Proteomics: a major new technology for the drug discovery process

  • Published on
    19-Sep-2016

  • View
    216

  • Download
    2

Embed Size (px)

Transcript

  • research focus REVIEWS

    DDT Vol. 4, No. 2 February 1999 1359-6446/99/$ see front matter Elsevier Science. All rights reserved. PII: S1359-6446(98)01291-4 55

    Among the major pharmaceutical and biotechnol-ogy companies, it is clearly recognized that thebusiness of modern drug discovery is a highlycompetitive process. All of the many steps in-

    volved are inherently complex, and each can involve ahigh risk of attrition. The players in this business strivecontinuously to optimize and streamline the process; eachseeking to gain an advantage at every step by attemptingto make informed decisions at the earliest stage possible.The desired outcome is to accelerate as many key activitiesin the drug discovery process as possible. This should pro-

    duce a new generation of robust drugs that offer a highprobability of success and reach the clinic and marketahead of the competition.

    There has been noticeable emphasis over recent yearsfor companies to aggressively review and refine theirstrategies to discover new drugs. Central to this has beenthe introduction and implementation of cutting-edgetechnologies. Most, if not all, companies have now inte-grated key technology platforms that incorporate gen-omics, mRNA expression analysis, relational databases,high-throughput robotics, combinatorial chemistry andpowerful bioinformatics. Although it is still early days toquantify the real impact of these platforms in clinical andcommercial terms, expectations are high, and it is widelyaccepted that significant benefits will be forthcoming. Thisis largely based on data obtained during preclinical studieswhere the genomic1,2 and microarray3,4 technologies havealready proved their value.

    However, there are several noteworthy outcomes that re-sult from this. Many comments are voiced that scientistsarmed with these technologies are now commonly facedwith data overload. Thus, in some instances, rather thanfacilitating the decision process, the accumulation of morecomplex data points, many with unknown consequences,can seem to hinder the process. Also, most drug compa-nies have simultaneously incorporated very similar compo-nents of the new technology platforms, the consequencebeing that it is becoming difficult yet again to determinewhere a clear competitive advantage will arise. Finally, inrecent years, largely as a result of the accessibility of thetechnologies, there has been an overwhelming emphasisplaced on genomic and mRNA data rather than on protein

    Proteomics: a major newtechnology for the drugdiscovery processMartin J. Page, Bob Amess, Christian Rohlff, Colin Stubberfieldand Raj Parekh

    Martin J. Page*, Bob Amess, Christian Rohlff, Colin Stubberfield and Raj Parekh, Oxford GlycoSciences, 10 The Quadrant,Abingdon Science Park, Abingdon, Oxfordshire, UK OX14 3YS. *tel: 144 1235 543277, fax: 144 1235 543283, e-mail: martin.page@ogs.co.uk

    Proteomics is a new enabling technology that is being

    integrated into the drug discovery process. This will

    facilitate the systematic analysis of proteins across any

    biological system or disease, forwarding new targets

    and information on mode of action, toxicology and sur-

    rogate markers. Proteomics is highly complementary to

    genomic approaches in the drug discovery process and,

    for the first time, offers scientists the ability to integrate

    information from the genome, expressed mRNAs, their

    respective proteins and subcellular localization. It is ex-

    pected that this will lead to important new insights into

    disease mechanisms and improved drug discovery

    strategies to produce novel therapeutics.

  • analysis. It is important to remember that proteins dictatebiological phenotype whether it is normal or diseased and are the direct targets for most drugs.

    Proteomics: new technology for the analysis of proteinsIt is now timely to recognize that complementary technol-ogy in the form of high-throughput analysis of the totalprotein repertoire of chosen biological samples, namelyproteomics, is poised to add a new and important dimen-sion to drug discovery. In a similar fashion to genomics,which aims to profile every gene expressed in a cell, pro-teomics seeks to profile every protein that is expressed57.However, there is added information, since proteomics canalso be used to identify the post-translational modificationsof proteins8, which can have profound effects on bio-logical function, and their cellular localization. Importantly,proteomics is a technology that integrates the significantadvances in two-dimensional (2D) electrophoretic separa-tion of proteins, mass spectrometry and bioinformatics.With these advances it is now possible to consistently de-rive proteomes that are highly reproducible and suitablefor interrogation using advanced bioinformatic tools.

    There are many variations whereby different laboratoriesoperate proteomics. For the purpose of this review, the

    process used at Oxford GlycoSciences (OGS), which usesan industrial-scale operation that is integral to its drug dis-covery work, will be described. The individual steps ofthis process, where up to 1000 2D gels can be run andanalysed per week, are summarized in Fig. 1. The incom-ing samples are bar coded and all information relevant tothe sample is logged into a Laboratory InformationManagement System (LIMS) database. There can be a widerange in the type of samples processed, as applicable toindividual steps in the drug discovery pipeline, and thesewill be mentioned later. The samples are separated accord-ing to their charge (pI) in the first dimension, using iso-electric focusing, followed by size (MW) using SDSPAGEin the second dimension. Many modifications have beenmade to these steps to improve handling, throughput andreproducibility. The separated proteins are then stainedwith fluorescent dyes which are significantly more sensi-tive in detection than standard silver methods and have abroader dynamic range. The image of the displayed pro-teins obtained is referred to as the proteome, and is digi-tally scanned into databases using proprietary softwarecalled ROSETTA. The images are subsequently curated,which begins with the removal of any artefacts, croppingand the placement of pI/MW landmarks. The images fromreplicate images are then aligned and matched to one

    REVIEWS research focus

    56 DDT Vol. 4, No. 2 February 1999

    Figure 1. Steps involved in analysing a biological sample by proteomics. MCI, molecular cluster index.

    Sample

    Nor

    mal

    2D gels andimaging

    Curation andinterrogation

    Differential analysis(Proteograph)

    MCIFold decreaseFold increaseComposite normal

    Composite disease

    Mass spectrometryand annotation

    00

    100

    200

    300

    400

    500

    600

    700

    800

    900

    1000

    1100

    Abundance (%)

    Mas

    s:ch

    arge

    100 50

    147

    197218

    196

    225

    331354371

    416438

    459526

    536544 545

    588611

    612669

    698699716

    738807810

    827

    852845

    917922958

    10511055

    258326

    130102

    84

    Dis

    ease

  • another to generate a synthetic composite image. This isan important step, as the proteome is a dynamic situation,and it captures the biological variation that occurs, suchthat even orphan proteins are still incorporated into theanalysis.

    By means of illustration, Fig. 1 shows the processwhereby proteomes are generated from normal and dis-ease samples and how differentially expressed proteins areidentified. The potential of this type of analysis is tremen-dous. For example, from a mammalian cell sample, in ex-cess of 2000 proteins can typically be resolved within theproteome. The quality of this is shown in Fig. 2, whichshows representative proteomes from three diverse bio-logical sources: human serum, the pathogenic fungusCandida albicans and the human hepatoma cell lineHuh7.

    Use of proteomics to identify disease specific proteinsIn most cases, the drug discovery process is initiated bythe identification of a novel candidate target almost al-ways a protein that is believed to be instrumental in thedisease process. To date, there is a variety of meanswhereby drug targets have been forthcoming. These in-clude molecular, cellular and genomic approaches, mostlycentred upon DNA and mRNA analysis. The gene in ques-tion is isolated, and expression and characterization of itscoded protein product i.e. the drug target is invariablya secondary event.

    With the proteomic approach, the starting point is at theother end of the telescope. Here there is direct and im-

    mediate comparison of the proteomes from paired normaland disease materials. Examples of these pairs are: (1) pu-rified epithelial cell populations derived from humanbreast tumours, matched to purified normal populations ofhuman breast epithelial cells, and (2) the invading patho-genic hyphal form of C. albicans, matched to the non-invading yeast form of C. albicans. When the proteomeimages from each pair are aligned, the Proteograph soft-ware is able to rapidly identify those proteins (each refer-enced as having a unique molecular cluster index, or MCI)that are either unique, or those that are differentially ex-pressed. Thus, the Proteograph output from this analysis isboth qualitative and quantitative.

    Proteograph analysis for a particular study can also beundertaken on any number of samples. For example, onemight compare anything from a few to several hundredpreparations or samples, each from a normal and diseasecounterpart, and have these analysed in a singleProteograph study. In this way, it is possible to assignstrong statistical confidence to the data and in some in-stances to identify specific subpopulations within the inputbiological sources. This feature will become increasinglysignificant in the near future, and there is a clear synergyhere whereby proteomics can work closely with pharma-cogenomic approaches to stratify patient populations andachieve effective targeted care for the patient. Whateverthe source of the materials, the net output of Proteographanalysis is immediate identification of disease specific pro-teins. This is shown in Fig. 3, which shows the results of a proteograph obtained by comparing untreated humanhepatoma cells with cells following exposure to a clinical

    research focus REVIEWS

    DDT Vol. 4, No. 2 February 1999 57

    Figure 2. Representative proteomes obtained from (a) human serum, (b) the pathogenic fungus Candida albicansand (c) the human hepatoma cell line Huh7.

  • cytotoxic agent. In this instance, only the top 20 differen-tially expressed MCIs are shown, but the readout wouldnormally extend to a defined cut-off value, typically a two-fold or greater difference in expression levels, determinedby the user.

    In a typical analysis involving disease and normal mam-malian material, in which each proteome would have~2000 protein features each assigned an MCI, the proteo-graph might identify somewhere in the region of 50300MCIs that are unique or differentially expressed. To capi-talize rapidly on these data, at OGS a high-throughput

    mass spectrometry facility coupled to advanced databasesto annotate these MCIs as individual proteins is applied. Asthese are all disease specific proteins, each could representa novel target and/or a novel disease marker. The processbecomes even more powerful when a panel of features,rather than individual features, are assigned. The relevanceof this is apparent when one considers that most diseases,if not all, are multifactorial in nature and arise from poly-genic changes. Rather than analysing events in isolation,the ability to examine hundreds or thousands of events simultaneously, as shown by proteomics, can offer real advantages.

    Identification and assignment of candidate targetsThe rapid identification and assignment of candidate tar-gets and markers represents a huge challenge, but this hasbeen greatly facilitated by combining the recent advancesmade in proteomics and analytical mass spectrometry9.Using automated procedures it is now possible to annotateproteins present in femtomole quantities, which would de-pict the low abundance class of proteins. The process ofannotation is similarly aided by the quality and richness ofthe sequence specific databases that are currently avail-able, both in the public domain and in the private sector(e.g. those supplied by Incyte Pharmaceuticals). In this re-spect, the advances in proteomics have benefited consider-ably from the breakthroughs achieved with genomics.

    From an application perspective, cancer studies provide agood opportunity whereby proteomics can be instrumentalin identifying disease specific proteins, because it is oftenfeasible to obtain normal and diseased tissue from the samepatient. For example, proteomic studies have been re-ported on neuroblastomas10, human breast proteins fromnormal and tumour sources1113, lung tumours14, colon tu-mours15 and bladder tumours16. There are also proteomicstudies reported within the cardiovascular therapeutic area,in which disease or response proteins are identified17,18.

    Genomic microarray analysis can similarly identifyunique species or clusters of mRNAs that are disease spe-cific. However, in some instances, there is a clear lack ofcorrelation between the levels of a specific mRNA and itscorresponding protein (Ref. 19, Gypi, S.P. et al., submit-ted). This has now been noted by many investigators andreaffirms that post-transcriptional events, including proteinstability, protein modification (such as phosphorylation,glycosylation, acylation and methylation) and cell localiz-ation, can constitute major regulatory steps. Proteomicanalysis captures all of these steps and can therefore pro-vide unique and valuable information independent from,or complementary to, genomic data.

    REVIEWS research focus

    58 DDT Vol. 4, No. 2 February 1999

    Figure 3. Table of differential protein expressionprofiles, referred to as a Rosetta Proteograph,between Huh7 cells with and without the cytotoxicagent 5-FU. Bars are quantized and do not representexact fold change values.

    Foregrounds: Huh7 cells treated with 5FUBackgrounds: Huh7 cells untreated

    Upregulated in Huh7 cells treated with 5FU with respect to untreated Huh7 cellsDownregulated in Huh7 cells treated with 5FU with respect to untreated Huh7 cells

    MCI Fold change pI MW3589 16.3 5.60 137184035 15.8 8.73 132244033 12.3 8.94 131174052 11.7 8.96 214344064 11.1 9.37 293893792 10.9 8.75 411623976 10.5 7.14 120613672 10.5 8.83 241213986 10.4 4.61 108533759 9.9 4.98 414204032 9.7 4.56 131171972 29.4 7.92 123963587 9.4 4.50 149542033 29.4 7.93 111263984 8.9 5.32 110902403 28.8 6.04 259503748 8.8 6.32 355132105 28.7 4.76 208033897 8.7 4.94 878424221 8.6 5.78 71963

  • Proteomics for target validation and signal transduc-tion studiesThe identification of disease specific proteins alone is in-sufficient to begin a drug screening process. It is critical toassign function and validation to these proteins by con-firming they are indeed pivotal in the disease process.These studies need to encompass both gain- and loss-of-function analyses. This would determine whether the activityof a candidate target (an enzyme, for example), eliminatedby molecular/cellular techniques, could reverse a diseaseph...