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Cell suicide is a normal process that participates in a widevariety of physiological processes, including tissuehomeostasis, immune regulation, and fertility. Physiological celldeath typically occurs by apoptosis, as opposed to necrosis.Defects in apoptotic cell-death regulation contribute to manydiseases, including disorders associated with cellaccumulation (e.g. cancer, autoimmunity, inflammation andrestenosis) or where cell loss occurs (e.g. stroke, heart failure,neurodegeneration, AIDS and osteoporosis). At the center ofthe apoptosis machinery is a family of intracellular proteases,known as ‘caspases’, that are responsible directly or indirectlyfor the morphological and biochemical events that characterizeapoptosis. Multiple positive and negative regulators of thesecell-death proteases have been discovered in the genomes ofmammals, amphibians, insects, nematodes, and other animalspecies, as well as a variety of animal viruses. Inputs fromsignal-transduction pathways into the core of the cell-deathmachinery have also been identified, demonstrating ways oflinking environmental stimuli to cell-death responses or cell-survival maintenance. Knowledge of the molecular mechanismsof apoptosis has provided important insights into the causes ofmultiple diseases where aberrant cell-death regulation occursand has revealed new approaches for identifying small-molecule drugs for more effectively treating these illnesses.

Addresses*The Burnham Institute, La Jolla, CA 92037, USA† IDUN Pharmaceuticals, Inc., La Jolla, CA 92037, USACorrespondence: John C Reed; e-mail: [email protected]

Current Opinion in Biotechnology 2000, 11:586–592

0958-1669/00/$ — see front matter© 2000 Elsevier Science Ltd. All rights reserved.

AbbreviationsAsp aspartic acidCARD caspase-associated recruitment domainER endoplasmic reticulumIAP inhibitor of apoptosis proteinsIL interleukinTNF tumor necrosis factor

IntroductionCell death can be physiological or pathological. Each second,for example, approximately one million cells perish in thehuman body. In fact, in the course of a typical day, the aver-age adult will produce and in parallel eradicate 50–70 billioncells, representing a mass of cells equivalent to an entirebody-weight over a year’s time. This massive flux of cell birthand death occurs in the self-renewing tissues of the body(skin, gut, bone marrow, sex organs), providing mechanismsfor rapidly regulating cell numbers by controlling the rates ofboth input and elimination. Physiological cell death playsimportant roles in a wide variety of normal processes, rangingfrom fetal development to immune system regulation.Usually, physiological (programmed) cell death occurs by

apoptosis, a morphological phenomenon characterized bychromatin condensation, nuclear fragmentation, cell shrink-age, and plasma membrane blebbing, with cells breaking intosmall membrane-surrounded fragments (apoptotic bodies)that are cleared by phagocytosis without inciting an inflam-matory response [1].

The morphological changes recognized as ‘apoptosis’ arecaused by proteases — specifically, activation of a familyof intracellular cysteine proteases that cleave their sub-strates at aspartic acid (Asp) residues, known as ‘caspases’for cysteine aspartyl-specific proteases [2,3]. These pro-teases are initially produced as inactive zymogens inessentially all animal cells, but are triggered into activa-tion generally as a result of their proteolytic processing atconserved Asp residues. The active enzymes consist ofheterotetramers composed of two large and two small sub-units, with two active sites per tetramer. Duringactivation, the zymogen pro-proteins are cleaved to gener-ate the large (~20 kDa) and small (~10 kDa) subunits,typically liberating an amino-terminal prodomain from theprocessed polypeptide chain.

Because caspases cleave their substrates at Asp residues andare also activated by proteolytic processing at Asp residues,these proteases can collaborate in proteolytic cascades, wherecaspases activate themselves and each other. Caspases can beviewed as either upstream ‘initiator’ caspases or downstream‘effector’ proteases [4]. The proforms of upstream initiatorcaspases possess large amino-terminal pro-domains, whichfunction as protein interaction modules, allowing them toassociate with various proteins that trigger caspase activation.In contrast, downstream effector caspases contain only shortamino-terminal prodomains, and are largely dependent onupstream caspases for their proteolytic processing and activa-tion. In humans and mice, ~14 caspases have been identifiedto date. Though most caspases are directly involved in apop-tosis, a few are not — at least in mammals and highereukaryotes where a subgroup of caspases (caspases 1, 4, and5 in humans) is involved in processing of pro-inflammatorycytokines, including pro-IL-1β and pro-IL-18 (reviewed in[5]). Though suppression of these caspases has potentialtherapeutic applications to inflammatory diseases, they willnot be discussed further here.

Defects in the physiological pathways for apoptosis makeimportant contributions to multiple diseases. In fact, it is esti-mated that either too little or too much cell death is involvedin over half of the diseases for which adequate therapies donot currently exist [1,6]. Though cell death occurs physio-logically in all self-renewing tissues, irreversible loss of cellsin terminally differentiated, post-mitotic cells of the brainand heart can be catastrophic. Conversely, a failure of normalcell-death mechanisms in self-renewing tissues leaves cell

Drug discovery opportunities from apoptosis researchJohn C Reed* and Kevin J Tomaselli†

Drug discovery opportunities from apoptosis research Reed and Tomaselli 587

production unchecked by cell turnover, resulting in cellaccumulation in cancer, restenosis, and other diseases. Thelist of illnesses where hyper- or hypo-activity of apoptoticmechanisms has been experimentally implicated includesAIDS, allograft rejection, Alzheimer’s disease, autoimmunity(lupus, type-I diabetes, rheumatoid arthritis), cancer, heartfailure, infectious diseases, inflammation, osteoporosis,Parkinson’s disease, restenosis, stroke, and trauma.Consequently, great interest has emerged in finding ways todevise therapeutic strategies for modulating apoptosis path-ways. In this review, we summarize recent findings that havesuggested random drug screening and rational structure-based approaches for obtaining small-molecule drugs thattarget apoptosis proteins. A wide variety of other types ofapplications of apoptosis-based technology will not be dis-cussed here, including protein therapeutics, antisenseoligonucleotides, gene therapy, cell engineering, bioproduc-tion, diagnostics, and crop protection.

Caspases: mechanisms of activation andimplications for drug discovery Though several ways of activating caspases have been docu-mented, probably the one most commonly employed bycells is the ‘induced proximity mechanism’ (reviewed in [7]).The induced-proximity model is predicated on the empiric

observation that the zymogen forms of unprocessed caspasesare not entirely inactive but rather possess weak proteaseactivity. When brought into close apposition through proteininteractions, the zymogens can trans-process each other, pro-ducing the fully active proteases (Figure 1).

Though many pathways for activating caspases may exist,only two have been elucidated in detail — both of whichutilize the induced-proximity mechanism (Figure 2). Oneof these pathways is represented by tumor necrosis factor(TNF)-family receptors, some of which use caspase activa-tion as a signaling mechanism (e.g. TNFR1/CD120a,Fas/APO1/CD95, DR3/Apo2/Weasle, DR4/TrailR1,DR5/TrailR2, DR6). Ligation of these receptors at the cellsurface results in the recruitment of several intracellularproteins, including certain pro-caspases, to the cytosolicdomains of these receptors, forming a ‘death-inducing sig-naling complex’ (DISC) that triggers caspase activationand leads to apoptosis (reviewed in [8,9]). The other apop-tosis pathway involves mitochondria, which releaseproteins such as cytochrome c into the cytosol, causingassembly of a multiprotein caspase-activating complex,referred to as the ‘apoptosome’ (reviewed in [10,11]). Thedeath-receptor and mitochondrial pathways for caspaseactivation are sometimes referred to as the ‘extrinsic’ and

Figure 1

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Caspase activation by induced proximity. In the induced-proximitymodel, pro-caspase zymogens are present as monomers that aresingle polypeptide chains with amino-terminal prodomains (Pro)followed by the regions corresponding to the large (L) and small (S)catalytic subunits. Bringing these zymogens into close proximity, eitherby interactions with adapter proteins that typically share sequence andstructural similarity with the prodomain regions of pro-caspases

(shown) or by over-expression of pro-caspases resulting in self-aggregation (not-shown), allows these proenzymes to trans-processeach other. This trans-processing is possible because the zymogenspossess weak protease activity. Once proteolytically processed,separating the large and small subunit fragments and (often) removingthe amino-terminal prodomain, the active protease assembles,consisting of a heterotetramer with two L and two S subunits.

588 Pharmaceutical biotechnology

‘intrinsic’ apoptosis pathways, respectively. Though capa-ble of operating independently, crosstalk between thesepathways can occur at multiple levels.

A wide body of experimental evidence, including geneablation (knockout) experiments in mice, has demonstratedthat caspase 8 represents the apical caspase in the TNF-family death-receptor pathway, whereas caspase 9 serves asthe apical caspase of the mitochondrial pathway [12–15].Without these caspases, the TNF/death-receptor and mito-chondrial pathways for apoptosis cannot function in mosttypes of cells. Consequently, caspases 8 and 9 haveemerged as attractive drug-discovery targets for interrupt-ing selectively either the extrinsic or intrinsic pathway atthe earliest step in these two protease cascades. The struc-tures of active caspase 8 and several other caspases havebeen solved by X-ray crystallography [16–21], providingcritical information for drug discovery, but a structure of thecatalytic domain of caspase 9 is still lacking at this time [22].

Data validating caspases as drug-discovery targets havecome from gene ablation studies in mice. For example,caspase 1 knockout mice exhibit marked resistance toendotoxin-induced sepsis [23]. Caspase 1, as well as cas-pase 2 and caspase 11, knockout mice also suffer less tissueloss in stroke models [24,25]. Inhibition of caspase 1 alsoslows progression in a mouse model of Huntington’s dis-ease [26]. The cytoprotective effects associated withablation of caspase 1 may, however, be secondary to ananti-inflammatory effect, given the important role of theseproteases in processing cytokine precursors. Cells fromcaspase 12 knockout mice display resistance to apoptosisinduced by amyloid β-peptide [27], a finding of potentialrelevance to Alzheimer’s disease. In this regard, caspase 12appears to be associated with the endoplasmic reticulum

(ER) and becomes specifically activated by ER stress, thuslinking ER damage to a caspase-activation pathway inde-pendent of the mitochondrial (cytochrome c) anddeath-receptor (TNF-family) pathways [27]. In addition,the β-amyloid precursor protein (βAPP) is a substrate ofcaspase 3, with caspase-dependent processing possiblyencouraging this protein to undergo subsequent aberrantprocessing by the secretases responsible for generation oftoxic β-amyloid peptides [28]. Though studied in themouse, human orthologs of caspases 11 and 12 remain elu-sive to date, and thus identification of the relevant humancaspases requires more effort.

Analysis of the structures of the active sites of caspases,experiments with combinatorial peptide libraries, andother data suggest that caspases recognize the Asp residuesthey cleave within the context of tetrapeptide motifs,where cleavage occurs at the peptidyl bond distal (carboxy-terminal) to the targeted Asp [29,30]. This informationabout the structures and mechanisms of caspases has beenexploited for developing small-molecule inhibitors, whichare finding their way into clinical trials for stroke, liver fail-ure, inflammatory diseases, and a wide variety of ailments[5,31]. Proof-of-concept data have been reported in a vari-ety of animal models using suboptimal peptidyl inhibitorsof caspases, such as carbobenzoxy–Val–Ala–Asp–fluo-romethylketone (zVADfmk). For example, peptidylcaspase inhibitors provide substantial protection in strokemodels of focal transient and permanent ischemia, as wellas myocardial infarction [32–35], and have been used todemonstrate in vivo efficacy in mouse models of hepaticinjury, sepsis, amylotrophic lateral sclerosis (ALS), and sev-eral other diseases [36–38]. All reported studies thus farhave employed broad-spectrum irreversible inhibitors thatform covalent adducts with the active-site cysteines of all

Figure 2

Pathways for caspase activation. Two of themajor pathways for caspase activation inmammalian cells are presented, the extrinsic(left) and intrinsic (right). The extrinsic pathwaycan be induced by members of the TNF-familyof cytokine receptors, such as TNFR1 and Fas.These proteins recruit adapter proteins to theircytosolic death domains (DDs), including Fadd,which then binds death effector domain (DED)-containing pro-caspases, particularly pro-caspase 8. The intrinsic pathway can beinduced by release of cytochrome c frommitochondria, induced by various stimuli,including elevations in the levels of pore-formingpro-apoptotic Bcl-2 family proteins such as Bax.In the cytosol, cytochrome c binds and activatesApaf-1, allowing it to then bind and activate pro-caspase 9. Active caspase 9 (intrinsic) andcaspase 8 (extrinsic) have been shown todirectly cleave and activate the effectorprotease caspase 3. Other caspases can alsobecome involved in these pathways (notshown), thus the schematic represents an over-simplification of the events that occur in vivo.

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known caspases. Such broad-spectrum agents may beacceptable and even preferred for acute injury whereshort-term administration is envisioned, but chronic dis-eases are likely to be best approached with selectiveinhibitors that target specific initiator caspases, such as cas-pases 1, 8 or 9. Differences in the specificities of theseinitiator caspases with respect to preferred tetrapeptidesubstrates and the structures of their active sites suggestthis may be possible (Figure 3).

Nucleotide-binding domains of caspaseactivators as drug targets A paradigm for caspase activation is embodied in CED-4/Apaf-1 family proteins. These proteins contain a caspase-associated recruitment domain (CARD), which bindshomologous structures within the prodomains of certaininitiator caspases [39], and a nucleotide-binding oligomer-ization domain, known either as a NB-ARC domain(nucleotide-binding domain homologous to Apaf-1, plantR gene products, and CED-4) or NACHT domain [40].Oligomerization of the NB-ARC domains of CED-4/Apaf-1-family proteins depends on ATP or deoxyATPbinding, and results in caspase activation by the induced-proximity method [41–43]. Though only hypotheticalmodels of NB-ARC domains are available (and thus struc-tural details are lacking), the nucleotide-binding site ofCED4/Apaf-1-family proteins makes for a promising drug-discovery target that could serve to prevent or decreasecaspase activation within certain pathways.

Gene ablation studies in mice indicate that Apaf-1 isessential for caspase activation via the intrinsic pathway[15]. Apaf-1 contains an autorepressing domain comprisedof several WD repeats, which renders it dependent on

mitochondria-derived cytochrome c for activation(reviewed in [10]). Not until binding cytochrome c can theNB-ARC domain of Apaf-1 oligomerize [42,43].Oligomerized Apaf-1 then binds via its CARD to the

Figure 3

Comparison of the X-ray structures of theactive sites of human caspases 1, 3 and 8.Each active site is complexed with a differentpeptide inhibitor (caspase 1–Ac-WEHD-aldehyde; caspase 3–Ac-DEVD-aldehyde;Caspase 8–z-EVD-methylketone). Arrowspoint to structural differences in the caspaseactive sites that contribute to the specificitiesin substrate and inhibitor recognition amongthe three caspases. Structures producedusing [17,20,60] and data submitted to theBrookhaven data bank. Caspase 1

Caspase 8

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Figure 4

NMR structure of Bcl-xL complexed with a peptide derived from theBak BH3 domain. The Bak BH3 peptide assumes an α-helicalstructure and binds into a hydrophobic groove on the surface of Bcl-xL.Structure is reproduced from [51] with permission.


CARD of pro-caspase 9, forming a large multiprotein com-plex. This ‘apoptosome’ thus brings pro-caspase 9zymogens into close apposition, facilitating their trans-processing of each other. Unlike caspase 8, however, wherethe amino-terminal prodomain of the zymogen is cleavedoff and the active protease is released into the cytosol, thecaspase 9 enzyme remains bound to Apaf-1 via its CARD-containing amino-terminal prodomain.

The human genome contains at least three additionalCED4/Apaf-1-related genes, which contain CARDs incombination with putative nucleotide-binding domains([44,45]; JC Reed, unpublished data). The roles of theseproteins in apoptosis regulation, however, remain largelyunknown. Another putative nucleotide-binding proteinimplicated in apoptosis is DAP3 [46]. Mutation of thenucleotide-binding motif in DAP3 converts it to a trans-dominant inhibitor of apoptosis induction by TNF-familydeath receptors, suggesting that this protein may also offeropportunities for small-molecule drug discovery.

Bcl-2 family proteinsThe mitochondria-dependent pathway for apoptosis is gov-erned by Bcl-2 family proteins. Both pro-apoptotic andanti-apoptotic Bcl-2 family proteins exist, and many of theseproteins physically bind each other, forming a complex net-work of homodimers and heterodimers (reviewed in[47,48]). Though the precise mechanisms are debated, thechief function of Bcl-2 family proteins is to regulate releaseof cytochrome c from mitochondria, with pro-apoptotic Bcl-2 family proteins inducing and anti-apoptotic familymembers suppressing cytochrome c release by affecting thepermeability of mitochondrial membranes. The relativeratios of anti- and pro-apoptotic Bcl-2 family proteins dictatethe ultimate sensitivity or resistance of cells to various apop-totic stimuli, including growth factor/neurotrophindeprivation, hypoxia, radiation, anti-cancer drugs, oxidants,and Ca2+ overload. Not surprisingly then, alterations in theamounts of these proteins have been associated with a vari-ety of pathological conditions, characterized by either toomuch (cell loss) or too little (cell accumulation) cell death.These conditions include cancer, autoimmune disorderssuch as lupus (where a failure to eradicate autoreactive lym-phocytes occurs), immunodeficiency associated with HIVinfection, and ischemia-reperfusion injury during stroke andmyocardial infarction, among others [49].

In humans, 21 members of the Bcl-2 family gene familyhave been described, providing opportunities for tissue-specific selection of drug-discovery targets. Nearly allBcl-2 family proteins contain a dimerization domain calledBH3, representing a ~16 amino acid amphipathic α-helixthat inserts into a hydrophobic crevice on the surface ofanti-apoptotic proteins such as Bcl-2 and Bcl-XL [50,51](Figure 4). Proof-of-concept experiments using peptidessuggest that molecules that mimic BH3 can be used toinduce apoptosis, a strategy that is being pursued for can-cer therapy. At least one description of a non-peptidyl

compound that functions as a BH3 mimic has appeared[52] and others seem likely to appear in the near future.Several assays are available for screening compoundlibraries for antagonists of either pro-apoptotic (e.g. Bax,Bak, and Bok) or anti-apoptotic Bcl-2 family proteins,including dimerization assays and cell-based (functional)assays using either yeast or mammalian cells.

IAP family proteinsThe IAPs (inhibitor of apoptosis proteins) represent a fam-ily of evolutionarily conserved apoptosis suppressors(reviewed in [53,54]. IAPs contain the BIR (baculovirus iaprepeat) domains, which are zinc-binding folds importantfor their anti-apoptotic activity [55]. Although IAP-familyproteins may exhibit several functions, most bind andpotently inhibit activated caspases, including the effectorcaspases 3 and 7, as well as the initiator caspase 9 [53].Altered expression of IAPs has been associated with dis-eases such as spinal muscular atrophy (SMA), amotorneuron degenerative disease where the IAP-familymember NAIP is sometimes inactivated by hereditarymutations, and cancer, where the IAP-member Survivin iscommonly over-expressed [53,54].

A mammalian IAP inhibitor, Smac (Diablo), was recentlydescribed, which suggests approaches to pharmacologicalinhibition of IAPs. Smac binds multiple IAP-family mem-bers (XIAP, cIAP1, cIAP2, and Survivin), apparentlyfreeing caspases from their grip so they can induce apopto-sis [56–58]. Peptides as short as 7-mers from theamino-terminus of Smac are sufficient to reverse caspase-binding and inhibition by IAPs in vitro [58], suggestingopportunities for discovery of small-molecule drugs thatmight find applications for treatment of cancer. Analogousdata have been obtained for Drosophila cell-death proteinsthat inhibit IAPs in the fly (Rpr, Grim Hid), suggesting anevolutionarily conserved mechanism for IAP repression(reviewed in [59]).

ConclusionsAdvances in elucidating the molecular mechanisms andthree-dimensional structures of apoptosis proteins haverevealed strategies for pharmacological intervention in awide range of ailments where cell life and death are imbal-anced, including cancer, autoimmunity, immunodeficiency,inflammation, ischemic heart disease, stroke and neurode-generative diseases. In addition to directly targeting the corecomponents of the cell-death machinery (caspases,CED4/Apaf-1, Bcl-2, IAPs), opportunities exist to indirectlyaffect the apoptosis pathway by attacking kinases (Akt/PKB,IKKα/β, DAP-kinase), phosphatases (calcineurin), and lig-and-binding transcription factors (PPARγ, RAR, RXR) thatmodulate the function or expression of critical apoptosis reg-ulators (reviewed in [47]). This progress in identifyingapoptosis-relevant targets for drug discovery suggests richopportunities for new and more effective treatments formany medical illnesses currently lacking adequate treat-ments. This effort will be aided by a deeper understanding

of the physiological and pathological roles of individualmembers of the multi-gene families that comprise the apop-tosis machinery, so that undesired side-effects can beminimized and efficacy maximized.

AcknowledgementsWe thank R Cornell and S Roggo for manuscript and figure preparation.

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