Design of suicide substrates: an approach to the development of highly selective enzyme inhibitors as drugs

212

TIPS -.~4ay 1983

enzyme to become an alkylating agent. Thus, the enzyme would generate its own irreversible inhibitor at the active site and the enzyme would have the capacity to commit suicide. The specificity of this novel approach to drug design thus depends upon the eventual catalytic turnover of the suicide substrate by its target enzyme. By considering the available knowledge of the catalytic mechanism of important target enzymes, the pharmacologist has designed and synthesized a number of effective suicide substrates. In every instance, an electrophilic group within the substrate is generated by catalysis and is then attacked by a nucleophilic functional group residing at the active site. Four principal mutes to the enzymatic generation of electrophiles have been exploited with success in the design of suicide substrates: (a) enzyme generation of a Michael acceptor (see below); (b) enzyme generation of an electrophile by protonation; (c) enzyme generation of an electrophile by formation of an acyl-enzyme intermediate; and (d) enzyme generation of an electrophile via

Design of suicide substrates:

an a p p r o a c h to the d e v e l o p m e n t of highly selective enzyme inhibitors as drugs T. M. Penning Department of Pharmacology, University of Pennsylvania Medical School, Philadelphta, Pennsylvania, 19104, U S.A.

The concept that a pharmacologically important target enzyme can generate, via its normal catalytic mechanism, an irreversible inhibitor f r o m an innocuous substrate analogue and hence commit suicide offers a highly selective approach to drug design. This article describes the principles exploited by the pharmacologist in his endeavor to develop suicide substrates.

The aim of drug design is the development of highly selective agents and some success has been achieved by the synthesis of specific enzyme inhibitors. A widely trieff approach has been to introduce an electron deficient group or electrophile into the substrate, e.g. an o~-haloketone or an haloacetyl group. Upon binding to the enzyme active site, a nearby and appropriately oriented electron rich group or nucleophile present on the side chain of an amino acid (such as the hydroxyl group of serine or the e-amino group of lysine) can attack the electrophilic substrate analogue. As a result a stable co-

_#-~ -v,.v,

H÷

C'~.C~C=C

--> C ~ C - -

' C~H'~ ENZ-B:

~"

I

=C--->C--C~---C--C-

QJ/

HI

[ [ E N Z -Y-'~ •

C~carbanion _~--'~

> --C--C--C--C--

conjugated vinyl

inactivated

~_~c--c---,~--~--c_~c---,~222.~c

Y H

Y

H

~

n. I > --c--c_--c--c-i I

i

ENZ-B"

...I I I 1 ~'C~C'~'-C-- ~ C--C~C-- ~ -C--C--C(--/AI ~,x

i

J 1'-7 x®

I

I

H

enzyme

-a©gtlfione

Y® )H

~ | ENZ

ENZ

II

carbanlon

c o n j u g a t e d allene

inactivated

enzyme

x

F~g.1. ~lichaelacceptor, an electronicrelaysystem m which X = O, N or S and Y = Electron rich nucleophilicgroup on enzyme.

valent bond is formed between the enzyme and affinity label and the enzyme is irreversibly inactivated. The selectivity of this approach to drug design depends upon the specificity of the binding of the substrate analogue to its target enzyme. However, the major disadvantage is that the substrate analogue is usually a highly reactive species that is not innocuous. Thus, alkylation of other macromolecules can take place in vivo and, in addition, the analogue may be scavenged by ubiquitous low molecular weight nucleophiles, such as glutathione. Selectivity could be greatly enhanced if the substrate analogue were itself relatively innocuous and required activation by the normal catalytic mechanism of the target

II

H

TaH-c-"

II

H

II

S'~

H

ENZ-

5

H

!-~NZ

ENZ-B:

cff~¢avbanlnn

ketenlmine

Inactl'¢ated enzyme

. _-fluorinated • FGx .

.

.

.

ENZ-B:

ENZ ¢arbanlon

c o n j u g a t e d vinyl

inactivated

enzyme

Fig. 2. Michael acceptor formation via ~-carbanions, Enz-B refers to base that mediates proton abstraction c~to group X. Enz- Y refers to a nucleophilic functional group on the side chain o f amino acid present at the active site.

© 1983 ElsevierSoence PubhshersB V , Amsterdam 0165- 6147/83/$0100

213

T I P S - 'flay 1983 insertion of oxygen. The aim of this article is to emphasize these principles for the design of suicide substrates and illustrate them by examples of pharmacological interest and potential therapeutic importance.

Enzyme generation of a Michael acceptor: via carbanion formation Structures which allow a relay of electrons to an electron sink X are termed Michael acceptors and can be readily alkylated by a nucleophile Y- (Fig. 1). Substrate analogues which contain an electron deficient group fl to group X (e.g. fl,.y-vinyl, fl,'y-acetylene, fl-cyano- and /3-fluorinated derivatives) can be readily converted to Michael acceptors providing the target enzyme mediates the introduction of an electronegative carbon atom or carbanion a to the electron deficient group (Fig. 2). Thus subsequent rearrangement would result in the formation of the corresponding conjugated electrophiles, conjugated vinyl, conjugated allene and ketenimine. These groups can then readily undergo attack by an enzyme nucleophile to produce inactivated enzyme.

normal intermediate (pyridoxallmine)

I,, e

li,

o~ carbinlon

Michael addition

inactivated enzyme

(vlnyI-GABA pyridoxelimine adduct) ENZ-Y~-)

--

/Y~NZ

N

o~carbanlon Michael addition (4-amlnohex-E-vnoic acid pyrldozallmine idduct)

!

GABAminergic and DOPAminergic agents The routes to the enzyme generation of Michael acceptors described in Fig. 2 have been applied with considerable success to the inactivation of pyridoxal phosphate linked transaminases and decarboxylases ~. These enzymes are intimately involved in neurotransmitter metabolism. Indeed, the need to develop GABAminergic and DOPAminergic agents for the treatment of Huntington's chorea and Parkinson's disease has led to the rational design of vinyl GABA, 4-aminohex-5-ynoic acid and difluoromethylDOPA. The first two compounds are suicide substrates for GABA transaminase and can potentiate the effect of GABA, while difluoromethylDOPA is unable to cross the blood-brain barrier, and is thus a suicide substrate for orily peripheral DOPA decarboxylase. DifluoromethylDOPA will thus potentiate the central effect of 16vodopa in the treatment of Parkinson's disease. The common intermediate in these pyridoxal phosphate linked enzymatic reactions is the Schiff's base that forms between the aldehyde of pyridoxal and the amino group of the amino acid (i.e. a pyridoxalimine). Subsequent catalysis depends upon carbanion formation c~ to the Schiff's base (Fig. 3). Thus, inactivation is to he expected when an amino acid analogue possesses an IG group which is an olefin (as in vinyl GABA) or an acetylene (as in 4-aminohex-5-ynoic acid) or alternatively, a

~+

cart)anion

F

inactivated enzyme

F

ENZ

J

Michael addition

F

inactivated enzyme

(dltluoromathyi-DOPA pyridoxalimine cdduct )

PY (~) =

peosphate pyrldoxal

Fig. 3 Carbamon [ormatlon c~to the Schtff s base R, = H or C02, R ,

=

a/k_vl

emino-ecetylene_~e

e.z-,®

imino-acetylene Michael accepter

~(amlno-acetylena

-t.z

Inactivated enzyme ENZ-¥ @

Imlno-ecetylene

S amino-acetylene

__~--ENZ

Inactivated enzyme

Michael accepter (~ hydroxy-acetylenes ENZ-Y@

.Y-- ENZ

2H-I- 2 e - - ~ o( hydroxy-acetylene

conjugated acetylenlc kalone Michael accepter

Inectlvatecl enzyme

Fig. 4. Michael acceptor formation vta oxutauon

214

TIPS - May 1983 MONOAMINE OXIDASE R-- CH2--NH2

-'I-

FAO

•

n-- CH=

(9 NH2 -71--

FADH

l

2e'reduction

RCHO -~- NH3 R I

R

I

c

CH

[

..

.T (~

FADred

n--c=..~ H

INHIBITORS

c.s c%

~-~-~.-"--c.--c----c.

Dopronll

~

2

2

J

CH3

- -

DL-pargyllno

( c % ) i - . - c.~--c-=c. N,N-dlmethylpropargyllne

'P (A

.'P. ,"..c,.,+'

.c-¢S--c.___..l

(~)~ CH3 Michael acceptor oxidized N,N,-dimethylpropargyline

, ,r ,,)

'CHa

I" CH3

covalent adduct on inactivated enzyme

Fig. 5. ~onoamine oxidase, catalyses the oxidation o f monoamines to aldehydes via an imino intermediate, during the reaction there is a concomitant reduction o f the flavin coenzyme. The fl-aminoacetylenes Deprend, DL-pargyhne and N,N-dimethylpropargyline can be oxidized by the enzyme to the corresponding imino-acetylenes which form covalent adducts with reduced flavin to yield inactivated enzyme.

cide substrates of horse liver alcohol dehydrogenase4. Effective irreversible inhibitors of alcohol dehydrogenase could be of use in controlling certain intoxication episodes, e.g. prevention of formaldehyde formation following ingestion of methanol. Interestingly, only the a-hydroxy-olefin was a suicide substrate, being oxidized to the corresponding vinyl ketone which alkylates the enzyme. Unfortunately, the resulting enzyme-inhibitoradduct is rapidly hydrolysed to yield malonic dialdehyde. It should be emphasized that the 3-ethylthioprop-2-en-l-ol was a superior suicide substrate to allyl alcohol and this is presumably due to the added electrophilicity of the sulfur containing molecule. Enzyme generated electrophiles via protonation Substrate analogues which contain a diazoketone or oxirane ring can be employed as suicide inactivators of enzymes that catalyse reactions via protonation. Enzyme mediated protonation of these analogues would lead to the protonation of electrophiles (either a diazonium or strained oxirane intermediate) which would then alkylate the active site (Fig. 6). Several diazoketones have been found to be effective antipurines or antiandrogens. Antipurines. A principle of cancer chemotherapy is the restraint of DNA synthesis in rapidly growing tumor cells; purine analogues are of importance in this regard. Two antipurine agents which have undergone extensive trials in the treatment of certain tumors are the diazoketones azaserine and 6-diazo-5-oxonorleucine (Fig. 7). Azaserine which was originally isolated from the broth of Streptomyces and shown to have antibiotic properties now appears to act as a glutamine analogue and is an effective suicide substrate of formylglycinamide ribonucleotide (FGAR) aminotransferase5. 6-Diazo-5-oxonorleucine behaves in a similar fashion5. FGAR aminotransferase catalyses the conversion of the formylglycinamide ribonucleotide to the formylglycinamidine ribonucleotide (FGAM, see Fig. 7). The lust step in the reaction is the activation of the amide group of glutamine to ammonia via initial protonation. It is this 'glutaminase' reaction that azaserine and 6-diazo-5-oxonorleucine inhibit, since upon protonation they give rise to the electrophilic diazonium species which then mediates destruction of enzyme activity (Fig. 6).

halogen (as in difluoromethylDOPA) drugs may be effective antihypertensive which can rearrange to give the correspond- and antidepressive agents. In each case, the ing Michael acceptor. enzyme oxidizes the fl-amino-acetylenic group to produce a conjugated electrophile, Enzyme generation of a Michael the imino-acetylene Michael acceptor, aeceptor via oxidation which inactivates the enzyme. An intriguIn addition to carbanion formation, an ing consequence of the oxidation is that it is enzyme may generate its own Michael the reduced FAD cofactor that acts as the acceptor via oxidation. This can be illus- nucleophile in the alkylation event2 (Fig. trated by the oxidation of amino-acetylenes 5). It is of interest that many years before to imino-acetylenes or by the oxidation of the concept of suicide substrates was introeither c~-hydroxy-acetylenes or c~-hydroxy- duced, Erwin and Hellerman demonstrated olefins to the corresponding conjugated that DL-pargyline was an irreversible ketone (Fig. 4). monoamine oxidase inhibitor. They observed that a pre-requisite for enzyme Monoamine oxidase inhibitors inactivation was the reduction of FAD sugDeprenil, DL-pargyline and N,N- gestive of DL-pargyline turnovera. dimethylpropargyline are all fl-aminoacetylenic suicide substrates of mito- Alcohol dehydrogenase inhibitors chondrial monoamine oxidase (Fig. 5). The a-hydroxyacetylene, prop-2-yne- Antiandrogens Since this is the first enzyme in the 1-ol and the c~-hydroxyolefin, 3-ethylThe final step in androgen biosynthesis is metabolic cascade of catecholamines, these thioprop-2-en- 1-ol were developed as sui- the reduction of the double bond of testo-

TIPS

-

.'flay 1983

215

diazoketones

N

~

H-A-ENZ

peptides as substrate analogues. Formation of the acyl-enzyme intermediate with the nitroso-peptide would result in release of the nitrosamine with the eventual production of a carbonium ion, nitrogen and water. As the carbonium ion is produced directly at the active site it is in a position to cause rapid enzyme inactivation. This mode of inhibition has been described for the inactivation of a-chymotrypsin with the dipeptide derivative N'-isobutyryl-N-benzyl-N-ultrophenylalaninamide. In this instance, the enzymatically generated benzyl carbonium is alkylated by the polypeptide backbone and the resulting imidate hydrolyses to produce a clip in the polypeptide chain 7.

2

Y--ENZ

:Y-ENZ N

diazoketone

dlazonlum intermediate

inactivated

enzyme

oxiranes

E.zn

H C

H C---R

H~

r H

n I-- I--A

---~

\o/ oxirane

ENZ-A-H:

NZ

inactivated

H-A-ENZ strained oxirane

Enzyme generation of an electrophile by insertion of oxygen Mixed function oxidases are a family of enzymes which possess the ability to split molecular oxygen and insert one atom of oxygen into the substrate and one atom of oxygen into water. Two reactions are hydroxylation and epoxidation.

enzyme

acidic group on e n z y m e

Ftg. 6. Electrophileformation viaprotonation. sterone to 5a-dihydrotestosterone by the NADPH-dependent 5ot-reductase of androgen target tissues. Two androgen dependent diseases prevalent in man are prostatic cancer and benign prostatic hyperplasia. An effective suicide substrate of 5a-reductase could offer a great improvement over existing antiandrogenic drug therapy for these disorders, e.g. use of diethylstilbestrol in the treatment of prostatic cancer. With this need in mind, the diazoketone 5a,20R-4-diazo-21 -hydroxy20-methylpregnan-3-one was synthesized and found to he a suicide substrate for the reaction". The enzyme reaction normally proceeds via initial protonation of the carbonyl at C-3, followed by hydride transfer from the pyridine nucleotide cofactor to the allylic carbonium ion at C-5 (Fig. 8). Enzyme mediated protonation of the diazoketone would thus lead to inactivated enzyme with the expulsion of nitrogen. Enzyme generation of an electrophile by formation of an acyl-enzyme intermediate Serine proteases mediate peptide hydrolysis by formation of a tetrahedral acylenzyme intermediate (Fig. 9). Several of these enzymes are involved in blood clotting, e.g. thrombin and plasmin. Effective suicide substrates of these enzymes may be useful as potential anticoagulants and as thrombolytic agents. This activation could be accomplished by synthesizing nitroso-

o '~(CH

NI~

-)~-~ --~'COOH 22 ~IH

glutamine

2 O

H

N-----~ ~-',~--@

oOH

ezaserlne

2

N~H-~C----O

22

y' Im2 6 - d i a z o - 5 - o

¢oox |

pl

"

~

+

ATP.:'

rlbose

~

~OOX

FGAR

xonorleucine

glutamine

(CH2)2 ADP

rlbose

FGAM

91utemate

FGAR e m i n o t r a n e f e r e e e

!(~N)2 "5

,.z-v

> ENZ-Y

COOH

)

°

NH

3

ENZ-A-H = acidic group on enzyme Fig. 7. Antipurmes.

~

216

T I P S - .~lay 1 9 8 3 H

5(~,20R-4-diazo-21-hydroxy-20-methylprognan-3-one

H

H~

~

A'~'~

"~'H+

~:A-'E N Z

I

ENZ

allylic carbonium ion

testosterone

5~dihydrotestosterone

ENZ-A-H=acidic

group on enzyme Fig. 8. Antiandrogens

Mechanism for serlne p r o t e a s e

catalysis

O@ .%'H

1

%--"

NH--R

.

"': "2

1

ENZ

I ENZ

coo.

2

T

~H

HO-ENZ

ENZ

Mechanism for i n a c t i v a t i o n by n i t r o s o - p e p t i d e s

""

"

I

k.7"

O-ENZI

1

nitroso-peptide

t enzyme g e n e r a t e d carbonium ion

Fig. 9. Electrophile formation vta an acyl-enzyme intermediate.

Hydroxylation: RH + Oz + NADPH + H ÷ ----) ROH + NADP + + H20 Epoxidation: R1-CH = CH-R2 + 02 + NADPH + H ÷

,o

----~R--Ctt---CH-R + NADP + + I-I20

inhibitors Substrate analogues which are either dihalogenated on the carbon atom to undergo hydroxylation or which contain a cyano group ot to the carbon atom to receive oxygen are good candidates as suicide substrates for enzymes that conduct hydroxylation. This is because a difluoromethyl Hydroxylase

analogue would be activated by hydroxylation to a highly reactive acyl fluoride while the o~-cyano analogue would be converted by hydroxylation to a ketenimine (Fig. 10). Pharmacologically important hydroxylases include tyrosine and dopamine hydroxylase which are essential for the biosynthesis of catecholamines; cholesterol side chain cleavage enzyme which is a precursor step for the biosynthesis of all steroid hormones and aromatase which catalyses the final step in estrogen biosynthesis. Effective dopamine /3-hydroxylase inhibitors could increase the production of noradrenaline and adrenaline and may be of use in the treatment of phenochromocytoma and reducing sympathetic tone. A suicide substrate for this enzyme activity is p-hydroxybenzylcyanide which contains a cyano group ot to the carbon atom to be hydroxylated s. In this instance, p-hydroxymandelonitrile is the precursor of the enzymatically generated ketenimine which inactivates the enzyme. Breast cancer is one of the most prevalent diseases in women and 30-40% of these tumors are dependent upon estrogen for growth. An effective antiestrogen, in this case an aromatase inhibitor, could be of importance in the treatment of advanced disease. With this goal in mind, a number of suicide substrates for aromatase have been developed. Before discussing these, it is important to realize that during the aromatization of androst-4-ene-3,17-dione to estrone, the mixed function oxidase carries out three successive hydroxylation at C-19. The first two hydroxylations are known to give rise to a g e m - d i o l which eliminates I-GO to yield the 19-oxo-4androstene-3,17-dione (Fig. 11). Thus, suicide substrates have been developed which are activated by either one hydroxylation or two hydroxylations. 19,19Difluoroandrost-4-ene-3,17-dione is presumably activated by a single hydroxylation step to yield the acylfluoride which then alkylates the active site9. 10/3propargylestr-4-ene-3,17-dione, which was the first suicide substrate of aromatase to be described, may be activated by two hydroxylations to yield the corresponding conjugated acetylenic ketone, 10-(1-oxo2-propynyl)estr-4-ene-3,17-dione, which then inactivates the enzyme 1°. lnhibitors of epoxidation Substrate analogues which contain monosubstituted acetylenes or allenes are good candidates for suicide substrates of enzymes that conduct epoxidation since insertion of oxygen will give rise to the highly electrophilic oxirene or allene oxide, respectively, which can be attacked by an enzyme nucleophile (Fig. 12).

T I P S - 'flay 1 983

217

dlhaloganatad analogues F r

F

I CH--~

)

F

~--ENZ

~ --O'~H---~/q~¢~0 ~

F

) C-~O

ENZ

difluoromethyl analogue

hydroxylatod analogue

inactivated enzyme acyl-fluorlde

4~ cyano analogues H

N~C ----CH2--R

~--

ENZ

- " h--. U

cyano analogue

hydroxylatOdanalogue

[

ketenlmine ,Y----EN Z

Inactivated enzyme Fig. 10. Electrophile formation via insertion of oxygen, hydroxyiase intubitors.

andrOlt-4-ene-3,1 ?-dlone

901-111OI

1 a-@lt@-ImdlOSt-

ilcyl-fluorlde

~14

aeading

i n a c t i v a t e d eRzyme

~.

lO/~-pr opar g y l e s t r - 4 - e n e -

~ j-Y--ENZ

10-1- l-oxo-2-proDygyl)-

- 3,17-diane

inactivated enzyme

estr-4-ene-3,17-dlone

Fig. 11. Aromatase reacnon.

Q ENZ-Y

monosubstltututed acetylene

ENZ J

oxirene inactivated enzyme .....E N 7

allene

Conclusion From the four routes to e n z y m e generated electrophiles explored by the pharmacologist a vast array of potentially important c o m p o u n d s have been synthesized which may treat diseases as varied as neurological disorders (Parkinson's disease); hypertension; and hormonally dependent tumors (breast cancer). The author hopes that lifts review will sumulate the design o f appropriate molecules for the suicide inactivation o f specific target e n z y m e s and hence increase our armoury o f 'magic bullets'.

elt¢one

4-one-:l, 1?-(llone

19,19-dlfluoro-androet-4-enQ*3,17-diane

Inactivation o f c y t o c h r o m e P-450 by c o n t r a c e p t i v e steroids Acetylene gas or more complex monasubstituted acetylenes, e.g. the contraceptive steroids 17a-ethinylestradiol or norethisterone are suicide substrates for the phenobarbital induced cytochrome P - 4 5 0 o f rat liver microsomes. In every instance, an oxirene is believed to be formed and the attacking nucleophile becomes a substituent on the protoporphyrin ring n. These findings m a y have an important bearing on drug metabolism in users o f the contraceptive pill and in addition may raise doubts about the effectiveness o f some o f the aromatase inhibitors, e.g. the monosubstituted acetylene 10fl-propargylestr-4ene-3,17-diane.

allane oxide inactivated enzyme

Fig. 12. Electrophile formation via insertion of oxygen: inhibitors of epoxidation.

l Bey, P (1978) in Enzyme-Activated Irreversible lnhibuors (Softer, N., Jung, M. J and KochWesvr, J., ads), pp. 27-...41,North Holland, Amsterdam, London and New York 2 Maycock, A., Abeles, R , Salach, J 1. and Singer, T P. (1970) BiochemistD, 15, 114--125 3 Henerman, L. and Erwln, V. G (1968)J Blol Chem. 243, 5234-5243 4 Muclnnes, J , Schorstem, D. E, Suckling, C. J and Wngglesworth, R (1981)./. ChemSoc. Perkin Trans. 1103-1108 5 Buchanan, J. M (1978) in Enzyme-Acuvated Irreversible inhibaors (Seller, N., Jang, M. J. and Koch-Weser, J., ads), pp. 277-289, North Holland, Amsterdam, London anti New York 6 Blohm, T. R., Metcalf, B. W., Laughlin, M. E., Sjoerdsma, A. and Schatzrnan, G. L. (1980) Bu)chem. Biophys. Res. Commun. 95,273-280 7 White, E H., Perks, H M. and Roswell, D F (1978)J. Am Chem. Sac 100, 7421-7423 8 Baldom, J. M , Villafranca, J. J and Mallettee, M F. (1980) Fed Proc Fed. Am Sac. Exp. Biol. 39, 2568 9 Marcotte, P.A andRobmson, C H (1982)Biochemistry 21, 2733-2778 10 Covey, D. F., Hood, W. F. and Parikh, V D (1981)J Biol Chem. 256, 1076--1079 I l Odtz de Montellano, P. R., Kunz¢, K L.. Yost, G S. andMtco, B A (1979)Proc NatlAcad Scl. USA 76, 746-749 The author received his Ph.D m.Btochemtstrv from Southampton Umversitym 1976 and was then " a Postdoctoral Fellow in Pharmacology at the Johns Hopkins Umversity Medical School He is currently an Assa'tant Professor of Pharmacology at the University of Pennsylvania Medical School.