Inhibitory proteaz bakteryjnych

Inhibitory proteaz bakteryjnych, st. Biotechnologia podręczniki, Materiały - Biotechnologia

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Bacterial Protease Inhibitors
ClaudiuT. Supuran,
1
AndreaScozzafava,
1
BrianW. Clare
2
1
University of Florence, Dipartimento di Chimica, Laboratorio di Chimica Inorganica e Bioinorganica,
Firenze, Italy
2
Department of Chemistry, The University of Western Australia,
Nedlands W.A., Australia
!
Abstract:
Serine-, cysteine-, and metalloproteases are widely spread in many pathogenic bacteria,
where they play critical functions related to colonization and evasion of host immune defenses,
acquisition of nutrients for growth and proliferation, facilitation of dissemination, or tissue
damage during infection. Since all the antibiotics used clinically at the moment share a common
mechanism of action, acting as inhibitors of the bacterial cell wall biosynthesis or affecting
protein synthesis on ribosomes, resistance to these pharmacological agents represents a serious
medical problem, which might be resolved by using new generation of antibiotics, possessing a
different mechanism of action. Bacterial protease inhibitors constitute an interesting such
possibility, due to the fact that many specific as well as ubiquitous proteases have recently been
characterized in some detail in both gram-positive as well as gram-negative pathogens. Few
potent, specific inhibitors for such bacterial proteases have been reported at this moment except
for some signal peptidase, clostripain, Clostridium histolyticum collagenase, botulinum neuro-
toxin, and tetanus neurotoxin inhibitors. No inhibitors of the critically important and ubiquitous
AAA proteases, degP or sortase have been reported, although such compounds would presumably
constitute a new class of highly effective antibiotics. This review presents the state of the art in the
design of such enzyme inhibitors with potential therapeutic applications, as well as recent
advances in the use of some of these proteases in therapy.
2002Wiley Periodicals Inc. Med Res Rev, 22,
No. 4, 329–372, 2002; Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/med.10007
Key words:
AAA protease; antibiotics; anthrax lethal factor; botulinum neurotoxins; Clostridium
collagenase; cysteine protease; degP; metalloprotease; serine protease; sortase; tetanus neurotoxin
1. INTRODUCTION
Proteases (PRs)—also denominated proteinases or peptidases—constitute one of the largest
functional groups of proteins, with more than 560 members actually described.
1
By hydrolyzing one
Correspondence to: ClaudiuT. Supuran, University of Florence, Dipartimento di Chimica, Laboratorio di Chimica Inorganica e Bioi-
norganica, Via della Lastruccia 3, Room188, Polo Scientifico, 50019 Sesto Fiorentino, Firenze, Italy.
E-mail: claudiu.supuran@unifi.it
Contract grant sponsor : Italian CNR^Target Project Biotechnology
Medicinal Research Reviews, Vol. 22, No. 4, 329 ^372, 2002
2002 Wiley Periodicals Inc.
329
 330
*
SUPURAN, SCOZZAFAVA, AND CLARE
of the most important chemical bonds present in biomolecules, i.e., the peptide bond, PRs play
crucial functions in organisms all over the phylogenetic tree, starting from viruses, bacteria,
protozoa, metazoa, or fungi, and ending with plants and animals. Numerous practical applications in
biotechnology of such enzymes, and the understanding that PRs are important targets for the drug
design, ultimately fuelled much research in this field.
1
Whether much progress has been registered
in the design and clinical applications of viral PR inhibitors (mainly those targeted against the
aspartic protease of HIV),
2–4
not the same situation is true for the bacterial proteases.
5,6
PRs are
widespread in all types of bacteria, where they are involved in critical processes such as colonization
and evasion of host immune defenses, acquisition of nutrients for growth and proliferation,
facilitation of dissemination, or tissue damage during infection.
5,6
Even more subtle roles for
bacterial PRs have recently been evidenced in the interaction between host and the invading micro-
organisms: interruption of cascade activation pathways, disruption of cytokine network, excision of
cell surface receptors, and inactivation of host protease inhibitors (PIs).
5–8
Nevertheless, it is
surprising that there is little or no regulation of bacterial PRs by plasma-derived PIs, such as for
instance the serpins (serine protease inhibitors) which are present in relatively high concentrations
in plasma.
5
Even worse for the host is that bacteria developed strategies for neutralizing such
plasma-derived PIs, assuring in this way an efficient attack of the invaded organism.
5–8
Taking into
account all these facts, it is obvious that bacterial PRs may represent very attractive targets for the
development of novel types of antibiotics, since inhibition of such critical enzymes would pre-
sumably lead to the death of the invading pathogen.
5
Till date all the antibiotics used in clinical
practice share a common mechanism of action, acting as inhibitors of the bacterial cell wall bio-
synthesis or affecting protein synthesis on ribosomes and not intervening in more fundamental
metabolic processes of the pathogen. Considering the specific role that bacterial PRs play in such
critical steps for the successful invasion of the host
5,7
and the constant emergence of antibiotic
resistance,
9
it is crucial to develop bacterial PIs as a novel antibiotic class. Mention should be made,
that no drugs belonging to this class of pharmacological agents are available at present for clinical
use, and this review is also meant as to attract attention to this relatively unexplored and potentially
important field, in which some progress has nonetheless been registered ultimately. Thus, many
possible targets for the drug design will be discussed, together with the recent progress achieved in
understanding the PR types present in pathogenic bacteria, as well as the inhibitors for such
enzymes. In this review, the discussion will be restricted to PRs present in Eubacteriae, since the
Archeobacteriae are generally non-pathogenic. Also, PRs of viral, fungal, protozoan, or other para-
sitic origin will be not considered in this review.
Five catalytic types of PRs have been recognized so far, in which serine, threonine, cysteine, or
aspartic groups as well as metal ions play a primary role in catalysis. All these types of enzymes are
present in bacteria.
1
The first three types of PRs are catalytically very different from the aspartic and
metallo-PRs, mainly because the nucleophile of the catalytic site is part of an amino acid in the first
case, whereas it is an activated water molecule for the second group of such enzymes. Thus, acyl
enzyme intermediates are formed only in the reactions of the Ser/Thr/Cys PRs, and only these
peptidases can readily act as transferases.
1
The classification of PRs used in this review is based on that of Rawlings and Barrett,
1,10
in
which the catalytic type of the protein represents the top level in the hierarchical classification.
According to this rule, the PRs can be divided into clans based on three-dimensional protein folding
and into families based on evolutionary relationships of the primary sequence. It must be noted that
in the fore-cited classification, several enzymes in which threonine rather than serine residue forms
the nucleophile critical for catalysis, have been placed in the serine PR section. The terminology
used in describing the specificity of PRs depends on a model, in which the catalytic site is con-
sidered to be flanked on one or both sides by specificity subsites, each being able to accommodate
the side chain of a single amino acid residue, as originally proposed by Berger and Schechter,
11
and adopted thereafter by many researchers.
1
These sites are numbered from the catalytic site, S1,
BACTERIAL PROTEASE INHIBITORS
*
331
S2, ...Sn towards the N-terminus of the substrate, and S1
0
,S2
0
, ...Sn
0
towards the C-terminus. The
residues they accommodate are numbered P1, P2, ... Pn, and P1
0
,P2
0
, ... Pn
0
, respectively, as
follows (the catalytic site of the enzyme is marked ‘‘*’’, and the scissile peptide bond of the substrate
as ‘‘#’’). The same formalism is also valid for enzyme inhibitors that bind to the catalytic site:
Substrate/Inhibitor:
-P3-P2-P1#P1
0
-P2
0
-P3
0
-
Enzyme:
-S3-S2-S1*S1
0
-S2
0
-S3
0
-
For each enzyme with potential therapeutic use per se or for the development of inhibitors
useful as antibiotics, the main characteristics will be mentioned, such as the EC classification (when
available), the catalytic and family type (in the classification of Barrett et al.),
1
the organisms in
which it is present, the preferred scissile bond(s) and eventually the type of in vivo attacked
substrate, relevant for the pathogenic effects of the considered bacterial species. Furthermore, the
latest developments in the design of inhibitors for such PRs will be discussed, as well as possibilities
to consider such inhibitors as leads for the drug design of novel classes of antibiotics.
It must be mentioned that very few aspartic PRs have been isolated up to now in bacteria, and
those described so far seem to be not involved in pathologic processes connected with bacterial
infections.
1
This is the reason why no chapter dedicated to aspartic PRs is included in this review.
2. SERINEPROTEASES
PRs in which the catalytic mechanism depends upon the hydroxyl group of a serine residue, which
acts as the nucleophile attacking peptide bonds belong to the serine PR (SPR) class of enzymes.
1
The catalytic mechanism of serine PRs usually involves, in addition to the serine residue that carries
the nucleophilic attack, a proton donor/general base. In many SPRs, the proton donor is a histidine
residue, and there is a catalytic triad, because a third residue is also required, probably for orien-
tation of the imidazolium ring of the histidine, and this residue may be an aspartate, or another
histidine
1,12
(Fig. 1A). For other SPRs, catalysis is achieved by a catalytic dyad in which a lysine
residue plays the role of proton donor, and a third catalytic residue is not required (Fig. 1B). There
are other SPRs that have a Ser/His catalytic dyad. Finally, for several serine endopeptidases the
N-terminal residue itself is the attacking nucleophile, and they include the threonine-type PRs.
1,12
There are about 40 families of serine- and threonine-PRs, among which enzymes extremely
important for the homeostasis and vital functions of higher vertebrates, such as the digestive
Figure 1. Schematic representation of the serine protease amino acid residues involved in the proteolytic scission. A: Catalytic
triad (chymotrypsin numbering). B: Catalytic dyad (a lysine residue activates the hydroxyl group of the serine residue essential for
catalysis).
332
*
SUPURAN, SCOZZAFAVA, AND CLARE
enzymes trypsin, chymotrypsin, and elastase,
1
the enzymes involved in the blood coagulation
cascade (thrombin, coagulation factors VIIa, IXa, X),
13–16
the kallikreins, involved in blood pres-
sure regulation or smooth muscle relaxation,
17
or tryptase, an enzyme predominantly present in
mast cells, and involved in various normal and pathologic processes,
18
etc. Bacteria seem to possess
all the major types of serine PRs mentioned above (Table 1), and those with potential therapeutic
applications will be discussed in this article. Bacterial SPRs, which are not involved in pathogenesis
(such as subtilisins or proteases isolated from different lactobacilli) will be not included in this
discussion, although such enzymes are important as additives for detergents (subtilisins) or for the
cheese production (lactobacilli proteases).
A. Proteasesof theS1andS2Families
Trypsin-like proteases are widely distributed in streptomycetes (S. griseus, S. erythraeus,
S. exfoliatus, etc.) (Table I), in which they seem to play important roles related to morphological
differentiation.
19,20
Streptomyces trypsins play a major role in the metabolism of mycelial substrate
or nongrowing mycelial proteins for supporting the growth of aerial mycelium on surface cultures,
but the detailed physiological role of these enzymes is not well defined to date.
19
These enzymes
are inhibited either by typical trypsin inhibitors such as DFP (diisopropylfluorophosphate), Ts-Lys-
CH
2
Cl, benzylsulfonyl fluoride, or proteinaceous inhibitors such as leupeptin, soybean trypsin
inhibitor, and aprotinin among others.
19
The X-ray crystal structures of the enzymes isolated from
S. griseus. and S. erythraeus have been reported, being relatively similar to that of the bovine
trypsin, at least for the active site residues involved in the catalysis, but this did not lead to the
development of potent active site directed inhibitors.
19,20
Several other secreted or membrane bound bacterial endopeptidases, namely streptogrisins
(Table I), were isolated from Streptomyces spp. They are chymotrypsin-like PRs with broad
substrate specificity and readily cleaving peptide bonds on the carboxyl side of Phe, Tyr, Leu, and
Met.
21,22
In contrast to a-chymotrypsin, streptogrisin B is not inhibited by Ts-Phe-CH
2
Cl, whereas
the tripeptidyl chloromethane derivative Ac-Gly-Leu-Phe-CH
2
Cl acts as a potent inhibitor.
22
The
enzyme is also inhibited by 4-iodobenzenesulfonyl fluoride, 4-methoxy-3-chloromercuri-benzene-
sulfonyl fluoride, 4-chloromercuribenzenesulfonyl fluoride, Ac-Gly-Leu-Phe-CH
2
Cl, turkey ovo-
mucoid inhibitor OMTKY3, potato inhibitor I, Streptomyces subtilisin inhibitor and eglin c.
22,23
The
gene of streptogrisin B codes for a prepro-enzyme, the pre- and pro-peptides being 38 and 76 amino
acid residues long, respectively.
21–23
As in many other bacterial SPRs, the pre- and pro-regions are
responsible for the enzyme secretion across the membrane and for its correct folding. After
synthesis, the pre-region is cleaved off by a signal peptidase. The peptide bond connecting the
propeptide to the mature enzyme is cleaved by a self-processing event, when a Leu#Ile bond is
cleaved. A large number of mutant streptogrisins possessing different substrate specificities and
thermostabilities have been reported.
24,25
Some of them act as peptide-coupling enzymes, and show
interesting biotechnological applications.
25
The naturally occurring proteinaceous protease in-
hibitor chymostatin forms a hemiacetal adduct with the catalytic residue Ser 195 of streptogrisin
A.
26
The X-ray structure of this adduct has been reported by Delbaere and Brayer,
26
and was sub-
sequently used for the design of low molecular weight aldehyde inhibitors of the type Z-Arg-Leu-
Phe-H (Z
¼
benzyloxycarbonyl)
27
or Ac-Pro-Ala-Pro-Phe-H (Ac
¼
acetyl).
25
None of these inhibi-
tors have application as antibiotics at the moment.
Glutamate-specific SPRs have also been isolated from many bacteria (Table I).
28
The bacterial
glutamyl endopeptidases may be divided into three groups according to the source organisms and
sequence relationships: a staphylococcal group, a Bacillus group and a Streptomyces group.
29,30
Some forms of glutamyl endopeptidase I, especially protease V8, have found extensive use in
the specific fragmentation of proteins prior to amino acid sequencing.
29
Boc-Leu-Glu-CH
2
Cl
(Boc
¼
tert-butoxycarbonyl) is a potent inhibitor of glutamyl endopeptidase I, but no protein
BACTERIAL PROTEASE INHIBITORS
*
333
Table I.
Bacterial Serine and Cysteine Proteases, With Their Preferred Scissile Bond and Organisms From
Which Were Isolated
Preferred scissile bond
EC
Protease
Family
Organism(s)
(P1#P1
0
)
NC
Streptomyces trypsins
S1
Streptomyces spp.
Arg#Xaa; Lys#Xaa
3.4.21.81 Streptogrisin B
S2
Streptomyces spp.
Phe#Xaa; Tyr#Xaa
3.4.21.80 Streptogrisin A
S2
Streptomyces spp.
Phe#Xaa;Tyr#Xaa
3.4.21.19 Glutamyl endopeptidase I
S2 Bacillus subtilis;
Glu#Phe ; Glu#Val
Staphylococcus aureus
3.4.21.82 Glutamyl endopeptidase II
S2
Streptomyces spp.
Glu#Arg; Asp#Arg
NC
Exfoliative toxin A
S2
Staphylococcus aureus
Glu#Xaa
3.4.21.12
a
-Lyticprotease
S2 Achromobacter lyticus
Ala#Ala
Lysobacter enzymogenes
NC
DegP (protease Do)
S2 Bacillus subtilis; Brucella
Val#Xaa; Ile#Xaa
abortus
Campylobacter jejuni;
Chlamydia trachomatis;
Escherichia coli
Mycobacterium lepre;
M. paratuberculosis
Rickettsia typhi;
R. tsutsugamushi
Salmonella typhimurium;
Yersinia enterocolitica
3.4.21.50 Lysyl endopeptidase
S2 Achromobacter lyticus
Lys#Xaa
3.4.21.72 IgA1-specific serine
S6 Haemophilus influenzae
Pro#Ser ; Pro#Thr
endopeptidase
Neisseria gonorrhoeae;
N. meningitidis
NC
C5apeptidase
S8
Streptococcus agalactiae;
His#Lys
S. pyogenes
NC
Dichelobacter (sheepfoot-rot)
S8 Dichelobacter nodosus
Non specific PR
basic serine proteinase
NC
Trepolisin
S8
Treponema denticola
Phe#Xaa
NC
Tripeptidyl-peptidases
S8, S33 Streptomyces spp.
AlaProAla#Xaa
NC
Prolyl tripeptidyl-peptidase
S9
Porphyromonas gingivalis
XaaYaaPro#Xaa
3.4.11.5 Prolyl aminopeptidase
S33
Flavobacterium meningosepticum H
2
N-Pro#Xaa
Mycoplasma genitalium;
Escherichia coli
N. gonorrhoeae,
NC
Streptomyces K15
S11
Streptomyces K15
acyl -
D
-Ala-
D
-Ala#Xaa
D
-Ala-
D
-Ala transpeptidase
3.4.16.4 Streptomyces R61
S12 Streptomyces spp.
acyl -
D
-Ala#
D
-Ala
D
-Ala-
D
-Alacarboxypeptidase
3.4.21.89 Signal peptidase I
S26 E. coli; H. influenzae;
Ala#Lys
M. tuberculosis
Pseudomonas fluorescens;
S. typhimurium
Streptococcus pneumoniae
3.4.21.87 Omptin
S18 E. coli; Yersinia pestis
Arg#Arg; Arg#Lys
Salmonella typhimurium
3.4.21.92 Endopeptidase Clp
S14 E. coli; B. Substilis, Listeria spp Unspecific PR
3.4.22.10 Streptopain
C10 Porphyromonas gingivalis
Phe#Tyr
Streptococcus pyogenes
(continued)
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