our existing IP position along with development of
a robust assay for screening compound libraries would place MRC in a strong position for
translational opportunities. Between 2005-2007, a feasibility study to investigate difficulties
with development of SPP assays was undertaken with MRCT and they remain convinced of
the potential for therapeutic development.
HCMV
HCMV, a member of the Betaherpesvirinae family, infects between 60-90% of the world
population, typically establishing a latent infection. It is the most common cause of
congenital infection in humans and is particularly problematic in transplant recipients and
HIV-infected persons. To evade host immune defence systems, the virus encodes a suite of
immunoevasins that perturb antigen presentation. HCMV US2 is responsible for degradation
of MHC class I molecules by interacting with SPP although the mechanisms involved are not
known. Utilising the extensive knowledge and experience gained from studies on SPP
maturation of HCV core, there is an opportunity to expand MRC interests to investigate
whether SPP inhibitors may have an impact on immune recognition in HCMV infection.
Section 3: Description of Proposed Project (~800 words)
3.1 Scientific Background
Considerable effort is being devoted to develop compounds which target HCV functions but
development of resistance is acknowledged as a problem. A broader range of targets,
including cellular functions, is preferred to widen the availability of anti-viral therapies. Apart
from virus-encoded functions, few cellular factors are known to be essential for virus
propagation. Our interests in SPP date from 2000 when we established that HCV core protein
required SPP for maturation (McLauchlan et al., 2002, EMBO J 21, 3980-3988). Since it was
not possible to cultivate HCV in the laboratory, we tested the effect of blocking core
maturation by SPP in tamarins using GB virus-B (GBV-B), a close genetic relative to HCV, as
a surrogate model. Infection in animals was attenuated when SPP proteolysis of GBV-B core
was impaired (Targett-Adams et al., 2006, J Biol Chem 281, 29221-29227). Our
unpubl

Publications

10 25 50
 
Description MRC Developmental Pathway Funding Scheme (DPFS)
Amount £327,361 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start  
 
Title Cell lines - SPP 
Description Novel cell lines expressing wild-type and mutant forms of signal peptide peptidase under an inducible promoter have been created. 
Type Of Material Cell line 
Provided To Others? No  
Impact The material has been only recently created and therefore it is too early to describe any notable impacts. However, I consider that they will become highly beneficial to others studying sigal peptide peptidase. 
 
Title HCV materials 
Description My group has produced antibodies, constructs and developed biophysical approaches that have been utilised by a wide number of international groups. Characterisation of the antibodies and constructs has been completed and we continue to develop the biophysical methods. Other researchers are aware of our materials through publication of papers and presentations at conferences. A considerable number of man-years has been devoted to this area and the benefits have been acknowledged in publications from those who have received our materials. 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Our materials have facilitated the ongoing development of systems for studying HCV biology and also influenced the field related to lipid disorders. 
 
Title NGS Target Enrichment 
Description The technology enabled the enrichment of HCV-specific sequences in Next Generation Sequencing. It also increased the numbers of samples that could be included in a single sequencing run from about 10-12 to 90, which is much more cost effective. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The methodology is now being developed for diagnostic services and to establish an enrichment platform for flaviviruses circulating in the Philippines. 
 
Description IBCP 
Organisation Institute of Biology and Chemistry of Proteins (IBCP)
Country France 
Sector Academic/University 
PI Contribution My team performs all analysis of mutant signal peptides to design suitable synthetic peptides that may be substrates for signal peptide peptidase. We test these mutant signal peptides in various systems to establish their ability to be cleaved by the enzyme
Collaborator Contribution IBCP is an institute that carries out NMR studies on proteins. Through a longstanding collaboration with the group, we have determined the structure of the HCV signal peptide that is a substrate for signal peptide peptidase. This would represent the first example of a structure for a substrate for this protease. The group at IBCP also have a protein synthesis facility that has prepared a synthetic substrate for SPP
Impact This interaction gave rise to a publication (Oehler et al in the publication list).
 
Description Interviews 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact I have given interviews to journalists for newspapers.

There was no further contact with the journalists and so it is difficult to judge any impact.
Year(s) Of Engagement Activity 2006,2007
 
Description PCR workshop - Glasgow Science Centre 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact The workshop was organised by Dr D Bhella from the Unit and he will no doubt provide more comprehensive details. Both of the CDFs employed on the project volunteered their time and expertise for the workshop.

As noted above, Dr D Bhella will most likely address this question on behalf of the Unit.
Year(s) Of Engagement Activity 2009
 
Description presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I have given a presentation to a HepC support group and been involved in fund-raising activities.

Support groups are more aware of the activities funded by MRC to study HCV infection
Year(s) Of Engagement Activity 2006
 
Description presentations 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I have given practical demonstrations and talks at open days for schools.

There are now well-established contacts with local schools and teachers. Students have expressed a greater interest in pursuing a scientific career after the open day events.
Year(s) Of Engagement Activity 2006,2007,2008