Renin Inhibitors William J. Greenlee Exploratory Chemistry Department, Merck Sharp and Dohme Research Laboratories, Merck and Co., lnc., P. 0. Box 2000, Rahway, New Iersey 07065

. . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . , . 11. Renin-Angiotensin Pathway Physiology . . . . . . . . . . . . . . . . . . . . . . . . . 111. Properties of Renin (EC 3.4.23.15) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Structure and Mechanism of Renin ............. V. Purification and In Vitro Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

173 175 176 177 179 . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . 180 180 A. Renin antibodies.. . . . . . . . . . . . . . . . . . . . . . . . . . 181 181 197 197 197 200 203 I. Cyclic renin inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 207 J. Irreversible inhibitors of renin . . . . . . . . . . . . . . . . . . . . . . . . 207 K. Molecular modeling in renin inhibitor design L. Renin inhibitors from natural sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 210 210 ........................ 211 212 214 216 216 216 217 D. Chronic administration of renin inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 E. Oral administration of renin inhibitors .............................. 220 221 F. Renin inhibitors in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ . 222 223 ............................. 224 I. Biliary excretion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 ............................. 225 VIII. Summary. . . . . . . . . . . . 226 References . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. INTRODUCTION The renin-angiotensin system (RAS) plays a central role in the regulation of blood pressure and in the maintenance of sodium and volume homeostasis. The formation of the potent vasoconstrictor angiotensin 11, the end product of the RAS, is outlined in Figure 1. Possible modes of intervention in this Medicinal Research Reviews, Vol. 10, No. 2, 173-236 (1990) CCC 0198-6325/90/020173-64$04.00 0 1990 John Wiley & Sons, Inc.

GREENLEE

174 1

2

3

4

5 6

7

8

9

10111213

Asp-Arg-Val-Tyr-lle-His-Pro-Phe-His-~-lle-His--Protein Ps P4 P, Anaiotensinoaen

1

2

3

4 5 6

1

P* P,

P,' P,' P,'

Renin

7

8

910

Asp-Arg-Val-Tyr-lle-His-Pro-Phe-His-Leu Anaiotensin I

i

Angiotensin-Converting Enzyme

1

2

3

4

5

6

7

8

Asp-Arg-Val-Tyr-lle-His-Pro-Phe Anaiotensin II

Figure 1. Renin-Angiotensin System. Substrate side-chain (Psthrough P3') for angiotensinogen are illustrated using the notation of Schechter and Berger (ref. 51); Scissile bonds are underlined.

process include inhibition of renin formation or release, renin inhibition, angiotensin-converting enzyme (ACE)inhibition, and angiotensin I1 receptor antagonism. Peptide analogs of angiotensin I1 inhibit the effects of this hormone by competitive blockade at its receptors, but their experimental and clinical applications have been limited by partial agonist activity and limited oral absorption. l f 2Competitive, nonpeptide angiotensin I1 receptor antagonists have been reported r e ~ e n t l y ,but ~ , ~their therapeutic potential has not yet been established. Inhibition of ACE is an effective therapy for the majority of hypertensive patients and represents a major advance in the treatment of hypertension and congestive heart f a i l ~ r e .Nevertheless, ~-~ ACE is not specific for its substrate angiotensin I, since it also cleaves kinins and other endogenous peptides. Although inhibition of the formation of angiotensin I1 appears to be the most important action of ACE inhibitors, potentiation of kinins in plasma or in tissues may contribute to their antihypertensive effects. The therapeutic effectiveness of ACE inhibitors in both normal and highrenin hypertensive patients has contributed to the effort devoted to the development of inhibitors of renin. The cleavage of angiotensinogen by renin to form angiotensin I is the first and rate-limiting step in angiotensin I1 generation (Fig. 1). Since angiotensinogen is the only known naturally occurring substrate for renin, inhibition of this specific reaction may have advantages over ACE inhibition.lO,llSome of the (albeit rare) side effects of ACE inhibitors (e.g., cough, angi~ederna)'~-'~ might not be shared by inhibitors of renin. Other possible advantages of renin inhibitors have been summarized by Boger.l1 William J. Greenlee received his Ph.D. degree in chemistry wifh Prof. R. B. Woodward at Harvard University, and completed postdoctoral work with Professor Gilbert Stork at Columbia Uniaersity before joining the Merck Sharp and Dohme Research Laboratories in Rahway, New Jersey in 1977. He has been involved in a number of projects at Merck, including the design of angiotensin-converting enzyme and renin inhibitors. He is currently a Director in the Exploratory Chemistry Department.

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175

Several reviews of renin inhibitors have been published r e ~ e n t l y , " * ~and ~-~~ these may be consulted for a more complete description of some aspects of the field. The present review focuses on recent advances in the area, especially progress toward clinically useful renin inhibitors. The majority of the references in the current review are to work published during the past two years or to unpublished work, a fact which attests to the continuing effort in the renin area. 11. RENIN-ANGIOTENSIN PATHWAY AND PHYSIOLOGY Several excellent reviews describe the the renin-angiotensin system and its role in h y p e r t e n s i ~ n . ~The l - ~basic ~ elements of the RAS are outlined in Figure 1. Angiotensinogen, the initial substrate for the RAS, is an alpha-2-globulin synthesized in the liver and found in the general circulation. Renin, synthesized and released by the kidneys in response to decreased renal perfusion, cleaves the amino terminal decapeptide from angiotensinogen to yield angiotensin I (A-I) (Fig. 1).ACE, both in the plasma and on the outer membrane of endothelial cells, catalyzes the removal of the carboxy terminal dipeptide to form angiotensin I1 (A-11). This short-lived octapeptide, which is cleaved to inactive fragments, acts via its receptors to constrict the vasculature, to stimulate the release of aldosterone from the adrenals, and, in a direct negative feedback loop, to inhibit the further release of renin from the k i d n e ~ . ~ ' - ~ ~ Although some or all components of the renin-angiotensin system have been shown to exist in other tissues,26-z8their precise roles in the arterial wall or in the central nervous system, for example, remain controversial. Renin is synthesized in the kidney in the form of prorenin (MW = 47,000), an inactive precursor, which is converted to active renin (MW = 44,000) by cleavage of an Arg-Leu bond by a "renin-processing enzyme" (RPE).z9,30 This cleavage, which appears to occur only in the kidney, may be a regdatory mechanism for the RAS, and represents a novel target for blockade of the RAS. Renin is stored in the kidney and secreted, in response to decreased renal perfusion and other Work on the isolation of renin from human kidney has demonstrated that less than 1 mg of active enzyme is present in each kidney. Both prorenin and active renin are released by the kidney into the circulation, about 90% of circulating renin being inactive prorenin. Concentrations of renin in the blood vary, depending on several physiological factors including salt levels in the diet. As expected for an enzyme important in controlling a homeostatic process, renin is rapidly turned over, with the half-life for clearance of exogenously administered renin estimated at 13 minutes in rats.32Exogenously administered, recombinant human renin is rapidly cleared in monkeys, largely by the liver.33 Angiotensinogen, the initial protein substrate for the RAS (MW = 80,000), has considerable homology to oll-antitrypsin, antichymotrypsin, ovalbumin, and antithrombin III.34The plasma concentration of angiotensinogen varies, depending on the levels of both renin and A-II.35but is usually close to the K, value (1.5 pM at pH = 6.5),6 and greatly exceeds the concentration of renin in plasma (about 2.5 P M ) . The ~ ~ turnover number (KaJ for cleavage of human substrate by human renin (0.6/s) is sufficiently low that Poulsen and Jacobsen have suggested that angiotensinogen may once have been a pro-

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teinase inhibitor for renin, whose former (and more efficiently cleaved) substrate was lost during evolution.37Recombinant human angiotensinogen expressed in Escherichiu coli serves as a substrate for human renin.38 No physiological role for des-(angiotensin) I) angiotensinogen has yet been determined. 111. PROPERTIES OF RENIN (EC 3.4.23.15)-FORMERLY EC 3.4.99.19

Excellent reviews by Day and Haber39and by 1nagami4' on the biochemistry of renin have appeared recently. Renin is an aspartic proteinase, a group of enzymes which occurs in higher animals (e.g., pepsin and cathepsin D), fungi (e.g., pencillopepsin and Rhizopus pepsin), viruses, and plant^.^' Many aspartic proteinases play a role in digestion or the intracellular degradation of proteins. The recent discovery of the human immunodeficiency virus (HIV) proteinase, a dimeric aspartic proteinase comprised of identical 99-amino acid subunits, has provided a second important therapeutic target within this class of enzyme^.^',^^ Mature human renin has 304 amino acid residues, three intrachain disulfide bridges, and two glycosylation sites. Renin shows a high sequence homology to other aspartic proteinases, especially among the residues which comprise the active ~ i t e .The ~ - similarities ~ ~ in sequence among the aspartic proteinases and an analysis of the possible evolution of their active site regions have been reviewed by Tang and W ~ n g . All ~ ' possess two catalytic aspartic acids, which are believed to share one proton. The aspartic proteinases typically are inactivated by the active site-directed agents diazoacetyl-DL-norleucine methyl ester (DAN) and 1,2-epoxy-3-(nitrophenoxy)propane(EPNP), and are inhibited by the natural product pepstatin (Sect. VI.C.2). Renin differs from the other aspartic proteinases in two important respects. While the other aspartic proteinases show maximum activity at low pH (hence the former name acid proteases), and become irreversibly deactivated at higher pH, renin has its pH optimum (pH = 6.0) closer to physiologic pH, and functions in plasma at pH = 7.4. All aspartic proteinases except renin have an aspartic acid residue (residue number 304 in pepsin) which is located in a (mainly) hydrophobic environment near the active-site aspartic acids, whose ionization constants are believed to be lowered as a result. The hypothesis that the alanine found in this position for renin could be responsible for its high pH optimum49is supported by its shift toward the acidic side in a human renin mutant in which alanine 304 is replaced by aspartic acid.50 Although other members of the class have little specificity, renin has a remarkably narrow substrate specificity, limited to a single peptide bond in angiotensinogen. Renin has functionally important specificity subsites in the active site cleft which bind amino acids on both sides of the scissile bond. In this review, these are labeled using the notation (Sl, Sz, S3, Sl', S2', S3', etc.) of Schechter and Berger.51The shortest peptide cleaved by renin is an octapeptide. The scissile bond of human angiotensinogen is Leu-Val, while it is Leu-Leu for other mammalian angiotensinogens.s2~s3 Even this minimal substrate difference is significant, in that while the human enzyme will cleave human and other mammalian substrates, only human renin will cleave human

RENIN INHIBITORS

177

substrate.’l Site-directed mutagenesis of human renin may provide additional information about its unique specificity. 1V. STRUCTURE AND MECHANISM OF RENIN

Although the three-dimensional structure of renin has not yet been determined, those of related aspartic proteinases show the active site aspartic acids centered in a long cleft which is capable of binding at least eight amino acids. The two essential aspartic acids are held close together by a network of hydrogen bonds. In this arrangement, the highly conserved residues next to each aspartate (Asp-Thr-Gly-Ser/Thr) form hydrogen bonds that restrain the carboxyl groups so that they are close together and approximately coplanar.% Interestingly, for renin (only) the sequence Asp-Thr-Gly-Ala is found at Asp 235. The aspartic acids are hydrogen bonded to each other and (for penicillopepsin and Rhizopus pepsin) to an electron density believed to be a water molecule or an ammonium ion.55,56The ionization state of the catalytically active aspartic acids of renin at physiological pH is not known with certainty, but one is believed to be protonated. Renin, like the other aspartic proteinases, has a flap region which can open and close upon substrates or inhibitors, and hydrogen-bonding interactions between the flap and substrate (or inhibitor) may be important for binding. Especially interesting is an apparently essential tyrosine found in the flap region of all aspartic proteinases sequenced to date. Cyclic peptides with sequences derived from the the renin flap region have been established by nuclear magnetic resonance (NMR) studies to have a beta-hairpin loop structure. The ability of antirenin antibodies to bind these synthetic peptides suggests a similar conformation for the flap region of r e r ~ i n . ~ ~ Despite the importance of this information for the design of inhibitors, the general features of the mechanism for cleavage of amide bonds by renin and other aspartic proteinases have not yet been established (reviewed by Some aspartic proteinases, most notably pepsin (but not renin), have been shown to catalyze transpeptidation reactions under certain conditions. On the basis of these results, mechanisms have been proposed in which a covalent intermediate is formed by nucleophilic attack of an active-site aspartic acid on the substrate. However, experiments designed to establish the existence of an “amino-enzyme” or acyl-enzyme (mixed anhydride) intermediate have thus far led to equivocal results. X-ray crystallographic studies of enzyme:inhibitor complexes (Sect. 1V.K) suggests that involvement of an active site aspartic acid in a covalent intermediate is geometrically unfeasable. Rich has used 13CNMR to study the binding of ketone “pseudosubstrate” analogs (Sect. VI.C.10) to pepsin, and has demonstrated a stereospecific, pepsincatalyzed addition of water to the ketone, a result which supports a general acid-base mechanism.58It is reasonable to suppose that pepsin-catalyzed hydrolysis of peptide bonds occurs by a similar process, where enzyme catalyzes direct addition of water to form a tetrahedral intermediate (Fig. 2 ) with the active-site aspartic acids mediating the required proton transfers. A slow, ordered release of products could then explain data which had been interpreted as evidence for covalent intermediates. 58 Two mechanisms for aspartic proteinases have been proposed which are

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178

H

0

R,

I

H

H

Tetrahedral Intermediate

Peotide

Reduced PeDtide

. . OH R2 .

.

Hvdroxy lsostere

Ketone b.sExe X = -H

Dihvdroxv lsostere

Difluorok&y&3lsostere X = -F

Statine Analog R, = 4-butyl

Statone Analog Rl = -i-butyl; X = -H

ACHPA Analog R1 = -CH,Qh

DifluorostR, = 4-butyl; X = -F

PhosDhostatine Analog R1 = -i-butyl PhOSDhO-ACHPA Analog R1 = -CHpcyh

Amino A W l Analog

Aminostatine Analog R1 = -i-butyl

Norstatine Analog R, = -i-butyl Nor-ACHPA Analog R1 = -CH,Qh

Figure 2. Stereochemical relationships of substrate and tetrahedral intermediate for hydrolysis to peptide b o n d isosteres used as transition-state mimics. R1 a n d R1 are alkyl groups w h i c h correspond t o the L e u a n d V a l sidechains in substrate; c y h = cyclohexyl

based on x-ray crystallographic studies. In a mechanism for rhizopus pepsin proposed by Suguna et al., a water molecule bound between the aspartic acids serves as the nucleophile, and the hydroxyl of an active-site serine (which is also present in renin) donates a hydrogen bond to one oxygen atom of the tetrahedral intermediate.59A stereochemical proposal for the catalytic mechanism of penicillopepsin, based on an x-ray structure of native enzyme and an enzyme:inhibitor complex, has been reported by James and S i e l e ~ k i . ~ ~ James proposes that the water molecule bound between the active-site aspartic acids is displaced during binding of substrate, and that a second water molecule near the active-site aspartic acids serves as the nucleophile. The proton shared by the two aspartic acids functions as the electrophile to polarize and (possibly) protonate the scissile carbonyl; release of products is the rate-limiting step. A mechanism in which the active-site aspartic acids bring about a ”pushpull” general acid-base catalysis of peptide bond hydrolysis has been proposed by Polgar,@’ and is consistent in general terms with those described above. Arguments are made in favor of concerted proton transfers mediated solely

RENIN INHIBITORS

179

by the active-site aspartic acids during the formation and breakdown of a tetrahedral intermediate for hydrolysis. A quite different mechanism proposed by Pearl relies on the hydrogen bonding interactions between substrate and the enzyme flap to distort the scissile peptide bond, making it susceptible to attack by a water molecule ”polarized” by the active-site aspartic acids.61 This mechanism differs from the others in that it requires an oxyanion intermediate to be formed with concomitant protonation of both aspartic acids. A genera1mechanism for the aspartic proteinases will have to be consistent with the (collective) ability of members of this class to function at widely different pH (pH = 2 to 7.5) in spite of the expected changes in ionization state of the catalytic aspartic acids.

V. PURIFICATION OF RENIN AND IN VITRO ASSAYS Although multiple forms of enzyme and the glycoprotein nature of renin have complicated its purification, homogeneous enzyme has been obtained from a variety of sources including human dog kidney,67hog kidney,68rat kidney,69and the mouse submaxillary Affinity columns prepared using pepstatin and other renin inhibitors have been used. Recombinant human prorenin has recently been expressed in mammalian cells and pure, active, glycosylated enzyme isolated in q ~ a n t i t y . The ~ ~ -im~~ portance of the prosequence for initial folding of renin has been demonstrated in a study of recombinant prorenin.T7 The variety of in vitro assays used to determine activities of renin inhibitors has been reviewed recently.39Among these are human kidney renin (HKR) assays which use partially, or extensively purified enzyme and human angiotensinogen; other mammalian angiotensinogens and synthetic peptides have been used as substrates. The most widely used synthetic substrate for renin is a tetradecapeptide whose sequence is that of the amino terminal region of porcine angiotensinogen. Questions have been raised as to whether the synthetic substrate represents in all respects the natural substrate, and whether in vitro inhibitory data obtained using this substrate will predict inhibition in vivo. Synthetic tetradecapeptide and angiotensinogen yield different kinetic constants (K,,,,I Q for hydrolysis by renin, with human angiotensinogen being cleaved at only one-eight the rate of the tetradecapeptide.78Inhibitor affinity (and type of inhibition) can also differ depending on which substrate is used.79 Inhibitory potency may also differ substantially, depending on the pH at which the assay is p e r f ~ r m e d .In ~ ~some ! ~ ~cases, in vitro renin assays are run at the pH optimum (pH = 6) for cleavage of human substrate by human renin, and at this pH, binding of inhibitors is often tighter than at higher pH. The availability of recombinant human renin and human angiotensinogen in pure form should make possible an assay in which pure enzyme cleaves pure human substrate at the pH of plasma (pH = 7.4). In human plasma renin assays, angiotensin I, cleaved from endogenous substrate by endogenous enzyme in human plasma, is measured by radioimmunoassay. Since the concentration of enzyme and substrate are unknown, the results are expressed in terms of ”plasma renin activity” (PRA), and it is important for IC50values for inhibitors to be determined in reference to a standard inhibitor. In a variation of this assay, exogenous angiotensinogen

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180

is added to raise its total concentration above the K, value (making the reaction rate proportional to the concentration of renin), and the resuIts are expressed as “plasma renin concentration. ” Proteinase inhibitors are usually added to prevent degradation of the angiotensin I formed. A modification of the standard renin assay, which uses A-I trapping by an antibody, has been introduced by Poulsen and Jorgensen.81Here, the A-I formed is protected by the antibody, and addition of proteinase inhibitors is unnecessary. Unfortunately, no standard renin inhibitor assay has emerged. Instead, a variety of assays conditions (pH, concentration of substrate, presence of other proteinase inhibitors, detection method) are used, and in many cases inhibitor data are reported without reference to ”standard” inhibitors. As a result, comparisons between literature IC50values are difficult. In this review, IC50values determined using pure enzyme assays (regardless of substrate) are enclosed in brackets if run at pH = 7.0-7.4, and in parentheses if run at pH = 6, to differentiate them from those obtained in plasma renin assays. Literature references cited in this review should be consulted when differences in IC50values are interpreted.

VI. IN VITRO INHIBITION OF RENIN Knowledge of the substrate sequence of human angiotensinogen, mechanistic hypotheses based on structural information drawn from x-ray crystallographic studies of several aspartic proteinases, and the discovery of pepstatin, a naturally occurring inhibitor of this class of proteinases have all contributed to the development of renin inhibitors. In the last several years, work on renin inhibitor design has yielded several classes of potent inhibitors, the best of which are active at (or below) nanomolar concentrations. Steady progress has been made toward lower molecular weight inhibitors which are more likely to become useful therapeutic agents. At the same time, the ”peptide character” of the inhibitors has decreased, and totally nonpeptide inhibitors have recently been reported. Inhibition of renin has been achieved with renin antibodies, substrate analogs, and several classes of transition-state analogs. In this review, the amino acid side chains (or their equivalent) of inhibitors are labeled using the notation (P3, P2, PI, PI’, P2’, P3’)of Schechter and Berger51to indicate their relationship to the scissile bond of substrate.

A. Renin Antibodies Inhibitory renin antibodies provided the first evidence that interference in the action of the renin-angiotensin system had therapeutic potential. Although these first renin antibodies were impure, recently polyclonal, monoclonal, and FAB antibodies prepared using purified renins have shown potent (Ki = 0.01-1.0 nM) renin and hypotensive effects when administered i.v. to salt-depleted monkeys (Sect. V1I.B).Antibodies have been raised to peptides which were predicted by inspection of a human renin model to be potential epitopes (including peptides with sequences derived from the flap region), and some of these antibodies bind the enzyme and inhibit its activity.87In some cases, monoclonal antibodies, although they inhibit the cleavage of angiotensinogen, either fail to inhibit completely the cleavage of tetradecapeptide substrate, or actually stimulate its cleavage. These antibodies

RENIN INHIBITORS

181 Table I Peptide Inhibitors of Human Renin”

Min. Substrate (1-1) Human Substrate (1-2) RIP (1-3) RI-103 ( 1 4 ) 1-5

His-Pro-Phe-His-Leu-Leu-Val-Tyr His-Pro-Phe-His-Leu-Val-Ile-His Pro-His-Pro-Phe-His-Phe-Phe-Val-Tyr-Lys Phe-(4-I)Phe-Val-Tyr-Lys (N-Et)Phe-D-Met-Gly-Phe-NH(2-Ad)

1-6

Boc-Leu-Ly s-Arg-Met-Pro-OCH3

IC&M)

Ref.

200,000 313,000

286 286 91 93 94 95

[2,000] [-2,000]b 90,000 16,600

aIC5flvalues from pure enzyme assays are enclosed in brackets if determined at pH = 7.4 and in parentheses if determined at pH = 6; all others are from plasma renin assays run at pH = 7.4. Amino acids (or other elements) at PI and PI’ positions are underlined. The following abbreviations are used in the tables: Boc = t-butoxycarbonyl; Cbz = carbobenzyloxy; Ibu = isobutyryl; Iva = isovaleroyl; EtOC = ethoxycarbonyl; Ac = acetyl; Cyh = cyclohexyl; Et = ethyl; i-Pr = isopropyl; t-Bu = t-butyl; MeBu = methylbutyl; NA = 1-naphthyl; Ph = phenyl; Bz = benzyl; AMP = -NHCH2(pyridin-2-yl); 2-Ad = 2-adamantyl. Other abbreviations are indicated. bReported to be equal in potency to RIP (1-3).

appear to bind at some distance from the active site (possibly near the flap), since binding of a small peptide inhibitor to the enzyme does not prevent binding of the antibody. A conformational change induced by the antibody, allowing increased access of tetradecapeptide, or more-rapid release of product, may explain its stimulatory effect on enzynie activity.88

B. Substrate Analogs and Other Peptide Inhibitors The success achieved in development of substrate analogs as renin inhibitors has provided a basis for recent work on potent transition-state inhibitors. Skeggs was the first to demonstrate in 1964 that the (6-13) octapeptide of horse angiotensinogen (1-l), althQugh hydrolyzed slowly by renin, was a weak competitive inhibitor (Table I) .89 Later, Kokubu and co-workers showed that the minimum sequence still capable of inhibiting renin was the tetrapeptide (Leu-Leu-Val-Tyr) which represents residues 10-13 of angiotensin~gen.~~ Beginning with the minimum substrate sequence, Burton and colleagues synthesized a series of peptide inhibitors including a decapeptide (13), which they named the “renin inhibitory peptide” (RIP). The amino terminal proline and carboxy terminal lysine of 1-3 contribute to water solubility, and the substitution of Phe-Phe for Leu-Leu prevents cleavage by renin.91,92 More recently, this approach has led to shorter peptides (e.g., 1-4), which were reported to have in vitro potency equal to that of RIP.93Several peptides such as 1-5, which have structures related to those of stabilized enkephalin analogs, are competitive inhibitors of human renin with modest potency.94Peptide 1-5 has been suggested to bind in such a way that the carboxy terminal amide bond replaces the scissile bond of substrate. Peptides such as 1-6, which are derived from the prosegment of human prorenin, are competitive inhibitors of human reni~~.~~ C. Transition-State Inhibitors of Renin

The design of enzyme inhibitors which resemble the transition state for the enzyme-catalyzed reaction has been described by W ~ l f e n d e nThe . ~ ~transition

182

GREENLEE Table I1 Reduced Peutide Inhibitors of Human Renin” Ic50

Ref. 2-1 H-110 (2-2) 2-3 H-76 (24) H-77 (2-5) H-117 (2-6) H-113 (2-7) H-142 (2-8) 2-9

400,000b 820 420 1,000 1,000 370 190 10 (8.6)

98 286 287 286 286 286 286 286 106

”See Table I footnotes, An R over a bond indicates a reduced peptide isostere (Fig. 2). bICg determined with hog renin. Cal = 3-cyclohexylalanine.

state is the molecular arrangement of highest free energy along the reaction pathway. It is believed that the acceleration of reactions brought about by enzymes is a result of tight binding and stabilization of the transition state species by the enzyme, and thus it is expected that inhibitors whose designs approximate the transition state will bind more tightly than substrates or products. For peptide-bond hydrolysis, a putative tetrahedral intermediate (Fig. 2), which would result from hydration of the peptide bond carbonyl, is presumed to approximate the transition state, and is the model on which transition-state inhibitor designs are based. 1. ”Reduced Peptide” lsostere”

Using the transition-state inhibitor approach, Szelke and co-workers designed potent inhibitors of renin in which the scissile bond of substrate analogs is replaced by a ”reduced peptide” isostere (-CH2NH2-) as illustrated in Figure 2.97 Although the ”reduced peptide” isostere shares the tetrahedral geometry of the tetrahedral intermediate, it lacks both hydroxyl groups. A reduced peptide analog (2-1) of Kokubu’s tetrapeptide inhibitor (Leu-LeuVal-Phe-OCH3) was reported by Szelke in 1972 (Table 11), and was the first renin inhibitor to prove effective in lowering blood pressure when tested in v~vo.~ Although * this inhibitor had only moderate potency, a dramatic improvement in potency was achieved when the reduced peptide isostere was incorporated into Skegg’s octapeptide (l-l)/giving the analog H-76 (2-4). When the N-terminal sequence of human substrate became known, peptides were synthesized which incorporated the human cleavage site (2-6) and human substrate sequence (2-7), and to which solubilizing groups haci been added (2-8). This last ”reduced peptide” analog, H-142 (2-8), is 30,000 times more potent than the human octapeptide substrate analog (1-2) from which it was derived.99The high potency for inhibitors containing this isostere may be the result of an electrostatic interaction between the basic amine and the active site aspartic acids. X-ray crystallographic studies of reduced peptide inhibitors complexed with endothiapepsinlOO,lO1 and Rhizopus pepsin59(Sect. V1.K) are consistent with this hypothesis. Recently it was reported that in-

RENIN INHIBITORS

183

Table 111 Statine-Containing Inhibitors of Human Renin" ICsa (nM)

Pepstatin A (3-1) SCRIP (3-2) SR-42,128 (3-3) 3 4 ES-305 (3-5) CGP-29,287 (3-6) ES-1005 (3-7) 3-8 L-363,564 (3-9) 3-10 3-11

Iva-Val-Val-Sta-Ala-Sta

22,000

4 28 Iva-Phe-Nle-Sta-Ala-Sta 13 BNMA-His-Sta-Leu-NH-Bz 9.2 BNMA-His-Sta-NH-Z(S)-me thylbutyl Cbz-(Arg)2-Pro-Phe-His-Sta-Ile-His-Lys(Boc)-OCH~ 1 BNMA-H~S-S~~-L~U-NH(CH~)~-CH(NH~)CH~~H [4.5] 16 Boc-Phe-His-Sta-Leu-NH( l-Bz-piperidin-4-yl) 25 Boc-His-Pro-Phe-His-Sta-Leu-Phe-NH2 [2.3] Iva-His-D-Pro-Phe-His-Sta-Leu-NH-Bz [150] Boc-Phe-His-S ta-Leu-NH(CH&NHC(=NH)NHz 64

Iva-His-Pro-Phe-His-Sta-Leu-Phe-NH2

Ref. 95 110 113 288 119 118 180 229 196 220

224

"See Table I footnotes. BNMA = bis(naphthylmethy1)acetyl.

hibitors in which the -CH2NH- is replaced by -CH2S-, -CH2SO-, or -CH20are 50 to 100-fold less active,, supporting the existence of this electrostatic interaction. lo3r104 The x-ray crystal structures also demonstrate that the water molecule bound between the aspartic acids in the native enzymes is displaced by inhibitor.'OO,'O1 As has been proposed by Richlo5for statine-containing inhibitors of aspartic proteinases (Sect. VI.C.2), the displacement of this water molecule may make an entropic contribution to the overall binding energy (see below). Although the use of the "reduced peptide" isostere in renin inhibitors has been investigated less thoroughly than some other inhibitor designs, the high potency of inhibitor 2-91°6 demonstrates its effectiveness in lower molecular-weight inhibitors. 2 . Inhibitors Containing Statine

The naturally occurring aspartic proteinase inhibitor pepstatin A (3-1)lo7is an exceedingly potent (K, = 0.05 nM) inhibitor of pepsin,lo8 but a much weaker inhibitor of human renin (Table III).95The central statine [4(S)-amino3(S)-hydroxy-6-methylheptanoicacid] element of pepstatin A (Fig. 2) has been ~~ proposed to serve 8s a mimic of the transition state for h y d r o l y s i ~ 'or, alternatively, as a "collected substrate" analog, where the substrates are peptide and water.'" The importance of the 3s hydroxyl group of statine in statine-derived inhibitors is demonstrated by the 1000-fold drop in inhibitory activity upon changing to the 3R configuration.110Rich has proposed that an enzyme-bound water molecule is displaced by the hydroxy group of statine upon binding of inhibitor.lo5The resulting gain in entropy may contribute to the overall binding energy. X-ray crystallographic studies of complexes of pepstatin analogs with Rhizopus pepsin56 and peni~illopepsin,~~ and of L363,564 (3-9) with endothiapepsin"' confirm that the active-site water molecule i s displaced upan binding of statine-containing inhibitors, and suggest a system of hydrogen bonding interactions between the 3s hydroxyl and the active-site aspartic acids (Sect.VI.K).

184

GREENLEE

Derivatives of pepstatin with improved water solubility have been prepared as inhibitors of human renin, the best being pepstatin-Glu with a Ki of 5.8 p,M. '12 More recently, the Sta-Ala-Sta sequence has been incorporated into highly potent pentapeptides such as SR-42,128 (3-3).1'3,1'4 Radiolabeled SR42,128 is a competitive inhibitor of human renin (substrate = human tetradecapeptide) with a Ki values which depends on pH [Ki = 0.35 nM, pH = 5.7; Ki = 2 nM, pH = 7.4].l15 A QSAR study of renin inhibitors containing the Sta-Ala-Sta fragment has been published. '16 Since renin is a highly specific proteinase, Boger and co-workers incorporated statine into the angiotensinogen sequence hoping to find more potent renin inhibitors.ll' Powers et al., noting similarities between the structure of pepstatin and substrates of aspartic proteinases, suggested that statine replaces both amino acids around the cleavage site,l17a conclusion also reached by Boger et a1.l" using molecular modeling methods (Sect. V1.K). Peptides such as "SCRIP" (3-2; statine-containing, renin inhibitory peptide), synthesized on the basis of this hypothesis, are potent inhibitors of human renin and are selective for renin versus other aspartic proteinases (Sect. VI.N).lI0 Incorporation of statine modeled as a single amino acid replacement gave less-potent inhibitors. Inhibitors such as CGP-29,287 (3-6) have demonstrated that ICs0 values of 1 nM can be obtained with statine-containing renin inhibitors. 118 Continual progress has been made toward potent lower molecularweight inhibitors containing statine. These include dipeptide inhibitor ES-305 (3-5), which incorporates the bis-napthylmethylacetyl (BNMA) group.'19 3. Statine Side-Chain Modifications

In a molecular modeling study in which an x-ray crystal structure of a complex of Rhizopus pepsin with pepstatin was used as a model for human renin (Sect, VI.K), Boger and co-workers noted the expansive S1 subsite of the enzyme and predicted that inhibitors containing statine analogs with larger P1 side chains would show improved potency. Although a statine analog with a benzyl sidechain, referred to as "AHPPA" [4(S)-amino-3-hydroxy5-phenylpentanoic acid], gave human renin inhibitors of potency only comparable to those containing ~ t a t i n e , ~the ~ ' cyclohexylmethyl ,~~~ analog, named "ACHPA [4(S)-amino-5-cyclohexyl-3(S)-hydroxypentanoic acid] gave a substantial increase in potency against renin (Table IV). The ACHPA-containing analog of SCRIP, named "ACRIP" (4-1) was fourfold more potent,12' and one inhibitor series has been described'*' in which a 60-fold enhancement in potency has been achieved by replacement of the isobutyl by the cyclohexylmethyl substituent (compare 15-1 to 12-4 in Table XV). The ACHPA-containing inhibitor SR-43845 (4-5), has been reported to have exceedingly high potency (IC50 < 16'' M) against human plasma renin.Iz2 The cyclohexyl modification has contributed to the reduction in molecular weight achieved in several recently reported inhibitor series. Thus ACHPA-containing inhibitors as small as tripeptide analog 4-6 are highly Further reduction in size, however, results in a substantial drop in potency (4-7; 4-8).120,125 An inhibitor (4-14) which incorporates an analog of statine with a shortened side chain is less potent that the statine analog (4-13).Iz6 An inhibitor (4-17) containing an analog of ACHPA in which the cyclohexyl ring is replaced by

RENIN INHIBITORS

185

Table IV Inhibitors Containing ACHPA and Other Side Chain Analogs of Statine" Icso

Ref. ACRIP (4-1) 4-2 4-3 L-364,210 (4-4) SR-43,845 (4-5) 4-6 4-7 4-8

ES-6864 (4-9) 4-10 4-11 4-12

Iva-His-Pro-Phe-His-ACHPA-Leu-Phe-NH2

0.9 0.6 (12) BNMA-His-ACHPA-Leu-NH-Bz 1.0 Boc-Phe-His-ACHPA-Leu-NHCH2Ph(3-CH2NH2) 10,000 >10,000

129 129 129

12-12 18-10 18-11

Ac-PheAc-(N"-Me)PheAc-(C"-Me)Phe-

173 173 173

18-12

173

18-13

173

&

EtOC-N

18-14

"See Table I Footnotes. bIC50value for RS mixture.

(15)

173

RENIN INHIBITORS

203

ample, in the inhibitor A-64662 (25-1). "' Although incorporation of either *,~'~ (C*-methy1)Phe or (N"-methy1)Phe at P3 results in loss of p o t e n ~ y , ~ ~inhibitors 18-10 and 18-11 are stable to chymotrypsin. '" Conformationally constrained amino acids have also been incorporated at P3, again giving inhibitors (18-12; 18-13; 18-14) which are stable to ~hymotrypsin.'~~

H. Toward Nonpeptidic Inhibitors: Additional Amino Terminal Modifications That the carboxy terminal amino acids of peptide-derived renin inhibitors can be replaced with nonpeptide elements has been amply demonstrated by the high potency of inhibitors described above (Sect. V1.E). Substantial progress has also been made toward minimizing the "peptide character" of the amino terminus of renin inhibitors by eliminating peptide bonds and introducing conformational constraints. Potent inhibitors have been described which incorporate P3 and P4binding elements and eliminate the amide or carbamate moiety which is usually found between them. An example is the bis-naphthylmethylacetyl (BNMA) group of ES-305 (3-5)119 and ES-1005 (3-7)I8O Unfortunately, some other bis (heteroary1)acetylgroups which might contribute to higher aqueous solubility, yield a substantial loss of potency.140Other effective P3 groups are the amino terminal morpholine amide of KRI-1314 (7-7)142 and ES-6864 (4-9),'77 the sulfone group of CGP-38,560A (5-lo), and related structures illustrated in Table XIX. 13' Although N-methylation of phenylalanine (e.g., in 18-9) leads to a substantial loss of potency, phenyllactic acid has been incorporated successfully at P3 as an ester (19-1),'31 an ether (19-2),'" or a carbamate (19-3).179The high intrinsic potency of the "hydroxy" isostere has made possible the discovery of "dipeptide" inhibitors of human renin in which the amino terminus comprises of simple acyl group (19-9; 1910; 19-11).131 Many attempts have been made to design renin inhibitors in which the cleavable Phe-His amide bond is modified or eliminated. Inhibitors in which this amide bond is replaced with a "reduced peptide" isostere,'40,181an ether,'= or a thioether linkage'" suffer a substantial loss of potency, suggesting that the carbonyl of the amide bond is involved in an important hydrogen-bonding interaction. Evans et al. used the "hydroxy" isostere to replace this peptide bond in inhibitors 19-12 to 19-15 (Table XIX), and (surprisingly) found inhibitors with R and S configuration at the hydroxy-bearing carbon to have comparable potency against human renin. '83 Elimination of the cleavable Phe-His bond has also been achieved by introducing conformational restraints in the P3-P2 region, an approach which has been applied to other peptides in attempts to obtain increased specificity and stability toward proteolysis.184 Previous work had shown that N-methylation of the P3-P2 peptide bond could be carried out without loss of potency in inhibitors containing the "hydroxy" isostere (e.g., 5-3; 17-5), suggesting that the peptide bond N-H in this series is not involved in a critical hydrogenbonding interaction with the enzyme. Using a model of human renin constructed by Carlson et al.,185Thaisrivongs et al. noted that this N-H bond for inhibitors in their (predicted) bound conformation is nearly eclipsed with the alpha-carbon C-H bond of phenylalanine, and synthesized inhibitors in-

GREENLEE

204 Table XIX Inhibitors with a Nonpeptidic Amino Terminus”

Ic50(d) Ref.

is-Cal-OH-Val-NH-n-Bu

7

19-1

131

FOG

His-Leu-OH-Val-lle-AMP 2.3

19-2

101

179

19-3

Rd H i:s - C a l - O H - V a l - N H - n - B u P ‘h 194 19-5 19-6 19-7 19-8

R t-BuCO(Et0)zPOt-BUSt-BUSO-

131 131

t-BuSOr

..L

131 131

150 10 3

132 132 132

R-His-Cal-OH-Val-NH-n-Bu

19-9 19-10 19-11

R PhCHzOCO(I-tetralon-3-y1)CO(Indol-2-yl)CO-

RENIN INHIBITORS

205 Table XIX Continued

19-12 19-13 19-14 19-15

Configuration 2(R or S),4S,5S 2(S or R),4S,4S 2(R or S),4R,5S 2(S or R),4R,5S

[2,2001 [481 1I0001 1381

183 183 183 183

12

186

Y o Ph~N~Leu-oH-Val-lle-A PM

"1 19-16

H. PhCH2 0~ N ~: L e ~ - o H V a l - l l e - N H 2

14b 27b

19-17

Cbz-NH

H

186 186

0

Ph~h~Leu-oH-Val-11e-NH2

26

19-18

186

Cbz-NH Ph C H 2 q N

19-19

Le~-~~-Val-lle-NH,

6.5'

186

(continued)

GREENLEE

206 Table XIX Continued

19-20

0.65

186

19-21

350

186

-10,000 -10,000

186 186

o 19-22

i

Sta-l le-AMP

"See Table I footnotes. bICsovalues are for the two diastereomers. cIC50for a mixture of diastereomers. An OHover a bond indicates the hydroxy isostere (Fig. 2).

which a lactam ring links the two. 186 The resulting inhibitors (19-17; 19-19) have potency equivalent to their acyclic counterparts, although none is as potent as the N-methylated analog (19-20) itself. Interestingly, the (P4)Cbzamino group appears to make no contribution to the potency of inhibitors 19-18 and 19-19 (compare with 19-16 and 19-17). The amino terminal portion of lactam 19-17 is a fairly simple acyl group, and the expected increase in proteolytic stability could contribute to improved bioavailability. As was observed for N-methylation of the Phe-His amide bond, introduction of the lactam constraint greatly reduces potency of the statinezcontaining inhibitor 19-22.186

I. Cyclic Renin Inhibitors

Several cyclic renin inhibitor designs have been reported. Possible advantages of cyclizations include stabilization of the resulting structures toward p r ~ t e i n a s e s and ' ~ ~ entropic advantages for binding. The oral bioavailabiIity of cyclosporin,18' a cyclic undecapeptide, provides encouragement that cyclic

RENIN INHIBITORS

207

renin inhibitors could show enhanced oral absorption. Naturally occurring cyclic peptides have been reported which incorporate statine-like elements (including pyridomy~in~~’ and didemnin B190,191), but little is known about their oral bioavailability. Early cyclic inhibitor designs used 18-membered ( 2 0 3 ; 20-4) or 20-membered (20-5) rings to enclose a Pro-Phe beta-turn postulated to occur in bound substrate, and yielded inhibitors with (moderate) potency comparable to acyclic analogs (20-1; 20-2). 192~193 Pairs of cysteine side chains (P2-P2’ and P4-P2’ pairs) have been linked in cyclic structures which are based on the Pl-P1’ Phe-Phe sequence (Sect. VLB), statine, or a reduced peptide isostere (Table XX). Only the cyclic peptide inhibitors (20-7; 20-9) have potency comparable to acyclic analogs.lY4 Two cyclic inhibitor designs investigated by Boger et al. were derived from molecular modeling using the Rhizopus pepsin:pepstatin x-ray crystal structure of Bott et al.56Cyclic disulfides (20-18; 20-19) constructed from P2 toward the carboxy terminus had potency comparable to that of an acyclic analog. An amino-terminal cyclic disulfide inhibitor (20-22) made by connecting P5 and P2 homocysteine side chains encloses a Pro-Phe beta-turn, as had inhibitors 20-3 and 20-4. As predicted, the optimal ring size for a P5-P2 cycle is found in the 16-membered ring inhibitor 20-22, and three other disulfide cycles with cysteine at either P5 or P2 (or both), were substantially less potent.195,196 A model of human renin constructed at Abbott (Sect. V1.K) has been used to design a series of inhibitors in which the P1 side chain of a ”reduced peptide” inhibitor is cyclized onto the alpha-nitrogen atom of alanine at P2.197 The 14member ring inhibitor 20-26 was equipotent to its acylic counterpart 20-23 and was substantially more stable to the action of chymotrypsin. The 10- and 12-member ring inhibitors 20-24 and 20-25 were much less active, a result which NMR and molecular modeling suggest is due to a cis amide bond between phenylalanine and the N-alkylated alanine.198 J. Irreversible Inhibitors of Renin

Renin, like other aspartic proteinases, is inactivated by the active-site directed reagents diazoacetyhx-norleucine methyl ester (DAN)and 1,2-epoxy3-(nitrophenoxy)propane (EPNP), which react with the active-site aspartic acids. Although no specific irreversible inhibitors of renin or other aspartic proteinases have been reported, inhibitors which could in principle react to form covalent bonds with renin have been reported. These include acrylate 5-17,296 azido analogs such as 15-15 and inhibitors with epoxide-containing ~ide-chains.’~~ Mechanism-based irreversible inhibitors of renin will be difficult to design if (in fact) water is the nucleophile in the aspartic proteinasecatalyzed hydrolysis of peptide bonds. The exceedingly high specificity of renin, which requires a substrate comprising at least eight amino acids, further complicates the task.

K. Molecular Modeling in Renin Inhibitor Design Although a crystal structure of renin is not yet available to serve as a basis for molecular modeling studies, exciting progress has been made toward this goal. Preliminary x-ray crystallographic data on mouse submaxillary gland

G REENLEE

208 Table XX Cvclic Inhibitors of Human Renin and Related Inhibitors"

IC50 (nM) 20-1 20-2 20-3 204 20-5 20-6 20-7 20-8 20-9 20-10 20-11 20-12 20-13

His-Pro-Phe-His-Pro-Phe His-Pro-Phe-His-Pro-D-Phe His-Pro-Phe-His-Pro-Pie His-Pro-Phe-His-Pro-D-Pde Cis-His-Pro-Phe-His-C&NH2

49,000 38,500 57,000 53,100 7,000 8,300 2,900 500 190 0.38 4,300 1,500 6 Inactive 760

Ref.

Bo~-Phe-Ala-Ser-~-Val-Ile-His-OCH3

V.71 [991 [2001 [3w 7.7b 9.2b 19b (1,400)

192 192 192 192 193 194 194 194 194 194 194 194 194 194 194 196 195 196 196 196 196 196 197

n 3 5 7

(~100,000) (69,000) (2,700)

197 197 197

Ac-His-Pro-Phe-His-Phe-Phe-Val-Trp(for)-NH* Ac-His-Pro-Phe-dys-Phe-Phe-C$s-Trp(for)-NHz Ac-Trp(for)-Pro-Phe-His-Phe-Phe-Val-Trp( for)-NH* Ac-Trp( for)-C&'he-His-Phe-Phe-C~s-Trp(for)-NH2

-

Ac-Trp(for)-Pro-Phe-His-Sta-Val-Trp(for)-NH2 Ac-Trp(for)-dys-Phe-His-Sta-C;s-Trp(for)-NHz Ac-Trp(for)-Pro-Phe-Cys-Sta-Cys-Trp(for)-NH2 Ac-Trp( for)-Pro-Phe-His-Phe-R-Phe-Val-Trp(for)-NH2 Ac-Trp( for)-Pro-Phe-C!ys-Phe-R-C;s-Trp(for)-NH2 Ac-Trp(for)-C!ys-Phe-His-Phe-R-Phe-Cis-Trp(for)-NH2

20-14 20-15 20-16 20-17 20-18 20-19 20-20 20-21 20-22

Ibu-His-Pro-Phe-His-Sta-Leu-Phe-NH2 Ibu-His-Pro-Phe-Ala-S ta-Leu-Phe-NHz Ibu-His-Pro-Phe-HCys-S ta-Leu-Phe-NH( CH2)Z-S Ibu-His-Pro-Phe-Cis-Sta-Leu-Phe-NH( CH2)2-S His-Pro-Phe-His-Sta-Leu-Phe-NH2 Abu-Pro-Phe-Abu-Sta-leu-Phe-OCH3 HC)s-Pro-Phe-HCis-S ta-Leu-Phe-NH2

20-23

20-24 20-25 20-26

"See Table I footnote. bICm value for hog renin. An R over a bond indicates a reduced peptide isostere (Fig. 2). For peptide inhibitors, amino acids believed to represent PI and Pi are underscored. Trp(for) = N(im)-formyltryptophan; HCys = homocysteine.

renin and inhibitor complexes have been described by Navia et ~ 1 1 .Groups l~~ at California B i o t e ~ h n o l o g y and ~ ~ , ~at~ L J p j ~ h nhave ~ ~ , reported ~~ obtaining crystals of recombinant enzyme and enzyme-inhibitor complexes, but as yet no x-ray crystal structure data have appeared in the literature. X-ray crystal structures of the related aspartic proteinases pepsin,200Rhizopus p e p ~ i n , ~ ~ , ~ ~ ~

RENIN INHIBITORS

209

Table XXI X-Ray Crystal Structures of Aspartic Proteinase: Inhibitor Complexes” ha-Val-Val-Sta-OEt

21-1

H-256 (21-2) Inhibitor

Pepstatin (3-1) 21-1

H-142 (2-8) H-256 (21-2) L-363,564 (3-9) H-261 (5-2) 2-3 13-7

Pro-Thr-Gly-Phe-R-Phe-Arg-Glu Type

Enzyme

Statine Statine Reduced peptide

Rhizopus pepsin Penicillopepsin Endothiapepsin

Reduced peptide Statine Hydroxy isostere Reduced peptide Amino alcohol

Endothiapepsin Endothiapepsin Endothiapepsin Rhizopus pepsin Endothiapepsin

Reference(s) 56 55,102 100,111 127,202 100,101 111 127 59 165

“An R over a bond indicates a reduced peptide isostere (Fig. 2).

peni~illopepsin,~~ and e n d ~ t h i a p e p s i nhave ~ ~ been determined. X-ray crystal structures of several aspartic pr0teinase:inhibitor complexes have been described (Table XXI). These include inhibitors incorporating statine,55,56,102,111 a “reduced peptide” i ~ ~ ~ t e r e , the ~ “hydroxy” ~ , ~ ~ isostere,lZ7 ~ , ~ ~and ~ , ~ ~ ~ the amino alcohol i ~ 0 s t e r e . Information l~~ gained from study of these structures has allowed hypotheses to be made about their mode of binding and the mechanism of aspartic proteinases (Sect. IV). X-ray crystal structures of two P1-P3’ substrate fragments (Leu-Leu-ValTyr-OCH, and PhOCH2CO-Leu-Val-Tyr-OCH,) reveal extended conformations similar to those observed for inhibitors in complexes with aspartic p r o t e i n a s e ~ .Conformational ~~~,~~~ analysis of pepstatin and the two related renin inhibitors 4-13 and 4-14 by NMR spectroscopy (in both dimethyl sulfoxide and pyridine) suggests that the three inhibitors also display extended conformations.lZ6 Models of mouse reninZo5 and human renin102,106,185,206-209 h ave been derived from their known primary sequences and one or more of the x-ray crystal structures listed in Table XXI. Renin models have played an important part in the design of renin inhibitors. The use of modeling based on the Rhizopus pepsin: pepstatin structure of Bott et al.56in the prediction that statine would serve best as a dipeptide mimic (Sect. VLC), in the design of ACHPA (Sect. VI.F), and in the design of conformationallyrestricted cyclic statine-containing inhibitors (Sect. VI.1) has been described.195~196A detailed description of uses of a renin model in visualizing enzyme-inhibitor complexes has been reported by Raddatz et a1.208A proposed network of hydrogen-bonding interactions between enzymes and the peptide backbone of a renin inhibitor has been derived from a human renin model at Abbott.lo6This model was used to design Abbott’s cyclic renin inhibitors (Sect. VI.1). The human renin model of Carlson et al.lS5was used in the design of conformationally restricted inhibitors at Upjohn (Sect. VI.H).186A model of human renin was used to choose peptide sequences as potential epitopes for renin-inhibitory antibodies .210 Additional contributions of modeling to inhibitor design are anticipated, especially when an x-ray structure of human renin with an inhibitor bound becomes available.

GREENLEE

210 Table XXII Inhibition of Renins of Other Species”

Ref. 22-1 22-2 22-3

CP-71,362 (22-4) 22-5

213 214 214 215 216

Ac-His-Pro-Phe-Val-Sta-Leu-l’he-NH2 Boc-His-Pro-Phe-His-AmSta-Val-Ile-His-NH2 Boc-His-Pro-l‘he-His-Leu-oH-Leu-Val-Ile-His-NH~ Boc-Phe-His-Cal-oH-Val-Lys-l’he Boc-Phe-His-Leu-AA-Leu-Val-Phe-OCH3 (R) +

IC50 (nM)

Renin source Human plasma Human kidney Dog plasma Rat plasma Monkey plasma Guinea pig Hog kidney

22-1

22-2

22-3

27

5

22-4

225

23 550

o.0033 30

12 -

1

3.3 16 52

370 4.4 -

”See Table I footnotes. An AA over a bond indicates an amino alcohol isostere; An OH over a bond indicates an “hydroxy” isostere. at hydroxyAmsta = aminostatine (Fig. 2); Cal = 3-(cyclohexyl)-alanine;(+)-Configuration bearing carbon of amino alcohol isostere.

L. Renin Inhibitors from Natural Sources Although screening of natural products has led to the discovery of interesting new inhibitors of ACE, few inhibitors of renin have come from natural product sources since the discovery of pepstatin. Pepstatin-like analogs named ahpatinins have been isolated from a Streptornyces and shown to inhibit renin. 211,212

M. Inhibition of Renins of Other Species Several inhibitors which are unusually potent against renins of specific species have been reported (Table XXII). Although human renin inhibitors containing statine usually show only weak inhibition of rat renin, inhibitor 22-1 has good potency against the rat enzyme.213Potent rat renin inhibitors which contain aminostatine (22-2) or the ”hydroxy” isostere (22-3)have been reported.’14 The lysine-containing inhibitor CP-71,362 (22-4) also inhibits rat renin, and has an exceedingly high potency against dog renin.’15 The high specificity for hog renin of the amino alcohol-containing inhibitor 22-5 has also been reported.216Consistent with their high degree of homology, h-uman and primate renins usually show similar ICs0 values for inhibitors. N. Specificity of Renin Inhibitors Versus Other Enzymes

Clinically useful inhibitors of renin would be considered optimal if they did not inhibit other proteinases, including the related aspartic proteinases pepsin and cathepsin D. In vivo inhibition of these or other aspartic proteinases could result in side effects for human renin inhibitors. Since pepstatin itself is a

RENIN INHIBITORS

211

Table XXIII Specificity of Inhibitors for Renin Versus Other Aspartic Proteinases"

Ic50 InM)

Inhibitor

F2 AAb

CGP-29,287 (33-6) ES-305 (33-5) ES-1005 (3-7) SR-43,845 (4-5) ES-6864 (4-9) U-71,038 (5-3) CGP-38,560A (5-10) KRI-1177 (7-1) KRI-1230 (74) 11-11 15-8

HIS His His His Thize (N"-Me)His His His His His His

20-26

Ala'

Renin

Pepsin

1 9.2 4.5

>10,000

40,000

Cathepsin D

Ref. 118 119 180 122 177 129 131 292 141 156 166 197

CO.01

>2,000

6.9 0.26 0.4 90 7.8 7 (7.6) 27 2,700

>10,000 6,800 8,200 >100,000 100,000 NTd

40,000 >10,000 16,000 5,000 >10,000 6,300 3,200 15,000 3,000 >lOO,OOO >lOO,OOO

>100,OOO

>100,000

8,000

"See Table I footnotes. bAmino acid at P2position of inhibitor. 'N"-alkylated alanine within a cyclic renin inhibitor. dm= not tested. Thiz = 3-(thiazol-4-yl)alanine.

substantially more effective inhibitor of pepsin than of human renin, statinebased renin inhibitors began with a specificity disadvantage to overcome. Nevertheless, high specificity for human renin versus pepsin and cathepsin D has been reported for inhibitors containing statine or other peptide bond isosteres, as .illustrated in Table XXIII. The presence of an aromatic amino acid (or an equivalent) at the P3 position appears to be an important determinant for this specificity. Although Rich has demonstrated the contribution of His at the P2position to the specificity (renin versus pepsin) described for many inhibitors,217high specificity has been reported for inhibitors (e.g., 2026) with neutral P2 amino acids (Table XXIII). Inhibitors which are based on transition-state designs other than statine, and which incorporate nonpeptidic (or cyclic) elements may be more likely to achieve high specificity for human renin.

0. Stability of Renin Inhibitors Stability toward peptidases in vitro has been reported for several inhibitors, suggesting that they may prove to be stable toward cleavage in vivo. Renin inhibitors have been subjected to the action of organ homogenates (pancreas, liver, intestine, kidney), or purified enzymes (chymotrypsin, trypsin, elastase), and structures showing stability toward these have been reported. ACHPA-containing inhibitor 4-10 was stable for 24 hours to freshly prepared rat liver homogenate.218KRI-1230 (7-4) showed some (10%)cleavage by liver homogenate,141but U-71,038 (5-3) which incorporates a metabolically stable N-methylated Phe(8)-His(9) bond, was inert.lZ9An inhibitor similar in structure to U-71,038, but which incorporates a Boc-(alpha-methy1)Pro-Phe-His sequence replacing Boc-Pro-Phe-(N-methyl)His, is stable to chymotrypsin,

212

GREENLEE

elastase, porcine pepsin, and rat liver h ~ m o g e n a t e . ~ Inhibitor '~ 15-8 is stable to liver, kidney, and intestinal homogenate.166Although other bonds are cleaved, the amide bonds in inhibitor 3-10, which involve the statine moiety, are stable toward cleavage during treatment of inhibitor with rat liver homogenate or after intravenous administration to dogs.220Thus the stability to proteinases reported for statine-containing inhibitor ES-305 (3-5),119 for example, is not surprising. The lability of the Phe(8)-His(9) bond of renin inhibitors to chymotrypsin has been mentioned, and various approaches to stabilization (Sects. 6.19 and 6.20) or elimination (Sect. V1.H) of this bond have been described. Inexplicably, the Phe-His bond of inhibitor 4-2 is completely stable for 24 hours to chymotrypsin, while this bond is totally cleaved when the carboxy terminus is basic, as in inhibitor 4-1L2I8 Studies of the stability of cyclic renin inhibitors such as 20-18 have shown that, although the portions of the cyclic structures enclosed by a cycle were stabilized to proteolysis, degradation could still occur on the linear portion of the inhibitor."' Cyclic inhibitor 20-26 has been reported to be stable to the action of ~ h y m o t r y p s i n . ~ ~ ~ Little information is yet available to confirm that renin inhibitors which are stable to homogenates are resistant to proteolysis in vivo. Enzymes other than those present in the organ homogenates used, or which are deactivated before or during in vitro assays, could play an important role. Thus, stability toward peptidases can be assured only in a wholly nonpeptide renin inhibitor. Other potential sites of metabolism are also of concern. A study of the metabolism of radiolabeled inhibitor 15-7 showed that after i.v. administration to rats, much of the 50% of radiolabel which appeared in the bile during 24 hours was associated with components other than the fragments expected to result from proteolysis.221The poor oral absorption and rapid biliary excretion of most renin inhibitors has made meaningful evaluation of their in vivo stability difficult. Moreover, reports that stabilized renin inhibitors do not show improved oral bioavailability (Sect. VI1.J) or duration of action after intravenous administration (Sect. VII.1) suggest that stabilizing inhibitors to proteinases will not in itself solve either problem. P. Physical Properties of Renin Inhibitors It is not yet clear what physical properties should be sought in renin inhibitors in order to improve their bioavailability. If it is assumed that a highmolecular-weight (MW > 600) inhibitor must be absorbed from the gastrointestinal tract by passive diffusion, then a lipophilic inhibitor with some water solubility would be desirable. Lipophilic side chains are preferred in the S3, Sz, S1, and Sl', binding sites of renin, and inhibitors based on the current transition state designs must make favorable binding interactions in at least the first three of these sites in order to achieve high (nanomolar) potency. The resulting low aqueous solubility for many inhibitors is likely to limit their oral bioavailability.2" Although a nonaqueous formulation enhances oral absorption of the highly lipophilic cyclic peptide cyclosporin,18xit is unclear whether a similar strategy would be effective and practical for an insoluble renin inhibitor.

RENIN INHIBITORS

213

In general, incorporation of less lipophilic (polar and/or ionizable) side chains into renin inhibitors to increase aqueous solubility results in an unacceptable loss in potency. An exception is histidine, which is accepted at P2, and does contribute to solubility, especially when present in the form of a salt. Thus, while inhibitor 12-8 has aqueous solubility low enough (0.0003 mg/ml) to make its in vivo evaluation difficult, the corresponding His analog 12-5 is nearly 100-fold more soluble (0.026 mg/ml).’” Moreover, it is clear that introduction of other polar and/or ionic groups in appropriate locations can yield potent inhibitors with high water solubility. Amino acids with ionizable side chains may be incorporated at remote sites (P5,P3’, P4’) without loss of potency. Analogs of pepstatin (3-1) with increased potency and solubility have been derived by addition of Glu or Arg at the carboxy terminus.*12 The contributions of N-terminaI Pro and C-terminal Lys to the solubility of RIP (1-3) have been mentioned. The nonapeptide CGP-29,287 (3-6) which contains two amino-terminal arginine residues is also quite soluble in water (100 mg/ml).”8 Arginine or lysine have been incorporated at P3’,as in the potent inhibitors 24-1 (solubility > 17 mg/m1)223 and 24-2,I4O and, with some loss of potency, at Pz‘ in inhibitor 4-11 (>lo0 mg/ml in 0.1 M citric (Table XXIV). Solubilizing amide groups may be incorporated at the carboxy terminus of some renin inhibitors without loss of potency (Table XXIV). Solubilities for a series of potent statine and ACHPA-containing “tetrapeptide” inhibitors incorporating polar and/or ionic groups at the carboxy terminus have been r e p ~ r t e d . ”As ~ expected, the most effective groups are those which are ionized at neutral pH (amines, guanidines, sulfonic acids, etc.). An example is inhibitor 3-11 (18 mg/ml in H20).224Other examples of inhibitors with C terminal solubilizing groups include SR-43,485 (4-5)’” and ES-1005 (3-7).I8O A solubilizing 2-pyridylmethyl amide (”AMP”) is found at the carboxy terminus of many inhibitors described in this review. High potency has also been obtained for shorter inhibitors where the carboxy terminus comprises an amide which contains a basic amine (Table XXIV). Inhibitors with C-terminal basic heterocycles are expected to show improved solubility (e.g., 1519; 19 mg/ml in 0.1 M citric acid).= Inhibitors which incorporate aminostatine14 (Sect. V1.J) or the amino alcohol isostere16’ (Sect. VI.C.12) have increased solubility compared with the corresponding statine analogs, and diol inhibitors would be expected to be more soluble than those containing a single hydroxyl. Solubilizing groups have also been incorporated at the amino terminus of inhibitors (Table XXV). Particularly effective in increasing solubility are basic amines as in A-64662 (25-1) (solubility of diacetate salt = 100 mg/m1),225 25-8 and 25-9. Also effective are sulfone groups as found in inhibitor 25-7226 and in CGP-38,560A (5-10).’32 Amino terminal acyl groups which contain either a primary amine or a carboxylic acid group give potent inhibitors when the ionizable groups are placed at an optimal distance, as in inhibitors 25-10 to 25-13.’35 Other inhibitors with amino terminal solubilizing groups include SR-43,845 (4-5)”’ and a series of amino terminal acyl, ureido, and carbamate groups (25-2 to 25-4; 19-3) reported by workers at Abbott.179 In addition to solubilities, measurement of partition coefficient has been

214

GREENLEE Table XXIV Inhibitors With Carboxy Terminal Solubilizing Groups" G o

24-1 24-2

Boc-Phe-His-ACHPA-Ile-Lys-NHZ Boc-Phe-His-ACHPA-Leu-Arg-NH2

24-3

So1.b

(d) ( m g W

Ref.

1.2

290 140

>17

5.2

140

1

140

n Boc-Phe-His-!

~

H 24-4

24-5 24-6 24-7 24-8

R -NH2 -OH -N(CH3)2 -NH=C(NHz)CH3

OH 0

3 33 38 5 19

>14 0.86 >30

140 293 124 293 293

"See Table I footnotes. bSolubility in 0.1 M citric acid.

reported for a series of renin inhibitors.224Although log P values have been determined for over 300 renin inhibitors at Merck, no correlation of these values to extent of oral absorption or duration of action has been possible.227 Q. Plasma Binding

In several cases, potency difference have been noted when human renin inhibition was compared in plasma renin and human kidney renin a ~ s a y s . ~ ~ J ~ l Degradation of inhibitor in plasma or differences in assay conditions may be responsible for these differences. Inhibitor 3-8 and related analogs were re-

215

RENIN INHIBITORS Table XXV Inhibitors with Amino Terminal Solubilizing Groupsa

& .

RCO-AA-His-!

H

25-1 25-2 25-3 25-4

RCO

AA

NH2C(CH&CH2COHO-CH(CH2CH2)2NCOO(CH2CH2)2N-COO(CH2CH2)2NCO-

(p-0Me)Phe Phe Phe (p-0Me)Phe

25-5 25-6 25-7 25-8 25-9

X BOC-NH(morpholin-4-yl)COCH2(morpholin-4-y1)CONH(i-propyl)S02CH2(CH3)zNCHzCONH(piperazin-l-yl)COCH2-

25-10 25-11 25-12 25-13

NH2(CH&CONHz(CH&COH02C(CH2)4COHO2C(CH2)jCO-

4-6

OH

ICw(nM) 14 0.68 0.89 2.4

19 40 5 15 15 105

His His Leu Leu

(2.2) (5.5) (3) (6)

Sol. (mg/ml)

loob

1C

1.2' >14' >10N >25' 11'

Ref. 121 179 179 179

124 226 226 226 226 226

135 135

135 135

"See Table I footnotes. bSolubility in water of diacetate salt. 'Solubility in 0.1 M citric acid.

ported to have high potency in a pure enzyme assay, but remarkably reduced activity in a plasma renin assay. This led to the hypothesis that plasma factors bind inhibitor and prevent its interaction with renin."8 Although direct evidence for binding of 3-8 by plasma components was obtained, subsequent work has shown its high potency in the pure enzyme assay to be artifa~tual."~ It is possible that plasma binding of renin inhibitors, a subject not yet extensively explored, could be an important determinant for their pharmacokinet-

216

GREENLEE

ics. For example, most drugs which are extensively excreted into bile (Sect. VI1.J) are highly plasma bound.230 VII. IN VIVO INHIBITION OF RENIN A. In Vivo Models for Evaluation of Renin Inhibitors

There is no ideal animal model for in vivo evaluation of renin inhibitors. Although various rat models have been used extensively for the in vivo testing of ACE inhibitors, rat renin is insensitive to many of the potent classes of human renin inhibitors (see Sect. V1.M). Exceptions are the potent rat renin inhibitor 22-1, which has been studied in sodium-deficient rats213and inhibitors 22-2 and 22-3 which were evaluated in renal hypertensive rats (Sect. VII.C).214In some cases, exogenous renin (hog or human) has been administered to rats in order to determine the effects of inhibitors on plasma renin activity (PRA) and blood pressure (BP) or mean arterial pressure (MAP).1n,231,232 Although renin must be administered by infusion due to its rapid clearance, the recent availability of recombinant human renin should make assays of this type easier to achieve. Salt-depleted dogs,215,233-235 guinea pigs,215monk e y ~ , ~baboons,238-240 ~ ~ , ~ ~and ~ m - a~r m ~ ~~s e t s ' l ~have , ~ ~been ~ used for in vivo evaluation of inhibitors. In these salt-depleted models, the accompanying volume depletion results in activation of the renin-angiotensin system and increased plasma renin activity. Under these conditions, a component of systemic blood pressure is renin dependent, and inhibition of renin is usually accompanied by a reduction in BP. Marmosets have the advantage of small size (300 g), and the high degree of renin dependency which can be achieved in their BP.241Nevertheless, salt-deficient animals are not hypertensive (in fact, their BP is often lower than in the salt-replete state) and their relevance to hypertension in man is uncertain. Renin inhibitors have also been evaluated in models of renal hypertension. 160242 Unfortunately, renal hypertensive primates are difficult to maintain, and may be unsuitable for long-term invest i g a t i o n ~ . ~As ~ ~is, the ~ ~ case ~ - ~for ~~ in vitro renin assays, many confusing differences exist among the many assays which have been used to report in vivo activities of renin inhibitors. Foremost among these is the degree of renin dependence of the animals used, which depends on the extent and acuteness of their salt depletion, and will directly affect the response of BP to inhibition of PRA. Evidence that inhibiting renin-angiotensin systems in vascular or other tissues or organs may be important in control of hypertension26-28raises additional questions about basing the evaluation of inhibitors solely on their effecton plasma renin activities. Extensive evaluation in hypertensive humans may be necessary to assess accurately the potential of renin inhibitors and their value relative to ACE inhibitors.

B. Intravenous Administration of Renin Antibodies The intravenous administration of renin antibodies to inhibit renin has been discussed by Haber.246A bolus i.v. dose (10 pg/kg) of the exceedingly potent (I@50= 1.3 x M) monoclonal antibody R-36-16 in marmosets produced complete inhibition of PRA and reduction in blood pressure for 2 h o ~ r s . ~ ~ ~ , ~

RENIN INHIBITORS

217

Chronic administration of R-36-16 to marmosets over 14 days by infusion from an osmotic minipump (300 pg/kg/day) produced a sustained reduction in BP. Curiously, although antibody concentrations were in excess over renin in plasma, PRA was not fully inhibited, and had returned to pretreatment levels by Day 14. It was proposed that antibody might be present in a partially degraded form, or that renin release during chronic administration of inhibitor could have a slightly altered form unable to bind (intact) antib~dy."~ Why blood pressure remains suppressed is unclear. In an elegant experiment, sodium-depleted marmosets immunized against pure human renin were shown to develop a high titer of renin antibodies which inhibited the activity of both human and marmoset renins. In these animals, PRA was completely inhibited and systolic BP reduced from 125 ( ? 13) mmHg to 87 ( ? 8) mmHg. Successful blockade of the RAS persisted for up to 4 months, at which time sickness and death due to immunologicalrenal disease occurred.z49The possibility remains that renin activity can be chronically blocked by immunizing with synthetic renin epitopes, without inducing an autoimmune disease."' Although renin inhibitory antibodies are expected to have limited utility as therapeutic agents, their study has provided evidence that inhibition of renin in the plasma compartment is sufficient to provide sustained lowering of blood pressure.

C. Intravenous Administration of Peptide-based Inhibitors In vivo studies of peptide-based inhibitors of renin to 1985 have been reviewed by Bogerl' and by Hofbauer and Wood.25Early studies include those of human statine-containing renin inhibitors such as SCRIP (3-2), reduced peptide inhibitors such as H-142 (2-8), and inhibitors based on the hydroxy isostere, including H-261 (5-2). Effective inhibition of PRA and lowering of BE' have been obtained with these and more recently reported inhibitors in renin-dependent models, including primates, with administration by infusion or i.v. bolus. Duration of action is usually quite short for peptide-based inhibitors, but can be increased with larger doses. Rapid bile excretion and metabolism are believed to be factors important for the short plasma halflives of inhibitors (Sect. VI1.J). Direct secretion of inhibitors into the gastrointestinal tract or uptake into tissue may aIso contribute to their rapid clearance from plasma. Recent intravenous data have been reported for a number of inhibitors, and the results of some of these studies are summarized in Table XXVI. Although the use of different models, doses and assay protocols (Sect. VI1.A) makes direct comparisons of inhibitors difficult, some generalities can be drawn from the data. Suppression of PRA in sodium-deficient models usually lowers BP and A-I1 levels only when PRA is greater than 90% inhibited. This phenomenon, which was observed by Blaine et al. during study of infusions of SCRIP (3-2) in sodium-depleted dogs, led to the hypothesis that a dissociation exists between PRA and BP (Sect. VII.H).234Little or no effect on HR has been reported for the renin inhibitors listed in Table XXVI, as has been found to be the case for other agents (e.g., ACE inhibitors) acting on the renin-angiotensin system. Several of the inhibitors listed in Table XXVI exhibit duration of action after i.v. administration which is exceptional when the i.v. dose (pmol/kg) and

218

GREENLEE Table XXVI Intravenous Administration of Renin Inhibitors to Sodium-Deficient Animals" ICSO Inhibitor

(fl)

Modelb

Dose (mglkg)

SR-42,128A (33)

28

CGP-29,287 (3-6)

30' 5'

CyMonkey(C) Baboon(A) Marmoset(C) Marmose t(C)

SR-43,845 (4-5)