INFECTION AND IMMUNITY, Apr. 1973, p. 655-665Copyright © 1973 American Society for Microbiology

Vol. 7, No. 41Printed in U.S.A.

Inhibition of the Cathepsin D-Type Proteinase ofMacrophages by Pepstatin, a Specific Pepsin

Inhibitor, and Other SubstancesMARTHA H. McADOO, ARTHUR M. DANNENBERG, Jlt., CARA J. HAYES,

STEPHEN P. JAMES, AND JOHN H. SANNER1Departmnteit of Radiological Sciences, School of Hygienbe and Public Health, antd the Department of Pathology,

School of Medicinte, The Johns Hopkins University, Baltimore, Marylanld 21205

lReceived for publication 10 November 1972

The macrophage is the main cell participating in chronic inflammation. It con-tains an acid-acting, cathepsin D-type proteinase with the specificity of pepsin,which may release mediators of the inflammatory process. To find new pharma-ceutical inhibitors of this proteinase, we tested a variety of chemical compoundsin vitro. For this survey, the possible inhibitor (at a concentration of 0.4 mg/ml)was assayed with partially purified cathepsin D-type proteinase from beef lung (amacrophage-rich tissue) and hemoglobin as the substrate. Diazophenylbutanone,three acetophenones, two barbiturates, a gold salt, a copper chelate of a substitutednicotinic acid, a hexapeptide containing a D-amino acid, and Pepstatin inhibitedthis enzyme; over 200 other potential inhibitors did not. By far the most activeand specific inhibitor found to date is Pepstatin, a pentapeptide with two a-NHlinkages, two a-OH groups, and five branched aliphatic side chains. Banyu Pharma-ceutical Co., Tokyo, Japan, produces this nontoxic compound for the treatment ofpeptic ulcers. In vitro, as little as 4 ng of Pepstatin inhibits the acid-acting cathepsinD-type proteinase purified from beef and rabbit lung as well as the similar proteinaseof rabbit peritoneal and pulmonary macrophages.

Macrophages, one of the body's main defensecells against infectious agents, contain an activeproteinase that hydrolyzes hemoglobin at pH 3.9(10, 54). It resembles cathepsin D purified frombovine lung (12, 13), rabbit lung (0. Rojas-Espinosa, A. M. Dannenberg, Jr., P. A. Murphy,P. A. Straat, P. C. Huang, and S. P. James,manuscript in preparation) and spleen (38, 44,58), bovine uterus (79), and human, chicken, andrabbit liver (5). The presence of cathepsin D(EC 3.4.4.23) in macrophages was demonstratedhistochemically with fluorescent antibody tech-niques (57) and antiserum to purified livercathepsin D (17, 76).We have partially purified an acid-acting

proteinase from beef (12, 13) (and rabbit) lung(0. Rojas-Espinosa et al., manuscript in prepa-ration), which hydrolyzes the B chain of insulinat the same sites as pepsin (13) and cathepsin D(8, 38, 44). We feel that this proteinase comesfrom macrophages because of the large numbersof these cells present in lung and because of

1 Affiliation: Searle Laboratories, G. D. Searle andCo., Chicago, Ill. 60680.

similar pH optima and other common properties(10, 12, 13) including the inhibitor studies hereinreported.

Proteases may play an important role in theinflammatory process by releasing vasoactivepeptides (48, 66, 77). From the fifth componentof complement (C5) they also release a potentchemotactic factor (C5a) that can attract macro-phages and polymorphonuclear leukocytes to thesite of inflammation (63, 65, 75). Rabbit ho-mogenates and purified beef lung proteinase canalso produce C5a from C5 (64). Macrophageproteinase may therefore be an importantmediator of parts of the chronic inflammatoryprocess, and an inhibitor of macrophage pro-teinase might serve as a new type of anti-inflammatory drug.Over the last 6 years our laboratory has been

screening various compounds in an effort to findan inhibitor of macrophage proteinase that mayserve as a simple pharmacological agent to con-trol its action in vivo. This report describes theresults of such efforts. Over 200 compounds weretested. Some were substrate analogues, some wereknown pepsin inhibitors, and some were anti-

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inflammatory agents. Many had structures thatconceivably could bind to the hydrophobic sitesin the catalytic center of pepsin and cathepsini Dand thereby inhibit their proteolytic activity.During our search for such inhibitors, we dis-

covered that Pepstatin was available in Japan,aid we helped make it accessible to researchersin this country and in England. Pepstatin is byfar the most effective inhibitor of pepsin andcathepsin D known to maii. It was discovered atthe Institute of Microbial Chemistry in Tokyo byH. Umezawa in culture filtrates of various speciesof actinomycetes of the streptomyces type (69,73). Pepstatin is a nontoxic pentapeptide contain-ing two y-NH linkages, two (3-OH groups, andfive branched 3- or 4-carbon aliphatic side chains(50, 73). It is produced by Banyu PharmaceuticalCo. in Tokyo for the possible treatment of pepticulcers (2, 73).

Pepstatin reduces carrageenan-produced edemain rats (2, 73), but no inhibitory effect on chronicinflammation was found in our preliminary ex-periments. A similar or less complex moleculemay still be found that does affect chronic in-flammation. The compounds listed in this reportshould be of help in such a search.

MATERIALS AND METHODSMacrophage and lung proteinase activity was

assayed with hemoglobin as the substrate by amethod (10) adapted from that of Anson (1). Bothurea-denatured and acid-denatured hemoglobinwere used.Urea hemoglobin. Bovine hemoglobin sub-

strate powder (2.2 gm) from Worthington Bio-chemical Corp., Freehold, N. J., was dissolved ina solution containing 82 ml distilled water, 40 g ofurea, and 8 ml of 1.0 M NaOH. The solution wasthen adjusted to pH 3.8 with 1.0 M citrate buffer(pH 2) so the final concentrations were 2% hemo-globin 6 M urea, and 0.1 M citrate.Acid hemoglobin. Hemoglobin powder (4.0 g)

was dissolved in 100 ml of 0.05 M HCI to make a4.0% solution at pH 3.15. (When it was dilutedwith an equal amount of 0.18% NaCl, the pH be-came 3.4.)

Inhibitors. Before beiug mixed with the ureahemoglobin substrate, 1.0 mg of inhibitor wasusually dissolved or suspended in 1.2 ml of 0.02 Mphosphate buffer (pH 7.3) and mixed with 0.10 mlof proteinase solution and preincubated for 20 minin a 38 C water bath. Before being mixed with theacid hemoglobin substrate, 1.0 mg of inhibitor wasusually dissolved or suspended in 1.2 ml of 0.18%NaCl (pH 5.6), mixed with 0.10 ml of proteinasesolution, and preincubated for 20 min at 38 C.

Acid hemoglobin was sometimes used instead ofurea hemoglobin, because urea tended to lower theactivity of the enzyme by hastening its denatura-tion at acid pH (cf. 5). Also the citrate present inurea hemoglobin would tend to bind Cu'+ when itwas added. In acid hemoglobin the enzyme pH

optimum was 3.4 (instead of 3.8), and its activitywas about 1.6 times higher. Certain inhibitors,poorly soluble in aqueous solutions, were dissolvedin dimethyl formamide (0.05 ml). The addition ofphosphate buffer or saline frequently precipitatedsuch inhibitors as a fine suspension or colloid. Aninhibitor, not soluble in dimethyl formamide (orethanol or propylene glycol), was used as anaqueous suspension. Most of the potential Cu2+chelate inhibitors from Sorenson were tested onlywith acid hemoglobin as the substrate. Most of theother potential inhibitors were tested with ureahemoglobin as the substrate.

Since we were unable to publish all of the in-effectual inhibitors we tested, we have preparedlists of them to be supplied on request. (Addressrequests to Dr. A. M. Dannenberg, Jr., JohnsHopkins School of Hygiene and Public Health,615 N. Wolfe St., Baltimore, Md. 21205.)

Potential inhibitors came from many sources.The largest number was from the Division ofMedicinal Chemistry (W. E. Rothe, Director),Walter Reed Army Institute of Research, Wash-ington, D.C. They were mostly aromatic com-pounds with configurations somewhat related tothe aromatic amino acids.The second largest number of potential inhib-

itors came from G. D. Searle and Co., Chicago,Ill. They were compounds showing suggestiveantipepsin activity during preliminary screening.Many were peptide derivatives. Searle is particu-larly interested in drugs that may be effective intreatment of peptic ulcers.The copper chelates were provided by J. R. J.

Sorenson, Kettering Laboratory, University ofCincinnati, Cincinnati, Ohio. Many of thesechelates were made while he was at G. D. Searleand Co. Cu'+, when chelated to a variety of anti-inflammatory drugs, has been shown by Sorensonto increase their anti-inflammatory effect multi-fold. The amount of Cu*+ involved is nontoxic andis in the range of normal daily intake.

Pepstatin and Sodium Pepstatin were suppliedby S. Itakura, Banyu Pharmaceutical Co., Ltd.,Tokyo, Japan. Most of the barbiturates weresupplied by D. P. Jacobus, Merck, Sharp & DohmeResearch Laboratories, Rahway, N.J. Gly-Phe-Leu-Gly-Phe-Leu (L) and Gly-Phe-Leu-Gly-Phe-D-Leu (rest L) were supplied by H. Keilov6,Czechoslovak Academy of Science, Institute ofOrganic Chemistry and Biochemistry, Prague,Czechoslovakia. 2-Diazo4'-bromoacetophenoneand 2-bromo4'-chloroacetophenone were suppliedby J. N. Mills, Oklahoma Baptist University,Shawnee, Okla. Ox lung proteinase inhibitor wassupplied by T. Astrup, James F. Mitchell Founda-tion, Institute of Medical Research, Washington,D.C. Phenylbutazone, chloroquine, hydrocorti-sone, p-aminobenzamidine, 6-mercaptopurine,and gold thiomaleate were supplied by R. J.Perper, Ciba-Geigy Corporation, Ardsley, N.Y.G. A. Hamilton, Pennsylvania State University,University Park, Pa., produced and tested 1-diazo-4-phenyl-2-butanone with our proteinase in hislaboratory.

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Abbreviations. When used, abbreviations arebased on the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclaturelisted in volume 2 of "Synthetic Peptides" (56)and also in Biochemistry (36). The configurationfor every amino acid in a peptide was listed inparentheses after the name as a single notation,e.g., (L) when all were (L).Cu*+ addition. Since phosphate buffer precipi-

tated Cu'+ and citrate buffer chelated Cu*+, inhib-itors tested with Cul+ were dissolved or suspendedin 1.1 ml of 0.18% NaCl. Then, 0.1 ml of 0.6 or0.06 mM cupric acetate (or 0.1 ml of 0.18% NaClas control), 0.1 ml of inhibitor, and 0.1 ml of en-zyme were added. The solution was incubated for20 min at 38 C before 1.3 ml of the acid hemoglobinsubstrate was added. Cul+ is known to enhance theinhibitory effect of certain diazo and ketone in-hibitors of pepsin and cathepsin D (see Discus-sion).Assay method. To 1.3 ml of the solution of en-

zyme (or enzyme plus inhibitor), 1.3 ml of theurea hemoglobin or acid hemoglobin substrate wasadded. At 0 h and 1 h, 1.0-ml samples were re-moved, mixed with 1.0 ml of 5% trichloroaceticacid, and centrifuged. (Trichloroacetic acid-sol-uble peptides are split from hemoglobin by theproteinase.) One milliliter of supernatant fluidwas diluted with 2.0 ml of distilled water, and theoptical density (OD) was read at 280 nm in 1-cmquartz cuvettes in a Beckman DBG spectro-photometer. With both urea hemoglobin and acidhemoglobin, the difference in OD between the 0-hand 1-h samples was linearly proportional toproteolytic activity up to an OD of about 0.400,i.e., 0.400 units of activity. (Note that these unitsare different from those published in reference 12.)Proteinase preparations. Most of the poten-

tial inhibitors tested were assayed at pH 3.8 witha partially purified cathepsin D-type proteinasefrom beef lung. This proteinase was partially puri-fied from beef lung by the method described byDannenberg and Smith (12), but one typographicalerror was found in their published directions. Theacetone-dried powder should be prepared by blend-ing below -10 C, 1 vol of ground lung with 2 volof 50% acetone in water, not 100% acetone. Afterthe ammonium sulfate step, the proteinase waspassed through a CM-Sephadex (C50) column,which further increased its specific activity to 1.3units/mg of protein (about 130-fold over the start-ing material of this preparation) (O. Roj as-Espinosa et al., manuscript in preparation).The cathepsin D-type proteinase from rabbit

lung was partially purified by a modification of theBarrett method (4, 5). Lungs (purchased frozenfrom Pel-Freez Biologicals, Inc., Rogers, Ark.)were homogenized in a Waring blender at 4 C with2.5 times their weight of 0.9% NaCl solution. Afterthe debris was removed by centrifugation, the pHof the supernatant suspension was lowered to 3.6with formate buffer and incubated overnight at37 C. After centrifugation, the protein present inthe supernatant fluid was concentrated by adding(NH4)2SO4 to 90% satuiration. The resulting pre-

cipitate, collected by centrifugation, was dialyzedagainst 0.05 M citrate buffer at pH 3.5. At thisstage the proteinase had been purified aboutthreefold, i.e., to 0.54 units/mg of protein (0.Roj as-Espinosa et al., manuscript in preparation).

Peritoneal macrophages were obtained by in-jecting 35 ml of mineral oil intraperitoneally intorabbits and, 5 days later, collecting the macro-phages in citrated saline solution (0.4% sodiumcitrate and 0.9% sodium chloride) (10, 11). The cellsuspension was centrifuged, resuspended incitrated saline, counted in a hemacytometer, andquickly frozen. After at least a week in the frozenstate, it was thawed, homogenized with a serolog-ical pipette, diluted to the appropriate concentra-tion with 0.9% saline, and used as a source ofproteinase with hemoglobin as the substrate.Pulmonary alveolar macrophages were obtained

by perfusing fresh rabbit lungs four times intra-tracheally with 30 to 40 ml of citrated saline solu-tion (11, 51). The pulmonary alveolar macrophageswere collected, counted, frozen, stored, and usedas a source of proteinase in the manner just de-scribed.

RESULTSThe major inhibitors found were diazophenyl-

butanone, three acetophenones, two barbiturates,a gold salt, a copper chelate of a substitutednicotinic acid, a hexapeptide containing a singleD-phenylalanine, and Pepstatin, a unique penta-peptide (Tables 1 and 2).

1-Diazo-4-phenyl-2-butanone (which is ratherunstable) was made by G. A. Hamilton (21, 22,26) and tested with our partially purified beef lungproteinase in his laboratory. In the presence ofCu2+ it proved to be an effective inhibitor.

2-Diazo-4'-bromoacetophenone, 2-bromoaceto-phenone, and 2-bromo-2-phenylacetophenone aremoderately effective inhibitors of beef lungproteinase. Cu2+ did not increase their inhibitoryproperties. Other acetophenones were tested withurea hemoglobin as substrate and found to benoninhibitory. They were 2-bromo-4'-chloro-acetophenone, 2-bromo-4'-phenylacetophenone,2-chloro-4'-phenylacetophenone, 2-diazo-3', 4'-dichloroacetophenone, 2, 4'-dibromoacetophe-none, 4'-chloroacetophenone. (2, 4'-Dibromo-acetophenone is frequently called p-bromo-phenacyl bromide in the older literature. Notethat the 2 position is on the CH3 group and the4' is on the ring of acetophenone. The CH3 groupis called position 2 and not position 1 in theliterature cited, and we are following this patternto be consistent.)

5,5-Dibromobarbituric acid and 5-benzylbar-biturate inhibited in the same range as the threeacetophenones. Cu2+ had little effect on theirinhibitory properties. To our knowledge, theinhibitory effect of certain barbiturates on

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TABLE: 1. Iinhibitors of the cathep8in D-type proleinase of beef lung

Compound

1-Diazo-4-phenyl-2-butanone

2-Diazo-4'-bromoacetophenone

2-Bromoacetophenonie

2-Bromo-2-phenylacetophenone

5,5-Dibromobarbituric acid

5-Benzylbarbituate

Gold sodium thiosulfate

Copper chelate of 2-(3-trifluo-romethylphenyl)nicotiniicacid

Gly-Phe-Leu-Gly-Phe-Leu (L)

Gly-Phe-Leu-Gly-Phe-D-Leu(rest L)

Amount inincubatedmixture(mg)a

0.10.1

1.00.50.2

1.00.40.1

1.00.2

1.00.4

1.0

1.00.4

0.60.40.2

2.01.0

2.01.0

Concn(mM)

0.20.2

1.70.90.3

1.90.80.2

1.40.3

1.40.5

1.9

0.80.3

0.40.30.1

1.20.6

1.20.6

Inhibition (% ofcontrol activity)

Ureahemo-globin

60

01460

3163

5179

46

2.530

Acidhemo-globin

491

556786

578596

6883

042

57

455

486292

6867

7387

Comment

With Cu2+ (0.05 mM)bWithout Cu2+ (0.05 mM)b

Cu2+ (0.6 and 0.06 mM) did notincrease the amount of in-hibition

Cu'+ (0.6 and 0.06 mM) did Inotincrease the amount of in-hibition

Cu2+ (0.6 and 0.06 mM) d!d notincrease the amount of in-hibition

Cu'+ (0.6creasedacid Hb

mM) slightly in-the inhibition in

Cu2' (0.6 and 0.06 mM) had noeffect

Effects of Cu2+ not tested

Without Cu2+ it has little orno effect

Effects of Cu2+ not tested

Effects of Cu2+ not tested

a Incubated mixture was 2.6 ml in size.b The assays involving 1-diazo-4-phenyl-2-butanone were performed by V. B. Valenty in the labora-

tory of G. A. Hamilton, Pennsylvania State University, University Park, Pa., with 3% hemoglobin assubstrate in 0.06 M acetate buffer at pH 3.8. The other assays were performed as described in Materialsand Methods.

cathepsin D or pepsin has Inot been previouslydescribed in the literature. Twenty-five otherbarbiturates, each of different structure thanthose listed in Table 1, had no inhibitory effect.Among these were barbituric acid, barbital,pentobarbital, and secobarbital.Gold sodium thiosulfate was a moderately

effective inhibitor. Various other anti-inflamma-tory agents had Ino effect, namely p-aminobenzamidine, e-amino -n - caproic acid,acetylsalicylic acid, chloroquine, 3-p-chloro-

phenoxypropanediol (Chlorphenesini, WallaceLaboratories, Cranbury, N.J.), cortisone acetate,N-2,3-dimethylphenylanthranilic acid, flu-fenamic acid, hydrocortisone, indomethacin(Indocin, Merck, Sharp & Dohme), 6-mercapto-purine, D-penicillamine, phenylbutazone, andgold thiomaleate. Note that gold thiosulfate wasinhibitory (see Table 1), whereas gold thiomaleatewas not.The copper chelate of 2-(3-trifluoromethyl-

phenyl)nicotinic acid was also a moderately

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TABLE 2. Inhibitory effect of Pepstatin on the cathepsin D-type proteinase of beef and rabbit lung andrabbit peritoneal and pulmonary macrophagesa

Amount of SodiumProteolytic Pepstatin Inhibition

Proteinase preparation Amtb activity of _ (% of controlcontrols activity)(in Units)" pug/2.6 ml of Concnassay mixture

Partially purified beef lung 1.43 units/mg of 0.230 1 0.5 AM 0proteinasec.d protein 0.1 0.05 PM 0

0.01 5.0 nM 500.001 0.5 nrM 600.0001 0.05 nM 95

Partially purified rabbit 0.54 uiits/ii/g of 0.185 1 0.5 JM 0lung proteinase protein 0.1 0.05 ;M 0

0.01 5.0 nM 550.001 0.5 nM 900.0001 0.05 nM 100

Pulmonary alveolar macro- 0.75 X 106 cells 0.220-0.260 10 5.0 pM 0phagese incubated 1 0.5 pM 0

0.1 0.05 FM 00.01 5.0 nM 800.001 0.5 nM 1000.0001 0.05 riM 100

Oil-induced peritoneal 3.6 X 106 cells 0.190-0.360 10 5.0 MM 0macrophagese incubated 1 0.5 MM 0

0.1 0.05juM 00.01 5.0 nM 600.001 0.5 nM 1000.0001 0.05 nM 100

a Assays were performed using acid-denatured hemoglobin as the substrate at pH 3.4. With the lungproteinases they were performed on three different occasions. With pulmonary and peritoneal macro-phages, cells from three different rabbits were assayed. The averages are listed in this table.

b A unit of proteolytic activity is the amount that should produce (if linearity held) an optical densitydifference of 1.000 under the conditions described in Materials and Methods. The number of cells listedwas present in 2.6 ml of substrate-enzyme solution.

c Similar results were obtained with urea-denatured hemoglobin as the substrate at pH 3.8.d CU'+ (0.6 mM and 0.06 mM) had no effect on the inhibitory activity of Pepstatin at any of the con-

centrations listed.e The cell populations contained 90 to 100% macrophages (10, 11). They were used as frozen and

thawed homogenates.

effective inhibitor. This substituted nicotinic acidgave little or no inhibition when copper wasabsent nor was this amount of copper inhibitoryby itself.

Other chelates were tested, namely the copperchelates of acetylsalicylic acid, e-aminocaproicacid, aspartic acid, 3,4,5,6-tetrahydro-f-carbo-line-5-carboxylic acid, 1-carboxyisoquinoline,3-carboxyindole, 3-p-chlorophenyl-3,4,5, 6-tetra-hydro-8-carboline-5-carboxylic acid, diisopropyl-salicylic acid, lysine, nicotinic acid, 1-phenyl-4-carboxyquinoline, salicylic acid, and tryptophan;the zinc chelate of anthranilic acid; and mixedchelates of pyridine and acetate, and pyridineand chloride. These chelates did not inhibit beef

lung cathepsin D. Some of the chelates testedshowed anti-inflammatory effects on carrageenanedema and adjuvant arthritis (J. R. J. Sorensen,unpublished data).

Gly-Phe-Leu-Gly-Phe-Leu (L) and Gly-Phe-Leu-Gly-Phe-D-Leu (rest L) were also moderatelyeffective inhibitors. The L form may not be atrue inhibitor but may merely compete as asubstrate. (Both hexapeptides are soluble in2.5% trichloroacetic acid, so their hydrolysiswould not be detected in our assay of hemoglobinhydrolysis.)By far the most effective and specific inhibitor

found to date is Pepstatin, a pentapeptide con-taining two 7-NH linkages, two ,-OH groups,

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and five branched 3- or 4-carboni aliphatic side nioted. Histological studies on the lesion biopsieschainis (50): remain to be done.

CH3 CH OH3 OCH3 CH3 CH3H CH3 CH3 CH3 CH3 HHOHH2 OH VH H2 H CH3 H

N-NH-IH-CO-NH-dH-CO-NH-6H-CH-CHOCONH-HN-COHN H-CH2-COOH(L) (L) (L)

As little as 0.01 gg (5 iiM) sh1ows inh1ibitoryeffects on the digestion of acid-denatured hemo-globin by partially purified, cathepsin D-typebeef and rabbit lung proteinase and the acid-acting proteinase present in rabbit peritonealmacrophages and pulmonary alveolar macro-phages (Table 2). Pepstatin inhibited thedigestioil of urea-denatured hemoglobin by thebeef lung proteinase at approximately the sameconcentrations as those listed for acid hemoglobinin Table 2.Among the other compouinds, 55 amino acids

and their derivatives and 45 compounds contain-ing aliphatic and aromatic amino acids in peptidelinkage had no effect on proteinase activity. Over100 miscellaneous compounds were also ineffec-tual. Among these were soybean trypsin inhibitor,Astrup ox lung inhibitor (3), N-acetylglucos-amine, amylopectin sulfate, chitodextran,chondroitin sulfate, heparin, starch, and D-trehalose. Carrageenan (0.4 mg/ml) inhibited theproteinase activity only 20%.Effect of Pepstatin on chronic inflamma-

tion. Two pilot studies were performed. In thefirst, 10 mg of Sodium Pepstatin was administeredsubcutaneously once a day to rabbits that wereinfected oIn the third day intradermally with theDanish strain of BCG. (Since Sodium Pepstatinwas not completely soluble in 0.9% NaCl solu-tion, we dissolved it in distilled water and addedan equal amount of 0.9% NaCl solution beforeinjecting it into the rabbits. This BCG strain[no. 1331] was supplied by K. Bunch-Christensen,Statens Seruminstitut, Copenhagen, Denmark.)Other dosage schedules were also tried: 10 mgtwice a day, 1 mg once a day, and 1 mg twice aday. The size of the resulting lesions was similarto controls during the subsequent 3 weeks. Theirtuberculin reactions also showed no differences.We concluded that the systemic administrationof Pepstatin once or twice daily had little or noeffect on chronic inflammation. Another experi-ment was performed in which approximately0.04 mg of Sodium Pepstatin was injected oncedaily directly into similar BCG lesions from 3 to24 days after their onset. Again no effect on thesize of the lesions and tuberculin reactions was

DISCUSSIONPeptide and protein inhibitors. There are

several classes of inhibitors for pepsin andcathepsin D. The most specific are unnaturalpenta- or hexapeptides like Pepstatin. Keilova(38, 39) found that Gly-Phe-Leu-Gly-Phe-D-Leucaused 89% inhibition of hydrolysis of Gly-Phe-Leu-Gly-Phe-Leu by cathepsin D. Hexapeptidescontaining a single D-Phe (instead of D-Leu) in the2 or 5 position were also inhibitory, but whenboth phenylalanines were in the D configurationno inhibition occurred. Evidently, two D aminoacids changed the tertiary structure toomarkedly.Our finding that Gly-Phe-Leu-Gly-Phe-D-Leu

partly inhibited the digestion of hemoglobin bybeef lung proteinase is consistent with Keilova'sresults. She used 0.35 mM of the L-hexapeptideas substrate and 1.0 mM of the D isomer andfound 89% inhibition. Our incubated solutioncontained 26 mg of hemoglobin and 1 or 2 mg ofhexapeptide inhibitor (Table 1). Even if theenzyme could combine with hemoglobin (prior toits hydrolysis) once for every 12 amino acids, westill would not have the favorable conditionsthat Keilova used. These figures illustrate whymany inhibitors effective in preventing thehydrolysis of peptide substrates may not be aseffective when protein substrates are employed.Other peptide inhibitors of pepsin, apparently

not tested on cathepsin D, are polylysines (37),Ac-Phe-OEt (L) (23), Ac-D-Phe-L-diiodo-Tyr(28), Z-L-His-L-Phe-D-Phe-OEt (34), Z-L-His-D-Phe-L-Phe-OEt (34), N-methanesulphonyl-Phe-OMe (L) (23), and N-methanesulphonyl-Phe-SMe (L) (23).

Lists of compounds (mostly amino acid andpeptide derivatives) that inhibited the hydrolysisby pepsin of Ac-Phe-Tyr (L) or Ac-Phe-Phe-Gly(L) and Ac-3,5-dinitro-Tyr-Phe (L) were pub-lished by Schlamowitz et al. (61) and Knowleset al. (42), respectively. Another list effectiveagainst Z-His-Phe(NO2)-Phe-OMe (L) was pub-lished by Inouye and Fruton (35). These threepapers did not mention whether these pepsininhibitors were effective with protein substrates.

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INHIBITORS OF MACROPHAGE PROTEINASE

A specific polypeptide inhibitor of pepsin is re-leased when pepsinogen is converted to pepsin(27, 62, 74). It has a molecular weight of about3,200 and is rich in lysine, leucine, and asparticacid (74). To our knowledge, it has never beentested on cathepsin D. The pepsin inhibitor fromAscaris lumbricoides (55) inhibits pepsin (40, 55)and cathepsin E (40) but not cathepsin D (40) orrennin (40). In contrast, Pepstatin inhibits all fourenzymes.Diazo and ketone inhibitors. Diazo-Ac-

norLeu-OMe (D,L), diazo-Ac-Gly-OEt, anddiazo-Ac-Phe-OMe (L) inhibit pepsin andcathepsin D in the presence of Cu2+ (5, 7, 31, 38,45, 59). The inhibition of our beef lung cathepsinby 1-diazo-4-phenyl-2-butanone in the presence ofCu2+ (see Table 1) is undoubtedly of a similarnature. Keilova (38), Bayliss et al. (7), andHamilton and co-workers (21, 22, 26) discussedthe possible mechanisms involved in inhibitionof this type of proteinase, e.g., blockage ofaspartic acid carboxyl groups in the active site.However, the exact mechanism still remainsunknown. Recently, an even more effectivepepsin inhibitor of this type was described byFruton's group (32). It is the bifunctional in-hibitor 1,1-bis(diazoacetyl)-2-phenylethane, abisdiazoketone.

2-Diazo-4'-bromoacetophenone (19, 31), 2-bromoacetophenone (18), and 2-bromo-2-phenyl-acetophenone (4, 18) inhibited pepsin and ourbeef lung cathepsin D. Barrett (4) found noinhibition of purified rabbit liver cathepsin D by2-bromo-2-phenylacetophenone. 2,4'-Dibromo-acetophenone (4, 18, 24, 31) and 2-bromo-4'-chloroacetophenone (18, 31) inhibited pepsin butnot cathepsin D (4, 38). Because of differentconditions of assay, these conclusions comparingthe effects of certain inhibitors on pepsin andcathepsin D from various sources must, however,remain tentative unless the comparison was madein the same laboratory.

Other diazo and ketone inhibitors of pepsin,not apparently tested on cathepsin D,are: N-diazoacetyl-N'-2, 4-dinitrophenylethyl-enediamine (with Cu2+) (67, 68), Z-Phe-diazo-methane (53), diphenyldiazomethane (14),1-diazo-4-phenyl-3-tosylamidobutanone (L) (withCu2+) (15), 2-bromo-1, 3-indanedione (18).Antibody and carbohydrate inhibitors.

Antibody, carbohydrate, and other inhibitors ofpepsin are reviewed by Cheret and Bonfils (9).Recently, Dingle, Barrett, and Weston (17, 76)found that antisera to purified human, chicken,and rabbit cathepsin D inhibited this pepsin-liketissue enzyme specifically and rather effectively.Our laboratory confirmed this finding with goatantiserum to purified rabbit lung cathepsin D.

(0. Rojas-Espinosa et al., manuscript in prepara-tion) Aoyagi et al. (2) found that heparin,carrageenan, and chondroitin sulfate (rather non-specific inhibitors of pepsin) were at least 25,000times less active than Pepstatin.Miscellaneous inhibitors. Other inhibitors

for cathepsin D in the literature are as follows.None of these are nearly as specific or effective asPepstatin. Six in this miscellaneous group areheavy metal ions (79), high salt concentration(79), vitamin A (71, 79), 3-phenylpyruvic acid(4), indolyl-3-pyruvic acid (4), and dithiothreitol(4, 79). It is of interest that indolyl-3-pyruvic acidpartially inhibits rabbit liver cathepsin D (4), butnot beef uterine cathepsin D (79), and thatdithiothreitol is more effective against rabbitcathepsin D than human and chicken cathepsin D(5). Bis(8-chloroethyl)sulfide (29) inhibits pepsinbut to our knowledge has not been tested oncathepsin D.

Tetranitromethane inactivates bovine spleencathepsin D (38), indicating that tryrosine isprobably a part of its active site or nearby. Thathistidine, serine, and free SH groups are probablynot involved in the active site of cathepsin D isindicated by the lack of inhibitory effect of L-1-tosylamido-2-phenylethyl chloromethyl ketone(4, 38), diisopropyl fluorophosphate (12, 38, 79),and sulfhydryl blocking agents (certain metalions, iodoacetate and p-chloromercuribenzoate)(4, 10, 38, 58, 79). (Additional lists of ineffectiveinhibitors for cathepsin D appear in references 79and 4.)Mechanism of action of pepsin and its

inhibition by Pepstatin. The mechanism ofaction of pepsin (and presumably that ofcathepsin D) is complex (see 14, 20, 41). It seemsto involve multiple cooperative intractions withthe substrate (30) at hydrophobic loci (20, 30, 34,70, 72) and at its two functional COOH groups(41), one of which is aspartic acid (7). It showsrelatively broad side chain specificity (20, 30).After the substrate is bound to the enzyme, bothCOOH groups function in splitting a single pep-tide bond in the substrate, and an amino enzyme(23, 41, 52) is formed. The addition of water re-sults in hydrolysis, but the addition of anotheramino acid NH2 group results in transpeptidation(23, 41, 52). These data are consistent with ahypothesis that a tetrahedral intermediate isformed which undergoes a reversible four-centerexchange reaction leading to the formation of theamino enzyme (14, 20, 41). Alternative hy-potheses exist, however (20).The active site of pepsin, therefore, contains

amino acids with hydrophobic side chains and atleast two with a COOH in the side chain. Theseamino acids would occupy an area in the enzyme

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molecule several peptide bonds in length (seereference 62).

Pepstatin is a pentapeptide with two -y-NHlinkages, two,-OH groups, and five branched3- or 4-carbon aliphatic side chains (50). Thispentapeptide could probably interact over theentire length of the active site of pepsin, whereassmaller inhibitors could not. Pepstatin's multiplelong aliphatic side chains would be tightly boundto the enzyme's hydrophobic region (43), and oneor both of its OH groups could form hydrogenbonds with the COOH groups of pepsin (2, 6,cf. 43). Pepstatin's gamma peptide linkages mayprotect the inhibitor from hydrolysis, but itsterminal COOH group does not seem to be im-portant (2).

Pepstatin binds tightly and pseudo-irreversibly(6, 43) to pepsin and cathepsin D in equimolarconcentrations (2, 6, 43, 78) with a dissociationconstant of less than 10-8 (2, 6, 43). Inhibitors ofsmaller size than Pepstatin are not as effective,apparently because they do not bind as firmly tothe enzyme's active site.The following "acid" proteinases are inhibited

by Pepstatin: pepsin (2, 6, 73), gastricsin (2),rennin (6), fungal proteinases (2), cathepsin Efrom rabbit bone marrow (6), and cathepsin Dpreparations from pig (33), rabbit (6), anid humanliver (6) and from bovine uterus (78). Pepstatinhas little or no effect on trypsin, chymotrypsin,papain, plasmin, thrombin, kallikrein, and certainother enzymes (6, 69). However, both in vitro andafter an intravenous injection, Pepstatin inhibitsrenin (25, 49), a "neutral" protease acting oInangiotensinogen.

Pepstatin's effect on chronic inflamma-tion. Pepstatin did not reduce the chronic in-flammation caused by the tubercle bacillus in ourtwo pilot experiments. Pepstatin may not havebeen effective in these in vivo experiments, be-cause it failed to enter macrophages or theirlysosomes, because it was rapidly destroyed, ex-creted, or inactivated (see 25 and 49), or becauseof reasons as yet unknown. Perhaps an analogueof Pepstatin would be more effective.The role of the proteinase cathepsin D in

chronic inflammation is unknown. That splitprotein products cause vasodilation and exuda-tion has been postulated ever since Menkin (48)described "leukotaxin." Current opinion incrimi-nates peptides of 8 to 15 amino acids in thesephenomena (66). Lung and macrophage pro-teinase act on the C5 component of complementto produce the chemotactic factor C5a (64).Cathepsin D may therefore increase the numberof cells (and their proteases) at the site of chronicinflammation. It can digest cartilage in vitro, andthis digestion is inhibited by Pepstatin (16, 78).

Thus, cathepsin D probably plays a role inrheumatoid arthritis, and a Pepstatin-like drugthat was effective in vivo might benefit patientswith this disease.Uses of Pepstatin yet to be evaluated. In

chronic inflammatory conditions, macrophageinfiltration frequently precedes fibroblast infiltra-tion and subsequent fibrosis. It is quite possiblethat their cathepsin D-like proteinase is a medi-ator of the fibrotic response. Thus the effect ofdrugs like Pepstatin onl the development ofpulmonary fibrosis and on the fibrosis associatedwith arthritis should be tested experimentally.Another possible use of such agents might be

the prevention of liquefaction of the caseous focusin tuberculosis, which may be caused by proteo-lytic enzymes. Liquefaction results in tremendousextracellular multiplication of tubercle bacilli withfrequent development of antibiotic-resistantmutants and spread of the disease throughout thebronchial tree and to other people (46, 47, 60).Any drug that could prevent or control liquefac-tioin would certainly be of great use, as liquefac-tion is a major factor in the perpetuation oftuberculosis in mankind. Preliminary experi-ments by Y. Yamamura, Osaka University,Osaka, Japan, have shown that Pepstatin par-tially inhibited this liquefaction process in rabbits(cf. 80).Macrophages seem to regulate the amount of

antigen presented to lymphocytes for the immuneresponse. This regulation involves the hydrolysisof "excess" amounts of antigen by enzymes inmacrophage lysosomes. Macrophage proteinaseinhibitors could decrease the hydrolysis of proteinantigens, so that more could be presented tolymphocytes. Depending on the antigenic dosepresented, the resulting immune response wouldbe enhanced or depressed. Thus proteinase inhibi-tors like Pepstatin (if they were effective in vivo)may find clinical use in the fields where an in-creased or decreased immune response would bedesirable, namely those of infectious disease,cancer, organ transplantation, arthritis, autoim-munity, and allergy.

ACKNOWLEDGMENTSThis investigation was supported by Public Health

Service grant no. AI-08876 from the United States-Japan Cooperative Medical Science Program of theNational Institute of Allergy and Infectious Diseases,Public Health Service grant no. HE-14153 from theNational Heart and Lung Institute for the JohnsHopkins Specialized Research Center on Lung, andby Contract DADA17-72-C-2187 with the ArmyMedical Research and Development Command.

S. Y. Wang, Professor of Biochemistry, JohnsHopkins School of Hygiene, provided the names ofmany of the compounds evaluated for this report.B. S. Schoenberg, W. M. Maniscalco, and R. L.Kamenetz performed some of the enzyme assays.

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D. P. Jacobus of Merck and Co., and W. R. Rothe,T. R. Sweeney, and J. A. Schafer of Walter ReedArmy Research Institute supplied us with many po-tential inhibitors for testing. S. Itakura of BanyuPharmaceutical Co., Ltd., supplied Pepstatin andSodium Pepstatin. J. R. J. Sorenson, University ofCincinnati, supplied the copper chelates. J. W. Cusic,C. A. Dornfeld, R. H. Mazur, M. J. Kalm, C. W.Marshall, R. C. Tweit, H. S. Lowrie, P. K. Yonan,J. M. Schlatter, E. F. LeVon, W. E. Coyne, A. H.Goldkamp, C. P. Krimmel, D. A. Jones, and S. Eichsynthesized many of the compounds supplied by G. D.Searle and Co. M. Ando, D. B. Dillman, A. E. Corrin,and A. M. Dannenberg, Jr., performed the pilotexperiments evaluating the effect of Pepstatin onchronic inflammation.

ADDENDUM

Due to a clerical error, our abstract, quoted byBarrett and Dingle (6), entitled "Inhibition ofthe Cathepsin D Type Proteinase of Macrophagesby Pepstatin, a Specific Pepsin Inhibitor by ArthurM. Dannenberg, Jr., Martha H. McAdoo, andStephen P. James, never appeared in FederationProceedings, vol. 31, 1972. The present manuscriptis the first publication from our laboratory onPepstatin.

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