Inhibition of IgAl Proteinases from Neisseria

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry

Vol. 265, No. 7. Issue of March 5, pp. 3738-3743, 1990

and Molecular Biology. Inc.

Printed in U.S.A.

Inhibition of IgAl Proteinases from Neisseria gonorrhoeae and Hemophilus influenxae by Peptide Prolyl Boronic Acids* (Received for publication,

September 11, 1989)

William W. Bachovchin$§, Andrew G. Plautsll, George R. Flentke$, Mary Lynchll, and Charles A. Kettner 11 From the *Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, the llCentra1 Research and Development Department, E. I. DuPont de Nemours Company, Experimental Station, Wilmington, Delaware 19898. and the VDeoartment of Medicine. Boston, Massachuskts 02111 ’

Division

of Gastroenterology,

Immunity to bacterial infections in man involves many factors, one of which is anti-bacterial antibody in both serum and secretions. IgA is the predominant form of antibody found in secretions bathing mucous membranes and in human milk and is widely regarded to comprise the first line of defense against infection (1, 2). Given this role of IgA in defending the sites of bacterial entry, it is significant that many species of human pathogens produce and release proteinases that cleave, with high specificity, a peptide bond in the hinge region of human IgA. Medically important IgA proteinaseproducing human pathogens include Neisseria gonorrhoeae and Neisseria meningitidis, which cause gonorrhea and meningitis, respectively; Hemophilus influenzae and Streptococcus pneumoniae, which cause bronchitis, pneumonia, otitis media, and meningitis; and Streptococcus sanguis and other Streptococcus and Bacteroides species which have been strongly implicated in periodontal diseases and dental caries (3-6). The IgA proteinase-catalyzed cleavage of IgA separates the Fc from the antigen-binding Fab regions of the molecule. Such cleavage would be expected to impair or abolish its * This work was supported by National Institutes of Health Grants DE 07257 and GM 27927. It was presented in part at the 28th ICARC, Los Angeles, CA, October, 1988. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be-hereb; marked “advertisement” in accordance with 18 USC. Section 1734 solelv to indicate this fact. § To whom correspondence and reprint requests should be addressed. 3738

England

Medical

Center

Hospital,

antimicrobial activity (7). This fact, coupled with the observation that non-IgA proteinase producing species of Neisseria and Hemophilus are not pathogenic, strongly implicates the IgA proteinases as a factor contributing to bacterial virulence. Very recently, Meyer (8) reported that IgA proteinases may also cleave CD8, an important protein antigen found on the surface of certain T-cells. Thus, IgA proteinase-mediated virulence may involve attack on the cellular as well as on the humoral immune system of the host. The IgA proteinases are therefore potentially valuable targets for antimicrobial agents. All IgA proteinases identified thus far cleave after a proline residue within the hinge region of human IgA. Nearly all cleave within a proline-rich 16-amino acid segment formed by duplication of the octapeptide sequence T-P-P-T-P-S-P-S. Proteinases from different sources cleave different prolyl-X bonds within this region as shown in Fig. 1 (3,9-E). Strains of N. gorwrrhoeae, N. meningitidis, and H. influenza.e produce one of two types of proteinases that have been designated type 1 and type 2. The type 2 enzymes cleave after the consecutive prolines in the second half of the repeat, but do not cleave the sequentially equivalent bond in the first half of the repeat. Curiously, other IgA proteinases, those from S. sanguis and S. pneumonia, have the reverse specificity, cleaving the bond following the consecutive prolines in the first, but not in the second, half of the repeat. The type 1 proteinases cleave following a single proline; the N. meningitidis and N. gonorrhoeae enzymes cut the same bond in the second octapeptide while that from H. influenzae cleaves after the last Pro in the first octapeptide (Fig. 1). The fact that each IgA proteinase cleaves in only one of the two available halves of the octapeptide repeat suggests that the sequentially identical octapeptide segments have different conformations or that the proteinases recognize structural elements distant from the cleavage site. A solved structure for human IgAl would of course settle the first conjecture and provide considerable insight regarding the second. Unfortunately, this structure is not yet available. Attempts to gain access to the active sites of IgA proteinases with small synthetic molecules, either as substrates or inhibitors, until now have been unsuccessful. The only known substrates are native macromolecules (i.e. native human IgA; the proenzyme forms of the proteinases from N. gonorrhoeae and H. influenzae, as these undergo autocatalytic processing (16); and perhaps the CD8 antigen on T-cells). The only known inhibitors are peptides sequentially homologous to the IgAl hinge octapeptides, and these are relatively weak inhibitors with I&,,, values in the millimolar range (17). The difficulty in developing synthetic substrates or inhibitors for the IgA proteinases may be partly attributable to the

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The a-aminoboronic acid analog of proline has been synthesized and incorporated into a number of peptides as the COOH-terminal residue. These peptide prolyl boronic acids are potent inhibitors of both the type 1 and type 2 IgA proteinases from Neisseria gonorrhoeae and Hemophilus influenzae, but not of the functionally similar IgA proteinase from Streptococcus sanguis. The best inhibitors synthesized thus far have Ki values in the nanomolar range (4.0 to 60 nM). These results indicate that the N. gonorrhoeae and the H. influenzae enzymes belong to the serine protease family of proteolytic enzymes while that from S. sanguis does not. As a group, the IgA proteinases have been noted for their remarkable specificity; thus, the peptide prolyl boronic acids reported here are the first small synthetic molecules to exhibit a relatively high affinity for the active site of an IgA proteinase and are therefore the first to yield some insight into the active site structure and specificity requirements of these enzymes.

New

of IgA

Inhibition

Proteinases

c. ramosum ( 13 I E.melaninogenicus (14) S.pneumoni‘?e ( 10) S.mitior ( 11) S.sanguis* (3)

I I

H.egypt/us (12) H.inf/uenzae,type 1: (10) N.meningitidis,type 2 (9) N.gonorrhceae,type 2* (3) H.inf/uenzae,type 2* (12) N.meningitidis,type 1 (9) N.gonorrhoeae,type l* (15) I , I I

I I

-C-P-“-P-S-T-P-P-T-P-s-~-s-~-p-p-~-p-s-p-~-c-

220

241

FIG. 1. Hinge region of the proline-rich human IgAl immunoglobulin heavy chain. The duplicated octapeptide is underlined, and arrows show the peptide bond cleaved by each known IgA proteinase. The numbers in parentheses in the figure are references documenting the individual cleavage sites. * indicates those enzymes tested with peptide boronic acid inhibitors.

standard proteinase inhibitors, and recent sequence analysis and hybridization studies of cloned IgA proteinase genes’ (1820) have shown that the type 1 and type 2 enzymes from N. gonorrhoeae and H. influenzae, although closely related to each other, are apparently unrelated to the enzyme from S. sang& Nevertheless, it is still not clear if any of the IgA proteinases

belong

to any

of the

four

known

proteinase

classes

as none of the available IgA proteinase sequences show obvious homology with proteinases of these classes. Thus, the IgA proteinases are either very distantly related to the established classes or they belong to new classes. We report here that peptides containing the a-aminoboronic

acid

analog

of proline

(boroPro)’

as the

COOH-terminal

residue

are potent inhibitors of the IgA proteinases from N. gonorrhoeae and H. influenzae, but not of the proteinase from S. sanguis. These results show that small, synthetic molecules can gain access to the active sites of IgA proteinases. They also strongly suggest that the N. gonorrhoeae and H. influenzae proteinases are serine proteases while that from S. sanguis is not, a finding which confirms the above supposition that the IgA proteinases are not likely to belong to a single protease class. Variation of the inhibition constant with the peptide portion of the boronic acid inhibitors provides insight into the specificity requirements and active site structures of the N. gonorrhoeue and H. influenzae enzymes. MATERIALS

Bacterial 32819 and

AND

METHODS3

Strains and Growth Conditions-N. 740 are clinical isolates yielding

type

gonorrhoeae strains 1 and type 2 IgA

1 J. Gilbert, A. Plaut, and A. Wright, unpublished results. * The prefix “boro” of -boroPro-OH is used to designate the analog of proline in which the -COOH group is replaced by B(OH)?. All natural amino acids are in the L-configuration. H-boroproline is in the DL-configuration. Other abbreviations used are: THF, tetrahydrofuran; H-Pro-OBz, the benzyl ester of proline; H-Thr(OBzl)-OH, the benzyl ether derivative of threonine; Boc, tertiary butyloxycarbonyl group; FABMS, fast atom bombardment mass spectrometry; DFP, diisopropyl fluorophosphate. 3 Portions of this paper (including portions of “Materials and Methods” and a scheme) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

Prolyl

Boronic

Acids

3739

proteinases, respectively. These strains were grown in defined media (21) at 37 “C in 5% CO,, 95% air, and the secreted IgA proteinase activity was harvested from the centrifuged culture supernatant by methods described previously (22). H. inflwnzae strains type b, ATCC 9795 yielding type 1 IgA proteinase, and type c, ATCC 9007 yielding type 2, were obtained from The American Type Culture Collection. Hemophilus strains were cultured at 37 “C in brain heart infusion broth (Difco) enriched with 10 pg/ml NAD and 10 rg/ml hemin in 5% CO,, 95% air. The Hemophilus proteinases were harvested using essentially the same method as described above for the Neisseria enzymes. S. sanguis, a Lancefield Group H strain (ATCC 10556) was cultured at 37 “C in Todd-Hewitt broth (BBL Microbiology Systems) in l-liter stationary flasks in air. IgA proteinase activity was harvested from the supernatant of completed cultures by precipitating the protein with 60% ammonium sulfate. The precipitate was dissolved in Tris-HCl buffer, 0.05 M, pH 8.1, and chromatographed on a BioGel P-100 polyacrylamide molecular sieve column from which the enzyme activity eluted in the void volume. The proteinase was used in this form without further purification. All IgA proteinase preparations used in these experiments fulfilled earlier criteria for specificity by cleaving human IgAl myeloma paraproteins into Fab, and Fc, fragments and by failing to hydrolyze human IgA2. This was verified for each enzyme preparation using analysis of digests by polyacrylamide gel electrophoresis, as previously described (9). Synthesis of the a-Aminoboronic Acid Analog of Proline and of Peptide Prolyl Boronic Acids-We report here the synthesis of the aaminoboronic acid analog of proline (boroPro) and of a series of peptide boronic acids containing boroPro as the COOH-terminal residue. The preparation of these compounds is described and discussed in the Supplementary MateriaLa Other Peptide Boronic Acids-Boc-Ala-Pro-boroVa1 and MeOSucAla-Ala-Pro-boroVa1 were synthesized as previously described (23). Preparation of IgA for Use in Determination of K,,, and Ki VuluesHuman IgA prepared as previously described tended to give kinetics resembling substrate inhibition in double reciprocal plots (upward curvature at high IgA concentrations). This effect was due to the presence of small amounts of some impurity (possibly IgG) acting as an inhibitor of the IgA proteinase. This material and its inhibitory effect is eliminated by further purification of the IgA as described below. One ml of Affi-Gel-Protein A (Bio-Rad) was held in place in a glass transfer pipette with glass wool. The column was flushed with 0.05 M Tris/HCl, pH 7.5, and 13.8 mg of Mor4 IgA was applied at room temperature. Approximately l-ml fractions were collected in 1.5-ml centrifuge tubes. The presence of protein was determined by absorption at 280 nm; fractions were analyzed with Ouchterlony plates using anti-human IgA, anti-human IgG (Atlantic Antibody), and anti-normal human serum (Meloy) antisera. Fractions testing positive for IgA and negative for IgG were combined and concentrated by ultrafiltration using an Amicon PM10 membrane. IgA prepared in this manner gave linear double reciprocal plots in kinetic studies with the S. sanguis, H. influenza 1 and 2, and N. gonorrhoeae 1 enzymes and accordingly was used in K, and Ki determinations involving these enzymes. For use in kinetics studies with the N. gonorrhoeae 1 enzyme, the substrate IgA required still further purification. This involved passing the Affi-Gel-Protein A-purified Mor IgA over a molecular sieving column. A 15-ml Bio-Gel P-300, 100-200 mesh (Bio-Rad) column (1.0 x 20.0 cm), was prepared in 0.05 M Tris/HCl, pH 7.5, and fitted with a Mariott flask at room temperature. The column was run at 8.0 cm/h linear flow rate after application of the human IgA. Fractions were assayed for protein by absorbance at 280 nm and for IgA by Ouchterlony. Fractions containing IgA were combined and concentrated as described above. For kinetic studies, the concentrations of the purified human IgA were determined using the Bio-Rad protein assay. Determination of K, and K, Values-IgA proteinase activity was assayed using Y-labeled (100,000 >400,000 >100,000 28 >100,000

651bOO 80;OO0 18,000 1,300 5,900 a b 3,260 250 7,000 >100,000

12fboo 73,000 4,500 13 30 47,000 b >100,000 50,000 19 25,000

33,000 55,000 7,200 26,000 a a a b a a a a

1.8

0.7

1.5

23

RM

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

>150,000 >180.000 >400;000 3,700 16 63 62,000 b 13.000 3;200 52 b

H-boroPro-OH Ac-boroPro-OH H-Ala-boroPro-OH Boc-Ala-boroPro-OH Ac-Ala-Pro-boroPro-OH Boc-Ala-Pro-boroPro-OH H-Ala-Pro-boroPro-OH Boc-Ala-Pro-boroVai-OH Boc-Pro-Thr-boroPro-OH Boc-Pro-Thr(OBzl)boroPro-OH MeOSuc-Ala-Ala-Pro-horoPro-OH MeOSuc-Ala-Ala-Pro-boroval-OH

-

Human

IeAl

substrate

(K-.

0.59

KM)

’ Indicates no inhibition * Not determined.

at 10m3 M or less.

>180,000 18.000 14;ooo 1,200


cpm Fd X 100 Fd + cpm heavy chain

RESULTS

Boronic

Ac-Ala-Pro-boroPro-OH and Boc-Ala-Pro-boroPro-OH, and the tetrapeptide, MeOSuc-Ala-Ala-Pro-boroPro-OH, are potent inhibitors of both the type 1 and type 2 enzymes from N. gonorrhoeae and of the type 2 enzyme from H. influenzae, having K, values in the nanomolar range (4-63 nM for the triand 19-52 nM for the tetrapeptide derivatives). These peptide boronic acids are somewhat less effective against the type 1 enzyme from H. influenzae with K, values only in the micromolar range, although these nevertheless still represent the most effective inhibitors of the type 1 H. influenzae available. The effectiveness of these tri- and tetrapeptide prolyl boronic acid as inhibitors of the IgA proteinases is about the same as that of other similar sized peptide boronic acids for their target enzymes. For example, &tic protease, a serine protease having a P1 specificity for alanine is inhibited by the tetrapeptide boronic acid MeOSuc-Ala-Ala-Pro-boroAla-OH with a K, of 67 nM and by the tripeptide boronic acid BocAla-Pro-boroval-OH of 0.3 nM (23, 25). The high affinity of the peptide prolyl boronic acids for the type 1 and type 2 Neisseria and Hemophilus enzymes indicates they are acting as transition state analog inhibitors of these enzymes. This in turn strongly suggests that these enzymes are serine proteinases because peptide boronic acids are much less effective against the other classes of proteases. The lack of inhibitory effect on the S. sanguis enzyme (Table I) likewise suggests that this enzyme is not a serine protease. In light of these results, and of the present uncertainty regarding classification of the IgA proteinases, we tested their susceptibility to inactivation by DFP. Under the assay conditions described above, 4 x 10m4M DFP was sufficient to fully inactivate both the type 1 and type 2 Neisseria and Hemophilus enzymes while it had no measurable effect on the S. sanguis enzyme. These results further support the above suppositions that the type 1 and type 2 Neisseria and Hemophilus enzymes, but not the S. sanguis enzyme, belong to the serine protease family of proteolytic enzymes. The amino acid sequence of the type 2 IgA proteinase from Neisseria has been reported (16), and it does not show obvious homology with either the trypsin or subtilisin families of serine proteases and thus does not offer strong confirmation of our conclusion that the Neisseria and Hemophilus enzymes are serine proteases. However, the Neisseriu sequence does show a g-amino acid segment that exhibits significant homology with a sequence that is highly conserved among the

esters to the free acids (23). Stock inhibitor solution of varying concentrations were prepared by serial dilution of the preactivated boronic acids into Tris-HC1 buffer, 0.05 M, pH 7.5. Unused solutions of inhibitor were stored at -70°C. In general, the reaction mixtures were 75 ~1 in total volume and consisted of 25 11 of the stock IgAl substrate solution, 25 ~1 of a stock inhibitor solution or buffer (as control), and 25 ~1 of one of the partially purified IgA proteinase preparations. The enzyme and inhibitor were preincubated for 30 min at 37 “C, and the reaction was started by the addition of the substrate. lo-~1 aliquots were removed from the reaction mixture at lo-min intervals over a IO-min period to assay for hydrolysis of IgAl. The aliquot was added to 100 ~1 of sample buffer and boiled 5 min. Sample buffer contained 12.5% glycerol, 1.2% sodium dodecyl sulfate, 1.2% fl-mercaptoethanol, and 0.001% bromphenol blue. Electrophoresis was carried out on 9% polyacrylamide gel electrophoresis gels. The gels were stained with 0.05% Coomassie Brilliant Blue, dried, and autoradiographed at -70 “C using Kodak XAR5 film with an intensifying screen. The developed autoradiograph was used as a template for cutting the dried gel into segments consisting of residual uncleaved IgA heavy chain and Fd (the (Y chain portion of the Fab fragment digestion product). Radioactivity in the uncleaved heavy chain and in the Fd product were measured on a Beckman Biogamma Counter after which the background counts from control digests were subtracted from each value. Quantification of IgA proteinase activity was expressed as percentage heavy chain cleaved, calculated using the formula:

cpm

Prolyl

Inhibition Amino

of IgA Proteinases

TABLE II acid sequences surrounding the active representative serine proteases

site serines

of

*, serine 195; +, serine 251. Protease

Elastase Bacterial trypsin Chymotrypsin (bovine A) CCP,” mouse Protease A, Streptomyces

sequence * -G-C-Q-G-D-S-G-G-P-L-H-C-T-C-Q-G-D-S-G-G-P-M-F-R-S-C-M-G-D-S-G-G-P-L-V-C-S-C-N-G-D-S-G-S-P-L-L-C-A-Q-P-G-D-S-G-G-S-L-F-A-

griseus

V8 protease, S. aureus IgA proteinase, N. gonorrhoeae

-T-T-G-G-N-S-G-S-P-V-F-N-G-V-L-G-D-S-G-S-P-L-F-A+

a Cytotoxic T cell protease.

5 Y. Fishman, A. G. Plaut, and A. Wright, unpublished

results.

Prolyl

Boronic

Acids

3741

improvements in K,. Removing the amino blocking group of 4 to yield 3, as expected, weakens the inhibition. A dipeptide with Pro in PZ instead of an Ala (as in 4) is likely to be substantially better than 4 as an inhibitor, and we are in the process of synthesizing this compound to test this prediction and to quantify it. Lengthening the peptide chain to three amino acids improves the inhibitor dramatically as shown by comparing inhibitors 5 and 6 with 2 and 4. Part of this improvement can probably be assigned to having Pro in P, in addition to lengthening the chain to three amino acids. The relative contributions of Pro in Pp and Ala in P3 cannot at the present time be defined. Why does lengthening inhibitor 2 by 2 amino acid residues to give compound 4 or 5 improve the affinity, against all four proteinases, by -lO,OOO-fold? The potency of boronic acids as inhibitors of serine proteases is widely attributed to the boronyl group’s ability to mimic the transition state and thus to take advantage of the transition state binding energy. In this view, it could be argued that Ac-boroPro-OH (2) is a surprisingly poor inhibitor (having a K; of only -2 X 1O-4 M for the type 2 Neisseria enzyme and even higher K, values for the other proteinases) considering that it incorporates the appropriate structures to make favorable contacts with both the S1 specificity subsite and the crucial transition state binding site. Essentially the same phenomenon has been observed previously with peptide boronic acid inhibitors of other serine proteases and may reflect, as discussed in detail previously, a synergism between the specificity subsites and the transition state binding site (27). Regardless of the explanation of this phenomenon, it is typical of serine proteasepeptide boronic acid interactions and therefore further supports the hypothesis that the type 1 and 2 Nezkeria and Hemophilw enzymes are serine proteases and that the peptide prolyl boronic acids are transition state analog inhibitors of these enzymes. The cleavage sites of the type 1 (after a P-T-P) and type 2 (after a T-P-P) proteinases (Fig. 1) predicts that R-Pro-ThrboroPro-OH derivatives should be more effective against the type 1, while R-Pro-boroPro-OH derivatives should be more effective against the type 2 enzymes. The data in Table I, however, show that the situation is not this simple. The RPro-Thr-boroPro-OH derivatives (9 and 10) are more effective against the type 1 enzymes than against the type 2 enzymes, as expected. However, the R-Pro-boroPro-OH derivatives (5,6, and 1 l), although the most effective inhibitors of the type 2 enzymes, as expected, are also the most effective inhibitors of the type 1 enzyme from Neisseria, having about lOOO-fold higher affinity for this type 1 enzyme than the putative type l-specific R-Pro-Thr-boroPro-OH inhibitors (9 and 10). They are also about equally effective as the type lspecific inhibitors in inhibiting the type 1 enzyme from Hemophilus. This raises the question of why the type 1 enzymes show no propensity for cleaving the hinge after the R-ProPro sequence in addition to cleaving after the R-Pro-Thr-Pro sequence. The answer may lie in the conformation flexibility of the peptides compared to conformation rigidity of sequences in the hinge or in recognition by the proteinases of structural elements distant from the cleavage site. Interestingly, etherification of the Thr of compound 9 (to form 10) substantially improves its affinity for the type 1 enzymes. In fact, inhibitor 10 is the most effective inhibitor of the H. influenzae enzyme and is nearly as effective against this enzyme as the R-Pro-boroPro-OH derivatives are against the type 2 enzymes. This suggests that a glycosylated Thr may be involved in substrate recognition by the type 1 enzymes.


chymotrypsin-trypsin family of serine proteases which include the active site serine (Table II). The 7-amino acid sequence, GDSGGPL, where the S is the active site serine, is nearly invariant among serine protease of the chymotrypsintrypsin family (26). This segment corresponds to Gly-193 to Leu-199 in the chymotrypsin numbering scheme. The type 2 proteinases contain the sequence GDSGSPL, which differs from this highly conserved sequence only in containing Ser in place of Gly at position 197 (252 in the IgA proteinase sequence). Moreover, there exist two other proteases assigned to the trypsin family of proteases which also have Ser at this position, the V8 Staphylococcus aureus and the cytotoxic T cell mouse proteases (Table II). Following Leu-199, the sequences of known serine protease become more variable. However, the Phe and Ala residues found following the putative Leu-199 in the IgAproteinases are also frequently found at positions 200 and 201 in the trypsin family of serine proteases (Table II, see Ref. 26 for a more extensive comparative listing of sequences found around the active site series of serine proteases). Site-specific mutagenesis experiments have recently been carried out in which this Ser in the H. influenzae type 2 proteinase was changed to a Cys. The mutated enzyme was catalytically inactive.5 Thus, the potent inhibition by peptide boronic acids, the above sequence homology, and the Ser-Cys mutagenesis experiment together strongly indicate that the type 1 and 2 Neisseria and Hemophilus IgA proteinases belong to the serine protease family of proteolytic enzymes. Thus, it is highly probable that the peptide boronic acid inhibitors of the IgA proteinases listed in Table I are acting as transition state analog inhibitors, and therefore that the variation in inhibitory constants with the peptide portion of the inhibitor contains valuable information about the structural requirements of the specificity subsites on the aminoterminal side of the cleavage site. The data in Table I show, not unexpectedly, that both the type 1 and type 2 enzymes exhibit a strong preference for proline at P1. Substitution of Ala or Val for Pro at this position abolishes the affinity for both type 1 and type 2 proteinases as shown by comparing compound 8 with 5 and 6, and 11 with 12 in Table I. That H-boroPro-OH, 1, the simplest possible prolyl boronic acid, is not an inhibitor of either the Neisseria or Hemophilus enzymes is readily explained. The unblocked free amino group carries a positive charge and this cannot be accommodated in the active site of these endoproteinases. Acetylating this compound, to yield 2, removes the positive charge and results, as expected, in a substantially improved inhibitor. Adding an amino acid to 2 to yield the dipeptide 4 results in further

by Peptide

3742

Inhibition

of IgA Proteinases by Peptide Prolyl Boronic Acids

REFERENCES 1. Mestecky, J., and McGhee, J. R. (1987) Adu. Zmmunol. 40,153245 2. Underdown, B. J., and Schiff, J. M. (1986) Annu. Reu. Zmmunol. 4,389-417 3. Plaut, A. G., Gilbert, J., Artenstein, M., and Capra, J. D. (1975) Science 190,1103-1105 4. Kilian, M., Mestecky, J., and Schrohenloher, R. E. (1979) Infect. Zmmun. 26, 143-149 5. Plaut, A. G. (1983) Annu. Reu. Microbial. 37,603-622

F’ Br-(CH,)&H-B,

,O-UCHJ, 0-WH,),

6. Mulks, M. H. (1985) in Bacterial Enzymes and Virulence (Holder, I. A., ed) pp. 81-104, CRC Press, Boca Raton, FL 7. Kilian, M., Mestecky, J., and Russell, M. W. (1988) Microbial. Reu. 52, 296-303 8. Pohlner, J., Halter, R., and Meyer, T. F. (1988) in Gonococci and Meningococci (Poolman, J. T., ed) pp. 427-432, Kluwer Academic Publishers Group, Dordrecht, Netherlands 9. Mulks, M. H., Plaut, A. G., Feldman, H. A., and Frangione, B. (1980) J. Exp. Med. 152, 1442-1447 10. Kilian, M., Mestecky, J., Kulhavay, R., Tomana, M., and Butler, W. T. (1980) J. Zmmunol. 124,2596-2600 11. Kilian, M., and Holmgren, K. (1981) Inject. Zmmun. 31,868-873 12. Kilian, M., Thompson, B., Peterson, T. E., and Bleeg, H. (1983) Mol. Zmmunol. 20, 1051-1058 13. Fujiyama, Y., Iwaki, M., Hodohara, K., Hosoda, S., and Kobayashi, K. (1986) Mol. Zmmunol. 23, 147-150 14. Mortensen, S. B., and Kilian, M. (1984) Infect. Zmmun. 45,550557 15. Mulks, M. H., and Knapp, J. S. (1987) Infect. Zmmun. 55, 931936 J., Halter, R., Beyreuther, K., and Meyer, T. F. (1987) 16. Pohlner, Nature 325,458-462 17. Burton, J., Wood, S. G., Lynch, M., and Plaut, A. G. (1988) J. Med. Chem. 31, 1647-1651 18. Gilbert, J. V., Plaut, A. G., Fishman, Y., and Wright, A. (1988) Infect. Zmmun. 56, 1961-1966 F. J., Plaut, A. G., and Wright, A. (1987) J. Bacterial. 19. Grundy, 169,4442-4450 J. M., and Falkow, S. (1984) Inject. Zmmun. 43, lOl20. Koomey, 107 S., and Bartenstein, L. (1980) Can. J. Microbial. 26, 1321. Morse, 20 Enzymol. 165, 117-120 22. Plaut, A. G. (1988) Methods 23. Kettner, C. A., and Shenvi, A. B. (1984) J. Biol. Chem. 259, 15106-15114 24. Plaut, A. G., Gilbert, J. V., Leger, G., and Blumenstein, M. (1985) J. Mol. Zmmunol. 7,821-826 25. Kettner, C. A., Bone, R., Agard, D. A., and Bachovchin, W. W. (1988) Biochemistry 27, 7682-7688 26. Brenner, S. (1988) Nature 334,528-530 W. W., Wong, W. Y. L., Farr-Jones, S., Shenvi, A. 27. Bachovchin, B., and Kettner, C. A. (1988) Biochemistry 27, 7689-7697 D. S., Jesthi, P. K., and Sadhu, vK. M. (1984) Orgon28. Matteson, ometallics 3, 1284-1288


The data in Table I reveal that the type 2 enzymes must have very similar active-site structures because the K, values for these two enzymes, although not identical, are similar and vary in parallel with variations in inhibitor structure. The type 1 enzymes appear to be less similar, but nevertheless must have some structural similarities because their K, values also show some parallel behavior. From this standpoint, the type 1 enzyme from Neisseria appears in some respects to be more closely related to the type 2 enzymes than to the type 1 enzyme from Hemophilus. Bacterial infections of man and other animals are highly species-specific. Although the underlying molecular mechanisms of this phenomenon, and pathogenicity in general, remain for the most part obscured by the complexity of the microbe-host interaction, it is not unlikely that IgA proteinase activity may be involved. If so, the specific inhibition of these bacterial proteinases may at least be sufficient to tilt the complex interaction between microbe and host in favor of the host. Antibiotic resistance among certain IgA proteinaseproducing pathogens such as H. influenzae, N. gonorrhoeae, and S. pneumonia, once thought securely vulnerable to drugs, is increasing. The inhibition of these proteinases offers an alternate approach to antimicrobial therapy which should be less susceptible to the development of such microbial resistance. With the development of the inhibitors reported here, this approach can now be experimentally tested.

Inhibition

of IgA Proteinases

by Peptide Prolyl Boronic Acids
