Substrates and inhibitors of proteolytic enzymes

Details Substrates and inhibitors of proteolytic enzymes Substrates and inhibitors of proteolytic enzymes

1999-11-03

2003-03-04

Ponnaluri, Padmashri (Department: 1627)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S004000, C435S007600, C435S007720, C435S091500, C436S172000

Reexamination Certificate

active

06528275

ABSTRACT:

The present invention relates to the field of compounds which are substrates or inhibitors of proteolytic enzymes and to apparatus and methods for identifying substrates or inhibitors for proteolytic enzymes.
Many therapeutically useful drugs act as enzyme inhibitors. In particular, proteolytic enzyme inhibitors have been the focus of much attention in the pharmaceutical industry, because they play a variety of roles in a multitude of biological systems. Their proteolytic activities are related to processes ranging from cell invasion associated with metastatic cancer to evasion of an immune response, as seen in certain parasitic organisms; from nutrition to intracellular signalling to the site-specific proteolysis of viral proteases and eukaryotic hormone-processing enzymes. However, the traditional random screening methods for the identification of lead molecules as inhibitors of proteolytic enzymes are often laborious and time-consuming. Therefore new and efficient methods which can accelerate the drug discovery process are greatly in demand.
We consider that proteases contain an active catalytic site which tends to become increasingly activated as the recognition pockets
1
(S
1
and S
2
etc) and (S
1
′ and S
2
′ etc) become better occupied. Therefore, it is important that those parts (P
1
and P
2
etc) (P
1
′ and P
2
′ etc) of the inhibitor that best fit into these pockets are identified as quickly as possible in order to design novel protease inhibitors. Therefore, we have devised a combinatorial method for the rapid identification of these binding motifs which will greatly expedite the synthesis of inhibitors of a variety of proteolytic enzymes such as aspartyl proteases, serine proteases, metallo proteases and cysteinyl proteases.
Proteases of interest include (but are not limited to):
1. Aspartyl proteases, such as renin, HIV, cathepsin D and cathespin E etc.
2. Metalloproteases, such as ECE, gelatinase A and B, collagenases, stromolysins etc.
3. Cysteinyl proteases, such as apopain, ICI, DerPI, cathepsin B, cathepsin K etc.
4. Serine proteases, such as thrombin, factor VIIa, factor Xa, elastase, trypsin.
5. Threonyl proteases, such as proteasome S.
The use of a fluorescence resonance energy transfer (FRET) substrate for the analysis of proteolytic enzyme specificity was first published by Carmel.
2
Since then many different quenched fluorogenic substrates for measuring enzyme inhibition have been described in the literature.
4-11
These substrates contain a fluorophore, F, in a P position (vide supra), which is quenched by another group, Q, present in a P′ position (vide supra) and separated from F by the scissile bond. The advantage of the positioning of these residues, F and Q, is that cleavage of a peptide bond occurs between the two natural residues and, therefore, represents a more natural hydrolytic event rather than the cleavage and release of a C-terminal chromophore.
For example, Bratovanova and Petkov
12
have synthesised fluorogenic substrates from peptide 4-nitroanilides. N-acylation of peptide 4-nitroanilides with the aminobenzoyl (ABz) group yielded substrates that are internally quenched by the presence of the 4-nitroanilide moiety. Upon hydrolysis of the aminoacyl-4-nitroanilide bond, the highly fluorescent N-ABz group is released attached either to an amino acid or peptide.
Immobilised libraries; where substrates are attached to a polymer or biopolymer support, have also been used for mapping protease binding sites.
13
Singh et al. reported recently that enzymatic substrate activity of 38 selected octapeptides attached via a linker to controlled pore glass is predictive of the same activity of similar peptides in solution. However, these results are preliminary and only for a specific example. Therefore, it is not clear whether immobilised substrates attached to polymers can reliably replace soluble substrates in mapping the hindered protease binding sites, especially since the hydrophilic or lipophilic nature of the polymer and the size of the interstices within the polymer are bound to influence the reaction between the enzyme and its substrates.
Mixtures of internally quenched, fluorogenic substrates have also recently been described in which the quencher group, Q, is 2,4-dinitrophenyl (Dnp) and is attached to the P side of the scissile bond, while the fluorogenic group, is N-methyl anthranilic acid (Nma) and is attached to the P′ side.
14
Examples of other Donor-Acceptor Chromophore Pairs that have been applied to Biological Systems are shown in Table 1.
TABLE 1
Donor-Acceptor Chromophore Pairs That Have Been
Applied To Biological Samples
Donor
Acceptor
Donor
Acceptor
Naphthalene
Dansyl
IAEDANS
TNP-ATP
IANBD
DDPM
&egr;-A
IANBD
IAEDANS
DDPM
NBD
SRH
DNSM
LY
ISA
TNP
IAEDANS
IANBD
Dansyl
ODR
E-A
F
2
DNB
DANZ
IAF
Pyrene
Bimane
FNAI
EITC
ANAI
IPM
NBD
LRH
IAANS
IAF
IAF
EIA
&egr;-A
F
2
DPS
FITC
ENAI
&egr;-A
DDPM
Proflavin
ETSC
IAEDANS
TNP
CPM
TNP-ATP
MNA
DACM
IAEDANS
IAF
PM
NBD
CPM
Fluorescein
FITC
TNP-ATP
IAEDANS
FITC
DANZ
DABM
FITC
TMR
NCP
CPM
IAF
TMR
NAA
DNP
CF
TR
LY
TNP-ATP
CPM
FTS
IAF
diI-C
18
&egr;-A
TNP-ATP
IAF
TMR
CPM
FM
FMA
FMA
LY
EM
PM
DMAMS
FITC
EITC
mBBR
FITC
IAEDANS
DiO-C
14
mBBR
DABM
IAF
ErITC
&egr;-A
NBD
FITC
EM
Pyrene
Coumarin
FITC
ETSC
IPM
FNAI
FITC
ErITC
IAEDANS
DABM
BPE
CYS
ANAI, 2-anthracene N-acetylimidazole; BPE, B-phycoerythrin; CF, carboxyfluorescein succinimidyl ester; CPM, 7-doethylamino-3-(4′maleimidylphenyl)-4-methylcoumarin; CY5,
# carboxymethylindocyanine-N-hydroxysuccinimidyl ester; diI-C
18
, 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine; DiO-C
14
, 3,3′-ditetradecyloxacarbocyanine; DABM, 4-
#dimethylaniniphenylazo-phenyl-4′-maleimide; DACM, (7-(dimethylamino)coumarin-4-yl)-acetyl; DANZ, dansylaziridine; DDPM, N-(4-dimethylamino-3-5-dinitrophenyl)maleimide;
# DACM, di-methylamino-4-maleimidostilbene; DMSM, N-(2,5-dimethoxystiben-4-yl)-maleimide; DNP, 2,4-dinitrophenyl; &egr;-A, 1,N
6
-ethenoadenosine; EIA, 5-(iodoacetetamido)eosin; EITC,
# eosin-5-isothiocyanate; ENAI, eosin N-acetylimidazole; EM, eosin maleimide; ErITC, erythrosin-5′-isothiocyanate; ETSC, eosin thiosemicarbazide; F
2
DPB, 1,5-difluoro-
#2,4′-dinitrobenzene; F
2
DPS, 4,4′-difluro-3,3′dinitropheylsulphone; FITC, fluorescein N-acetylimidazole; FTS, fluorescein thiosemicarbazide; IAANS; 2-((4′-
#iodoacetamido)anilino)naphthalene-6-sulphonic acid; IAEDANS, 5-(2-((iodoacetyl)amino)ethylamino)-naphthlene-1-sulphonic acid; IAF, 5-iodoacetamidofluorescein; IANBD, N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa1,3,diazole; IPM, 3(4-isothiocyanatophenyl)7-diethyl-4-methylcoumarin; ISA, 4-
#(iodoacetamido)salicylic acid; LRH, lissaminerho-2,1,3-benzoxadiazol-4-yl; NCP, N-cyc1ohexyl-N′-(1-pyrenyl)carbodiimide; ODR, octadecylrhodamine; PM, N-(l-pyrene)-
#maleimide; SRH sulphurhodamine; TMR, teramethylrhodamine; TNP, trinitrophenyl; TR, Texas red.
from: Wu, P. and Brand, L. 1994. Anal. Biochem. 218, 1-13.
The specificity of soluble peptide libraries have been determined.
15,16
Berman et al. described
16
an HPLC mass spectrometry technique in which 6 mixtures of 128 peptides were synthesised which were N-terminally labelled with the Dnp group in order to allow UV monitoring on the HPLC. The disadvantage of this approach is that each assay mixture has to be individually analysed, because no fluorogenic substrate is revealed, and that the effective concentration of each separate component is limited by the size of the mixture because of overall solubility factors. Drevin et al.
17
have suggested the use of individually synthesised fluorogenic substrates for the determination of enzyme activity using a chromophore which chelates lanthanide ions. Garmann and Phillips have suggested the use of FRET substrates in which the fluorogenic and quencher moieties are attached via thiol or amino functional groups after the peptide has been synthesised, but this has the disadvantage that they are not in library form and that th

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