The history of Z-VAD-FMK, a tool for understand- ing the significance of caspase inhibition*
Cornelis J. F. Van Noorden

Academic Medical Center, University of Amsterdam, Department of Cell Biology and Histology, Amsterdam, The Netherlands
Received 12 March 2001; accepted 26 March 2001


Dr. Robert Smith is one of the pioneers in histochemistry. One of his most important achievements is the recognition of proteo- lysis as a major physiological and patho- physiological process. As a consequence, he developed selective fluorogenic and chromogenic substrates and specific inhibi- tors of proteases that allow the (histochem- ical) analysis of protease activity. One of the latest successes is the design of Z- VAD-fluoromethylketone (FMK), the spe- cific caspase inhibitor, that is a key com- pound for studies on apoptosis. Its develop- ment was originally meant for therapeutic use but unforeseen cytotoxicity of a meta- bolic derivative of the FMK compound dis- abled its potential as a drug. However, as a

Correspondence to: Prof. Dr. C. J. F. Van Noorden, Department of Cell Biology and Histology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands;
tel: *31 20 566 49 70; fax: *31 20 697 41 56; e-mail: [email protected]
* Tribute to Dr. Robert E. Smith, one of the pioneers in histochemistry, at the occasion of his 70th birthday. http://www.urbanfischer.de/journals/actahist 0065–1281/01/103/3–241 $ 15.00/0

tool for fundamental research it is a great success. The history of Z-VAD- FMK is an example of the creative brain and the tireless perseverance of Ro- bert Smith for which histochemistry and cytochemistry owes him so much. This history of Z-VAD-FMK is a well-deserved tribute at the occasion of his 70th birthday.

Key wordr: proteases–proteinases–caspases-apoptosis–histochemistry–cytochem- istry–drug discovery–fluorogenic substrates–chromogenic substrates–protease in- hibitors

The history of Z-VAD-fluoromethylketone (FMK) is about the difficult routes in drug discovery. Z-VAD-FMK is a specific general inhibitor of caspases and as such it is used in studies on apoptosis. Originally, the compound was de- signed for therapeutic use. Unfortunately, it will never be applied as a drug due to the possible metabolic conversion of the FMK leaving group into the highly toxic fluoroacetate in vivo. However, fundamental research in apopto- sis has greatly benefitted from it. The fascinating history of Z-VAD-FMK is the history of the life of Dr. Robert E. Smith. Robert Smith is one of the pio- neers in histochemistry and one of his major achievements was the introduc- tion of fluorogenic and chromogenic substrates and selective inhibitors of pro- teases in a time that hardly anybody realized what the relevance of proteases in cell and tissue physiology and pathophysiology is. It started when he dis- covered crinophagy as a lysosomal process that is the most important regula- tory mechanism in peptide hormone-producing cells to keep the intracellular stock of hormone under control. This study (Smith and Farquhar, 1966) be- came a citation classic. It was basically an EM study in which lysosomes were demonstrated on the basis of acid phosphatase activity. Robert was able to deduct the process of crinophagy from the EM images that he made of pep- tide hormone-producing cells. However, he realized that the lysosomal en- zymes that were known at that time (phosphatases, esterases) could never be the enzymes that are responsible for lysosomal degradation of peptide hor- mones1. Therefore, he started to investigate proteases and founded his com- pany Enzyme Systems Products (ESP) to develop protease substrates and inhibitors. Every discovery of a protease substrate or inhibitor is an interesting story on its own, but the difficult road that led to the successful application of the caspase inhibitor Z-VAD-FMK is representative for the life of Robert Smith. His life was entirely devoted to scientific research in general and histo- chemistry and cytochemistry in particular. In 1990, Robert was honored with the prestigious A.G.E. Pearse prize of the Royal Microscopical Society to rec-
1It was 25 years later that the morphological concept of crinophagy was established by immunolo- calization of prolactin and cathepsin D (Kuriakose et al., 1989).

ognize his valuable contributions to histochemistry and in 2000, the Univer- sity of California in San Francisco created the Robert E. Smith chair in pathology, which has Professor Jim MacKerrow as the first holder, to memo- rize his contributions to the life sciences.

The history of Z-VAD-FMK
There is an interesting history to the development of fluoromethylketone (FMK) inhibitors for a family of cysteine proteinases called caspases. These inhibitors are tools to unravel the many mysteries of apoptosis and thus re- vealing the involvement of caspases in the development and progression of various diseases. Z-VAD-FMK (benzyloxycarbonyl-valine-alanine-aspartate- FMK) and related FMKs with aspartic acid in the P1 position have become extremely popular irreversible inhibitors of the 14 members of the caspase family that are known to date. The use of these inhibitors in basic and applied research in the 5 years since their introduction has been extensive as is evi- denced by the more than 600 provocative publications reporting their applica- tion. Unfortunately, FMK inhibitors will remain only helpful assisting in scientific research and will never become therapeutic agents, because of the FMK moiety that can produce the highly toxic fluoroacetate in vivo. Hope- fully, a second generation of compounds that is now being developed will be more suitable as drugs.
How did the FMK compounds come about at such an auspicious juncture? It becomes clear in this tribute that this was not by elegant planning, but rather by perception, persuasion, and perseverance, which are all strong as- pects of the character of Dr. Robert E. Smith.
The story starts in early 1983, when Dr. David Rasnick (senior synthesis chemist at Enzyme Systems Products (ESP), then in Dublin CA and now in Livermore CA) approached Robert with an idea to synthesize small peptide derivatives of FMKs as inhibitors for elastase (Rasnick, 1985). ESP was then a fledgling research reagent company and had only just moved from Indiana to California. The company was cash-strapped, trying to establish the rele- vance of synthetic substrates for the localization of serine and cysteine pro- teinases, based on the active site modeling concept of Schechter and Berger (1967). The practicality of the approach had been established in 1972 by Dr. Smith’s team while he was at Eli-Lilly, Indianapolis IN (Smith et al., 1972). According to Dr. Rasnick, it seemed reasonable that better peptide pro- tease inhibitors could be synthesized than the existing chloromethylketones and diazomethane peptide inhibitors of serine and cysteine proteinases be- cause these had serious limitations, essentially confining them to the applica- tion to dead cells and tissues in biochemical studies. Dr. Rasnick reasoned that peptide derivatives of FMK would be less alkylating than chloromethyl- ketones, thus allowing cell culture studies and permitting the discovery of how cells function when the activity of specific proteases is inhibited. However, it

appeared to be extremely difficult from a synthesis point of view to attach the desired amino acid to the ala-FMK to obtain a selective leukocyte elastase in- hibitor (Powers and Tuhy, 1973). An alternative was possible, but then a change of direction was necessary from inhibitors of serine proteinases to those of cysteine proteinases, and the much simpler inhibitor Z-phe-ala-FMK was synthesized (Rasnick, 1985). Z-phe-ala-chloroketone had been proven to be effective as an inhibitor of cathepsin B by Dr. Elliott Shaw and co-workers (Shaw et al., 1983).
Soon it was established at ESP that cathepsin B in crevicular and synovial fluids as well as cathepsin B purified from human liver could be irreversibly inhibited by Z-phe-ala-FMK. Furthermore, the compound showed very low toxicity and thus had great potential for in vivo use. The compound proved to be nonmutagenic and did not perturb the cell cycle. Moreover, an unexpected observation was made at ESP in kinetic studies: unlike chloromethylketone inhibitors which inhibited both serine and cysteine proteinases, FMKs effec- tively inhibited cysteine proteinases only (Rasnick, 1985). By early 1986, there was a strong appreciation for the potential of the compound as selective inhibitor and animal experiments were worth to be considered. The first ani- mal experiments were oriented towards the effectiveness of the various routes of administration and dose concentrations of the inhibitor and towards the ef- fects the inhibitor had on cathepsin B activity in vivo after administration of a single dose. By late 1986, rat adjuvant arthritis studies were underway.
By the end of 1987, the first studies of the effect of the inhibitor in a rat model of arthritis were completed with astonishing efficacy (Van Noorden et al., 1988). Everything looked bright for the newly established company Proto- tek of Dr. Smith that dealt solely with FMK technology for therapeutic inhibi- tion of parasitic proteinases (Rosenthal et al., 1993; Harth et al., 1993) and in- vasion and metastasis of cancer (Van Noorden et al., 1998, 2000). With the dawn of 1988, Prototek and Marion Laboratories, Kansas City MI, signed a research contract providing significant funding to support development of FMK inhibitors as candidates for treating rheumatoid arthritis (Van Noorden and Everts, 1991; Ahmed et al., 1992; Esser et al., 1994).
By remarkable coincidence of timing, studies of Dr. Roy Black and cowork- ers at Immunex were surfacing in the literature of an unknown protease of unidentified class that produced active interleukin-1β from an inactive precur- sor (Black et al., 1988, 1989 a, b). The salient discovery was that aspartic acid was an absolute requirement in the P1 position for hydrolysis by the enzyme that was optimally active at slightly acid pH in the presence of SH-groups, thus implying that the “converting enzyme” was cysteine-like in class. How- ever, it was not inhibited by E64, a general inhibitor of cysteine proteinases, and the bond hydrolyzed was not preceded with a pair of basic residues con- ventional for cathepsin B. Prototek became aware of Dr. Black’s work in late 1987. Dr. Rasnick contacted Dr. Black to determine whether FMK inhibitors could block proteolytic conversion of the inactive precursor of interleukin-1β. First, several inhibitors of cathepsin B were sent to Immunex but they all

failed to inhibit interleukin-lβ processing. Nevertheless, it was found that the converting enzyme was sensitive to iodoacetate, an inhibitor of most cysteine proteinases. In November 1988, Dr. Smith supplied Dr. Black with MeOSuc- phe-asp-FMK. In April 1989, Dr. Black reported that MeOSuc-phe-asp-FMK inhibited the enzyme generating mature human interleukin-1β but only at high concentrations. The Immunex paper, published in August 1990, established with certainty that the P1 position determinant was aspartate, and that the en- zyme had to be a novel and unique cysteine proteinase that was unlike the ca- thepsins (Sleath et al., 1990). In early 1990, Dr. Black informed Robert that Kodak/Sterling had purchased the Immunex interleukin-1β technology and that he would no longer be working in that area of research. As a conse- quence, the cooperation between Immunex and Prototek was terminated. Moreover, the synthesis of FMK inhibitors of interleukin converting enzyme (ICE) was discontinued at Prototek because of other priorities.
The paramount focus was on FMK inhibitors of the cathepsin subclass of cysteine proteinases. Moreover, a new method of synthesis had to be devel- oped, because the existing one resulted in FMK preparations which were thought to contain small residues of fluoroacetate that is highly toxic and pro- hibited in vivo use of FMK inhibitors. In vivo application of FMK inhibitors was then a serious option, and toxicity issues were considered but a pheno- menon known as “lethal synthesis” was not immediately recognized.
How the consequences of this phenomenon developed is an interesting story in disappointment and futility of a drug development program. The lethal aspects in conversion of FMK into fluoroacetate were discovered by Robert in 1990 on the basis of a histopathological study. The study showed a decrease in spleen size with loss of germinal centers, cardiomyopathy and lymphocyte infiltration of damaged muscular tissue of the left ventricle in rats that had been treated with FMK inhibitors for several weeks. Moreover, at Marion Laboratories 4 dogs had been given a single 25 mg/kg oral dose of a FMK inhibitor, a dose known to significantly inhibit cathepsin B. It killed 2 dogs within 18 h. The cellular toxicity was elucidated in 1991: fluoroacetate inhibited aconitase which damaged mitochondria of cardiomyocytes with loss of integrity of the outer membrane and subsequent release of cytochrome c, probably as a result of mitochondrial matrix swelling (Gribble, 1973). It was then established that conversion of ala-FMK to fluoroacetate and subsequent mitochondrial damage occurred in vivo but not in vitro. Eichold et al. (1997) at the Proctor and Gamble Health Care Research Center developed a highly selective and sensitive detection scheme and quantified fluoroacetate in rat tis- sues following administration of a single dose of Z-phe-ala-FMK. They found that fluoroacetate was metabolically produced in heart, kidney and liver. From that point, it was clear that, no matter how efficacious FMK compounds would be in inhibiting cysteine proteinases, FMKs would have little utility in any biological dimension and surely not as drug candidates.
There remained one academic reason for continuation of the FMK inhibi- tor program: Prototek had a significant supply of purified ICE and a specific

ICE substrate, Z-YVAD-AFC that had been synthesized by ESP, which was subsequently used for further purification of ICE (Kronheim et al., 1992). FMK peptide derivatives were evaluated for inhibition of ICE. In 1990, Dr. Jeff Prior completed the synthesis of MeOSuc-phe-asp-(OBz)-FMK which in assay with Z-YVAD-AFC showed no inhibition of purified ICE. Neverthe- less, after extended preincubations of crude preparations of blood monocytes in the presence of inhibitor, reasonable inhibition occurred. However, syn- thesis of better ICE inhibitors was at that point not a high priority in Prototek because the enzyme was considered to be a single aberrant of the large family of cathepsins in man, animals, plants and parasites. The prevailing view with- in Prototek was to remain focused on the cathepsins that were considered to be involved in diseases such as arthritis, cancer and parasitic infections (for review, see Roose and Van Noorden, 1995). Besides, there were no funds nor people available for such a single entity risk. Dr. Prior, whose expertise was in biochemistry (enzyme purification and kinetics), decided then to synthesize in his own spare time the ICE inhibitors that Robert had outlined, such as Boc- asp-(4Byl)-FMK, that was ready on June 27, 1990, Mu-phe-asp-(4Byl)-FMK on February 22, 1991, Boc-asp-(OMe)-FMK on March 21, 1991, and Mu-phe-asp-(OMe)-FMK, on May 8, 1991. Biochemical analysis proved that the inhibitors behaved above expectations. It was found that the unprotected free carboxyl group of aspartic acid in the P1 position reacted with the fluoro- methyl group which incapacitated the inhibitor. When the free carboxyl group of aspartic acid was protected by a methyl or ethyl group, the compound was stabilized. However, these groups could be removed in vitro and in vivo by esterases. Therefore, 4-benzyl or t-butyl blocks were introduced that could not be removed. In addition, these blocks appeared to assist in transporting the molecule through the cell membrane into the cytoplasm where ICE had been identified immunohistochemically.
One of the competitors of Prototek, Merck in Rahway NJ, was leading the way in ICE studies as was shown by Thornberry et al. (1992). This publica- tion was far more than a profound scientific contribution; it was an equally re- vealing commitment of a major pharmaceutical company to a single enzyme involved in inflammation. This made it look impossible for Prototek to be successful in the field of ICE inhibitors – truly a David-Goliath match. Furthermore, Smith’s own histopathological studies of FMK toxicity clearly put the company in harm’s way. Moreover, with the merger of Marion La- boratories with Merrill-Dow, this company now had its own research facilities to continue investigations without the help of and cooperation with Prototek. As a consequence, Marion Laboratories terminated its research contract with Prototek in 1991, leaving Prototek without 90 per cent of its financial base for research. A cut in the number of Prototek employees from 36 to 4 was the consequence, and these 4 took a significant cut in salary or no pay at all for over a year.
Uncertain of the ultimate fate of Prototek, strong-headed Robert convinced ESP to carry the flag and synthesize Z-YVAD-AFC, Z-YVAD-AMC and Z-

VAD-AFC as substrates for ICE. The in-depth reason was the use of these substrates to explore ICE further. Dr. Jamil Talhouk at ESP, a sugar chemist converted to peptide synthesis, was allowed to work on the project when time permitted. Dr. Talhouk synthesized Boc-asp-(OMe)-FMK (January 29, 1993) and Ac-tyr-val-ala-asp-(OEt)-FMK (February 17, 1993). Studies on the effects of these inhibitors on ICE activity showed that ICE had an extended hydro- phobic pocket at the P3 and P4 positions and that replacement of the tyr group by a Z group was actually an improvement in inhibition kinetics of the purified enzyme as well as an improvement in transport through the cell membrane. A similar phenomenon has recently been demonstrated for the N- terminal substituted 2-indole dipeptide ICE inhibitors of IDUN Pharmaceuti- cals (Grobmyer et al., 1999).
When it became evident in 1993 that ICE was a mammalian homologue of the product of the C. elegans cell death gene, Ced-3, speculation abounded that other cell death enzymes were present (Miura et al., 1993; Barinaga, 1994). This proved to be correct and now a family of aspartate-specific homol- ogous proteinases, the caspases, are known of which 14 have been purified at the moment. Their individual substrate specificity enabled the synthesis of specific substrates and more defined peptide and peptidomimetic inhibitors. By May 1995, a significant quantity of Z-VAD-FMK had been made by Dr. Irina Dynan at ESP. It was reasoned that it could be a good general cas- pase inhibitor. In vitro and in vivo studies of ICE activity indicated that the in- hibitor was efficacious. A license agreement was constituted between ESP and Prototek whereby ESP would have the exclusive right to market FMK in- hibitors for research applications, and Prototek would retain any potential, though unlikely, in their use as therapeutic agents.
It was decided to go as fast as possible outside ESP for validation of its ef- fects. Four laboratories were approached to evaluate the inhibitor. It was a risky venture but, on the other hand, it would have been a much larger under- taking requiring significantly more effort and time when the inhibitor had to be evaluated as a scientific conquest at ESP with all the jeopardy and delay of subsequent peer review. The inhibitor was presented for evaluation to the fol- lowing researchers in May 1995: Dr. Vishva M. Dixit (then, University of Mi- chigan; Clayton et al., 1997), Dr. Pierre A. Henkart (NIH; Sarin et al., 1996), Dr. Michael D. Jacobson (then, MRC for Molecular Cell Biology, University College London; Jacobson, 1998) and Dr. Eugene M. Johnson (Washington University in St. Louis; Cheng et al., 1998). The efficacy of the inhibitor ap- peared to be high in the hands of each investigator.
In 1996 at an IBC conference on protease inhibitors in San Francisco, Dr. David J. Livingston of Vertex Pharma presented studies on ICE and its in- hibition as a therapeutic strategy (Wilson et al., 1994). It appeared that the Z- VAD-FMK inhibitor did not work in their hands in in vitro studies. In Febru- ary 1997, a second conference on protease inhibitors took place in Washing- ton DC, and Dr. Livingston once again spoke on therapeutic strategies for ICE inhibition but now on the basis of in vivo studies in which Z-VAD-FMK

appeared to be effective. Removal of the ester group to activate the inhibitor was essential, which was accomplished in vivo but not in vitro by endogenous esterases. It was clear then that it could not be taken for granted that every user of the inhibitor would know that a free acid on the P1 aspartate is essen- tial for the inhibitor to be active. What was an axiom for Robert to use ester derivitization of an acid for protection and rely upon the ubiquity of esterases in vivo to appropriately activate the inhibitor, was not obvious for others. An- other lesson that had to be learned the hard way.
By the end of 1997, 8 homologues of ICE were recognized and the once thought aberrant cysteine proteinase was the first discovery of a new family, the caspases. Caspases, unlike their cousins, the cathepsins, are in signifi- cantly lower concentrations present in cells and tissues. As a consequence, the assay system for caspase activity requires greater sensitivity. Products of proteolytic cleavage of 7-amino-4-trifluoro-methyl coumarin substrates with their large Stoke shift possessed not only the greater sensitivity but also the lack of background blue fluorescence of fluorescent components in cells and tissues such as NAD(P)H and flavins. These substrates were available for al- most 20 years, but the sensitivity of detection was not required when used in assays of activity of cathepsins (Smith et al., 1980).
One of the first in vivo studies demonstrating inhibition of caspases and as a consequence inhibition of apoptosis was that of Dr. Pierre Vassalli and co- workers (Rodriguez et al., 1996). Z-VAD-FMK was shown to prevent liver damage resulting from apoptosis induced by administration of an anti-FAS antibody. Dr. Vassalli was so convinced of the efficacy of Z-VAD-FMK in the inhibition of liver damage that he stated in a personal communication with Robert that “if I had hepatitis C, I would not hesitate to take Z-VAD-FMK”. Thus, use of the compound in a life-threatening situation became credible. Subsequently, numerous studies demonstrated the in vivo effect of Z-VAD- FMK in preventing or attenuating cell death by apoptosis in brain, spinal cord, heart, blood vessels, liver and kidneys (Chandler et al., 1998; Namura et al., 1998; Yaoita et al., 1998).
What is to come? Some things are predictable, others less well-perceived (Graczyk, 1999). According to Robert, further grass-root investigations will likely identify new caspases, which will lead to the development of new spe- cific substrates and inhibitors. Moreover, many developments in caspase tech- nology will emerge in the near future. Peptidomimetic inhibitors will soon be available where the P2, P3 and P4 positions are replaced by an organic non- peptide molecule attached to the P1 aspartic acid, and where the P1‘ site will be unique heterocyclic traps that allow for the formulation of either reversible or irreversible enzyme inhibitors depending upon the pH in target cells. Other types of inhibitors will be developed, extensions to the N-terminal end will enhance selectivity in tissue localization and enzyme inhibition. For example, caspase inhibitors will be developed that can pass rapidly through the blood- brain barrier. Mechanisms will be identified that are responsible for activation of specific caspases relevant to (patho)physiological events. Understanding of

the interrelationships between caspases and cathepsins in certain diseases will lead to the development of bifunctional drugs. Identification and understand- ing of the significance of isoforms of caspases and their glycosylation and/or sialylation patterns in relation to their kinetic behavior (Smith et al., 1998) and their conformational configuration will become major issues as well. Fi- nally, questions have to be addressed, such as: ‘how long can an individual caspase in a specific cell or tissue be effectively inhibited?’, and ‘what trib- utes must be paid?’ as a part of the building an efficacy base (Nicholson, 1999; Salvesen, 1999).
Nevertheless, a firm conclusion can be drawn now with respect to Z- VAD-FMK as was done by Dr. Donald W. Nicholson, senior director of the Department of Pharmacology, Biochemistry and Molecular Biology of the Merck Frosst Centre of Therapeutic Research at a conference held in San Diego CA (1999): “If caspases are involved in apoptosis, Z-VAD-FMK will inhibit it”.

Acknowledgmentr. The editorial comments of Prof. Dr. Reinhart Gossrau, Free University of Berlin, and the careful preparation of the manuscript by Ms. Trees Pierik are gratefully acknowledged.

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