Fluorescent Probes for Proteolysis: Tools for Drug Discovery
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REVIEWS FLUORESCENT PROBES FOR PROTEOLYSIS: TOOLS FOR DRUG DISCOVERY Jacques Neefjes* and Nico P. Dantuma‡ Cells contain numerous proteases, which are found at many different locations. These proteases recognize an even larger number of different substrates and are involved in almost every process in the cell. Aberrations in proteolysis are linked to a plethora of diseases, such as cancer, inflammation, arteriosclerosis, neurodegeneration and infection. Because of their well-defined chemistry and key role in pathologies, proteases have been important targets for drug development. Recent progress in the development of fluorescent probes has opened up the possibility of visualizing protease activities in the natural environment of the cell. We will describe various strategies to follow protease activities in cells and organisms. PROTEASOME Proteolysis is a process crucial to the normal turnover One generally recognized function of proteases is A large barrel-shaped multi- of proteins in the cell. Proteases are not only involved the recycling of proteins into amino acids. Proteins can subunit complex, which in protein destruction, but also in activation, growth, be singled out for a number of reasons, including signs harbours several protease cell division, differentiation, migration, signalling and of malfunctioning or misfolding1.Although the vast activities. Substrates are threaded other cellular processes. In addition, many viruses use majority of proteins face destruction when old and into the barrel, where they are degraded by the proteases. cellular or viral proteases for various purposes, such as expired, a substantial fraction of newly synthesized pro- activation of their surface proteins or membrane teins undergoes a similar fate2,3,possibly because they fusion. Consequently, specific protease inhibitors are fail to adopt a proper conformation4.Because misfolded attractive drug candidates (TABLE 1).Although the differ- proteins tend to aggregate, the rapid recognition and ent specificities of proteases have been used during degradation of such aberrant proteins is crucial for recent years to assess their activities in vitro, their actions the survival of the cell5,6.Moreover, in many cases the have been difficult to determine under more-or-less destruction of proteins is crucial for the correct control physiological conditions. of cellular regulatory switches. In this case, their Proteases can be divided into a number of classes: the degradation is not a result of malfunctioning, and is in serine, aspartic, cysteine, threonine and metalloproteases. fact determined by specific spatially and temporally *Division of Tumor Biology, 7 The Netherlands Cancer This classification scheme basically refers to the amino controlled signalling events . Institute, Plesmanlaan 121, acid or metal that catalyses the nucleophilic attack on the The route that leads from a full-length intracellular 1066 CX, Amsterdam, substrate’s peptide bonds. Enzymes with proteolytic protein to its constituent amino acids is a multistep The Netherlands. activities have frequently evolved to carry out important process (FIG. 1).First, the processive degradation of ‡Microbiology and Tumor Biology Center, Karolinska tasks, which has resulted in large and divergent families of proteins by the PROTEASOME generates small peptide Institutet, Nobels väg 16, proteases. These often unrelated enzymes differ in many fragments8.This initial cleavage stage is generally suffi- S-17177, Stockholm, respects, but one characteristic unites them: they hydro- cient to abolish protein function and activity. Second, a Sweden. lyse peptide bonds and thereby cut proteins into small collection of proteases — among which are tripeptidyl Correspondence to N.P.D. pieces or a few distinct fragments. It is precisely this peptidase-II (TPP-II)9,leucine aminopeptidase (LAP)10 e-mail: 11 [email protected] chemical reaction that allows the design of probes to and thimet oligopeptidase (TOP) — trim these small doi:10.1038/nrd1282 selectively monitor protease activities. peptide fragments step-wise into single amino acids 58 | JANUARY 2004 | VOLUME 3 www.nature.com/reviews/drugdisc REVIEWS Table 1 | A few inhibitors and (potential) applications explain why the ubiquitin–proteasome system is impli- cated in a large number of diverse pathologies5,17,18. Target Inhibitor Disease References For example, aberrant proteasomal degradation con- Matrix metalloproteases Marimastat Cancer 86 tributes to malignant transformation19–21.The immune Prinomastat Tanomastat system relies on the ubiquitin–proteasome system for BMS-275291 the generation of antigenic peptides that are presented by 22,23 Cathepsin S Clik60 Autoimmunity 85 MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) CLASS I molecules . Proteasome Velcade (PS 341) Cancer 30 The proteasome also has a crucial role in the inflam- matory response, as it degrades inhibitors of nuclear Caspase-1 Pralnacasan Arthritis 125 factor-κB24.Given these functions, it is not surprising Caspase-8 IDN6556 Liver cirrhosis 126 that proteasome inhibitors have anticancer25,anti- HIV-1 protease Ritonavir HIV-1 infection 127 inflammatory26, angiostatic27 and immuno-modulating Nelfinavir 28 Saquinavir activity .Recently, the first proteasome inhibitor, Lopinavir bortezomib (Velcade; Millennium) — a boronic acid 29 HCV NS3 BILN 2016 HCV hepatitis 118 inhibitor (TABLE 1 and FIG. 2) — was approved after successful clinical trials in patients suffering from HCV, hepatitis C virus; HIV, human immunodeficiency virus. multiple myeloma29,30. Notably, two drugs that affect proteasomal degrada- that can be reused in the synthesis of new proteins. tion have been used in patients for some time without Endocytosed proteins do not usually enter the cytoplasm being recognized as such. These drugs are the human but are instead directed to lysosomal compartments immunodeficiency virus (HIV)-1 protease inhibitors (FIG. 1).In the lysosomes, another group of proteases, the ritonavir (Norvir; Abbot Laboratories) and saquinavir cathepsins11,degrades the internalized protein into (Fortovase; Roche), and have recently been shown to pieces. These fragments are subsequently reduced to affect proteasome activity as well31.It has been suggested single amino acids by lysosomal aminopeptidases like that inhibition of the proteasome by these compounds TPP-I and dipeptidyl peptidase-II (DPP-II)9.Given the could account for increased hyperlipidaemia32 and the broad substrate specificity of the proteasome and reduced incidence of Kaposi’s sarcoma33 in patients cathepsins, these proteases have to be compartmentalized treated with these drugs. Moreover, HIV-1 inhibitors to prevent the random degradation of ‘innocent by- can radiosensitize tumour cells for ionizing radiation standers’.This is achieved by placing the active sites of by blocking the proteasome34.Specific proteasome the proteases within large multisubunit complexes, in the inhibitors, such as peptide aldehydes1,boronic acids27, case of the proteasome, or within lysosomes, in the case vinyl sulfones35,epoxyketones26 or β-lactones36,have been of cathepsins. In addition to these broadly acting pro- used for many years in the laboratory (FIG. 2).So whereas teases, there are other proteases involved in many more drugs that target the proteasome are relatively new and specific processes, such as blood clotting12,apoptosis13, unexplored, proteasome inhibitors and modulators hold extracellular matrix degradation14,cleavage of trans- great promise as potential drugs for multiple disorders37. membrane proteins15 and precursor processing in the During the past few years several groups have devel- secretory pathway16. oped fluorescent probes for the ubiquitin–proteasome Clearly, proteases not only destroy proteins but also system that behave as authentic protein substrates (FIG. 3a). convert proteins into active moieties. In addition, pro- Only proteins, which are specifically recruited to the teolytic cascades regulate important cellular switches. proteasome, and subsequently unfolded and threaded It is therefore not surprising that proteases get a lot of into the core, can be degraded by this complex. The pro- scientific attention. A recent literature search revealed that teasome generally recognizes substrates by the presence MAJOR HISTOCOMPATIBILITY roughly 240,000 papers were published about proteases of a poly-ubiquitin chain conjugated to an internal COMPLEX CLASS I during the past 20 years. And this is yet another one, but lysine residue of the substrate38 or, less commonly, con- (MHC). A ubiquitously 39,40 expressed cell-surface one describing a timely issue: how can protease activity be jugated to the free amino terminus of the substrate . glycoprotein that presents a monitored in vivo? Given the chemistry of proteases, Proteins are modified by the addition of these poly- fragment of a protein to the various fluorescent substrates have been designed during ubiquitin chains by specific ubiquitylation enzymes41 immune system. This fragment, the past few years to test their activities as well as the that physically interact with degradation signals (such as which usually comprises nine effects of drug-mediated inhibition of these systems in misfolded domains, small motifs or post-translational amino acids, is generally derived 42 from intracellular proteins and cells or animals. Understanding the physiology of pro- modifications) that are detected in the substrate . MHC class I molecules, thereby teolytic processes inside the living organism