Enzymes as therapeutics
© Oldřich Farsa 2012
Hydrolases
Petidases
Fibrinolytics
Plasminogenogen activators
●catalyze cleavage of the endogenous plasminogen under formation of plasmin, a nonspecific
proteinase, which degrades the fibrine matter of the thrombus
t-PA activation
Alteplase
●fibrinolytic – (natural) tissue plasminogen activator (tPA)
●serine protease released from endothelium cells as one- or two-stranded molecule
●binds to fibrin fibers by means of binding sites on Lys and, being bound, activates
plasminogen to fibrin dissolving several hundert times faster than its free form in the blood
circulation
●572 AA
●glycoprotein
●Mr
: 59 050 without sugars attached to
Asn 117, 184 a 448, with sugars cca 65
000
●cleft by plasmin between AA 275 and
276 into chains A and B; they remain
linked by -S-S- bridge between Cys 264
and Cys 395
●both one- and two-stranded have
comparable fibrinolytic activity in vitro
●Alteplasum ad iniectabile EP must
contain at least 60 % of the two-stranded
form
●pI = 7 – 8
●preparation/production:originally isolated
from a human melanoma cell line;
produced by a recombinant technology;
enzyme produced on mammalian cells
has an optimal glycosylation; the onestranded
form is predominantly formed in
the Chinese hamster ovaries cells
Therapeutic usage
Actilyse®
lyophilizate for preparation of the infusion solution
●T1/2
= 5 min
●trombolytic treatment of acute myocardial infarction (heart attack)
●thrombolytic treatment of acute massive lung embolism accompanied with a haemodynamic insta
●fibrinolytic treatment of acute ischemic events (brain strokes)
Reteplase
●recombinant, produced in E. coli
●sequence consists of two shortes fragments of t-PA with 355 AA together (1 – 3 and 176 –
527)
●Mr
∼ 39 000
●removed 3 important domains: N-terminal „finger“, which is partly responsible for high
affinity to, EGF (epidermal growth factor) homologous domain and „kringle* 1“ domain
●
this + glycosylaton decrese ⇒ different (more suitable) both pharmacodymamic and
pharmacokinetic properties were expected
●T1/2
= 14 min
●greater fibrinolytic activity
●effectivities in coronary vessels passabling (reopening) and in lowering of mortality due to
heart attack are, however, comparable to those of alteplase
Rapilysin®
powder for preparation of solution or pre-filled syringe
Therapeutic indications
●thrombolytic therapy of a suspected acute myocardial infarction with persistent
ST elevation or recent left Bundle Branch Block within 12 hours after the onset of acute
myocardial infarction symptoms
* shipmaster´s rope loop
Tenecteplase
●glycoprotein produced by a recombinant technology in Chinese hamster ovaries cell lines
●(T = Thr, N = Asn, K = Lys; an analogue of alteplase, in which Thr103 was changed with
Asn, which gave a new glycosylation site, Asn117 was replaced with Gln, which removed the
mannose glycosylation site, the sequence Lys296-His-Arg-Arg299 was replaced with 4 Ala,
which increased both activity and resistance against plasmin activator inhibitor 1 (PAI-1)
●T1/2
= 20 – 24 min
Metalyse®
powder for preparation of injection solutions of 6, 8, 10 .103
i. u.
Therapeutic indications
●in adults for the thrombolytic treatment of suspected myocardial infarction with persistent ST
elevation or recent left Bundle Branch Block within 6 hours after the onset of acute
myocardial infarction (AMI) symptoms
●administration as a a single intravenous bolus over approximately 10 seconds
Desmoteplase
●syn. bat PA, vPA, DSPAα1
●saliva of the vampyre Desmodus rotundus contain contain 4 different plasminogen
activators (desmodus salivary plasminogen activators = DPSAs)
●the most important DSPAα1
= desmoteplase; the longest, the greatest (85%) homology
with human t-PA
●477 AA
●vPA has the finger domain, EGF domain and one kringle domain, lacks the second kringle
as well as the cleavage site needed two-stranded PA formation ⇒ the only PA, which
exists exclusively as one-stranded molecule with full catalytic activity
●T1/2
= 2,8 h
●prepared by recombinant technology
●clinical tests of phases 2 and 3 for acute ischemic (brain) stroke in USA, DE ...
Streptokinase
Streptokinasi solutio concentrata EP
●
originally isolated from the filtrate of the culture of β-haemolytic streptococci of the group C
●one-stranded polypeptide, 414 AA, Mr
∼ 47 000
●after binding to human plasminogen (complex 1 : 1), forms a plasminogen activator
●T1/2
= 23 min
●recombinant streptokinase produced in E. coli is currently predominantly used
●maximum activity at pH 7.5; pI = 4.7
●dissolves the fibrinous portion of exsudate also
●the most frequently used PA in Europe
Therapeutic indications
Streptase®
Systemic administration
●acute transmural myocardium infarction (not older than 12 hours) with persistent ST-elevation
or simultaneous left bundle block
●deep vein thrombosis not older than 14 days
●acute masive lung embolism
●acute or subacute peripheral arteries thrombosis
●chronic occlusive arterial diseases (not older than 6 weeks)
Local administration
●acute myocardial infarction for re-canalisation of coronary vessels (not older than 12 hours)
●acute, subacute and chronic thrombosis; embolisms of peripheral veins and arteries
Anistreplase
●syn. APSAC (Acylated plasminogen – streptokinase activator complex)
●a complex of streptokinase and the active site of human lysine plasminogen, in which, the
active proteolytic sites were inactivated by acylation – p-anisoic acid residues introduction
●Mr
∼ 131 000
●does not need circulating plasminogen for its activity
●after injection, the acyl group is slowly hydrolyzed, producing an activator that converts
plasminogen to plasmin, thereby initiating fibrinolysis
●T1/2
= 40 - 90 min
●if the treatment is initiated within 3 hours of onset of symptoms for acute myocardial
infarction, the drug preserves myocardial tissue and left ventricular function and increases
coronary artery patency (throughput)
Urokinase
EC 3.4.21.73
Urokinasum EP
●an enzyme isolated from human urine, which activates plasmonogen
●a mixture of low molecular (Mr
about 33 000) and high molecular (Mr
about 54 000)
forms: the high molecular one predominates in urokinase isolated directly from
kidneys, while the low molecular form in enzyme produced in kidneys cell lines
●recombinant, fully glycosylated form derived from murine cancer cells is also used
●activity at least 70 000 i.u./mg of protein
●T1/2
= 16 min
Rheotromb®
lyophilizate for preparation of the infusion solution, 500 000 i.u. (CZ)
Kinlytic ®
up to 250 000 i.u./vial (USA)
✗both discontinued
Therapeutic indications
Systemic fibrinolysis:
- arterial thrombosis
- massive embolism of pulmonary arteries
- deep venous thrombosis
Local fibrinolysis
- arterial thrombosis
- arterio-venous fistula closure
- Clogged cathteters reopening
Ancrod
EC 3.4.21.74, formerly EC 3.4.21.28
syn. venombin A
●protease isolated from the venom of Malaysian snake Agkistrodon rhodostoma
●glycoprotein
●selektively cleves bonds from/to Arg
●258 AA, Mr
= 29 145
●capable to activate the change of fibrinogen to fibrin (pro-clotting effect), but also fibrin
degradation (fibrinolytic), resulting effect depends on the species of recipient
●phase 3 clinical studies have not shown a significant difference in effect to improve survival
after acute myocardial infarction between ancrod and placebo
Alimentary tract peptidases
Pepsin
EC 3.4.23.1
●fundamental acid protease of stomach
●endopeptidase with wide substrate specifity
●
1st
discovered enzym at all – 18th
century, name given by T. Schwann 1825; 2nd
enzym
isolated in crystalline form (after urease – Northtrop 1930)
●
for distinguishing from minority gastric proteases: pepsin A (× pepsin B; pepsin C =
gastricsin)
●1 chain, 326 AA, Mr
∼ 34 600, acive sites formed by Asp32 and Asp215 (porcine)
●catalyses hydrolysis in pH range 1 – 6, optimum activity at pH about 3.5
●pKa
values in the active sitev 1.57 a 5.02
Quarternary structure of porcine
pepsin with marked N- and Cterminal
lobes; „random coils“ are
drawn by thin line, a substrate
beeing cleft is marked between
lobes
A (top): arrangement of the active site of pepsin: O
atoms are represented by bigger balls, H-bridges
are dashed, the biggest ball represnts water
molecule taking part in the nucleophilic attack (or
its O in particular)
B (bottom): proposed catalytic mechanism of
pepsin and other aspartate proteases: substrate
(top molecule) is oriented by the pair Phe-Phe to
the active site.
(a) -COOH of Asp32 is partially protonated, -COOH
of Asp215 is partially dissociated
(b) Molecule of H2
O is bound to (initialy) C=O of P1
– intemediate of hydrolysis with sp3
carbon
(c) Asp215 supplies H+
to N of substrate
(d) cleavage of the peptide bond
●pepsin is capable to cleave also Phe-Pro bond (in contrast to other peptidases)
●classical assay (of content/pepsine activity in given protein – Anson 1938): hydrolysism of
bovine haemoglobin at pH 2, adition of trichloroacetic acid, absorbance measuremet at
280 nm (in EP modified)
●roundworm Ascaris suum produces a specific peptide pepsin inhibitor (149 AA)
●Pepsini pulvis EP: from gastric wall of pigs, cattle or sheep
Pancreatic peptidase
●peptidases mixture used in drugs for support of digestion as a substitute for endogenous
peptidases : Gastrix ®
, Kreon ®
, Panzytrat ®
...
●a component of Pancreatinum = pancreatine [USAN] – a mixture of digestion enzymes
acquired from porcine pancreases (+ lipase, amylase)
Trypsin
EC 3.4.21.4
●1st
reported 1876 by Kühne as a proteolytic activity of pancreatic secretion; he distinguished
its activity from that of pepsin by higher optimal pH value
●name trypsin was later associated with cleavage of bonds LysCO-NH- , .
ArgCO-NH-.
●isolated in crystalline form 1931
●released as trypsinogen, activated
●
bonds to Lys and Arg are cleft 105
times more readily than to other natural AA, to Arg 2 –
5times readily than to Lys
●some AA in specific positions can inhibit its activity: Arg, Leu, Ile, Lys, Phe in P2 inhibits the
activity 2 – 16times, Pro in P3 lowers the activity 3 – 9times
●stable as the lyophilized powder or in a solution of pH 3, where it is inactive
●primary structures differ markedly from species to species, tertiary ones are very similar
●
one-chain peptide, in which AA residues with catalytic activity are linked with β-helix („βbarrel“)
sequences.
Chymotrypsin
EC 3.4.21.1
●minority constituent of pancreatic peptidase
●in < 50 % homologous with trypsin
●different substrate specifity: hydrophobic AA residues: PheCO-NH-, TrpCO-NH-,
TyrCO-NH●„catalytic
triad“: His57, Asp102 a Ser195
●released as chymotrypsinogen, activated
●A and B, 80% homology
●preparation: acid extraction, precipitation by (NH4
)2
SO4
, crystallisation (1955);
precipitation with acetone, chromatography on ion exchangers – possibility of
separation of A and B (1970); afinity chromatography (1996); gene transfer into a yeast
cell, cloning, recombinant chymotrypsinogen production (1996)
Tertiary structure of chymotrypsin. A: N-terminal β-helix (blue) is situated above C-terminal β-helix (red),
between them the active site. Zymogen-activating peptide (yellow) and C-terminal α− helix (green) stabilize
the entire strukture and active sites. Outer sites, active sites and Na+
binding site are spread on the whole
sufrace and are not in direct touch. B: Schematic of tertiary structure. C: Topology of chymotrypsin structure.
D: On an enzyme model oriented by the active site to a viewer, 8 loops, potential sites of interactions with
macromolecular substrates and inhibitors, are visible. E: Tertiary structure symetry. The terminus of every o βhelix
forms 3 loops on the side of the active site, and bents the back side of the molecule by the additional
two ones.
:
Glycosylases
Glycosidases, ie. enzymes hydrolyzing O- or S-glycosides
α-Amylase
syn. α-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1
●
catalyzes hydrolysis of α-(1,4)-glycoside bonds of components of starch, glycogen and various
oligosaccharides
●contained in salivary glands and pancreas secretions of mammals
●tertiary structure and ability of interactions with saccharides and inhibitors of protein type are
very similar in human and porcine amylase
●
endo-amylase: hydrolyzes internal α-(1,4)-glycoside bonds of amylose and amylopectin, which
leads to stepwise degradation of the substrate up to non-reducing terminus
●tertiary structure contains 3 domains: the ctalytic „heart“ domain consisted from (β/α)8
helix,
which contains an expanded loop inserted between the third β-chain and the third α-helix (called
B domain, residues 100 – 169) and C-terminal eigth-helical domain with β-helical arrangement
(domain C, residues 405 – 496)
●parts of A and B domains are involved in the architecture of the three sites with the most
important functions: the active site, Ca2+
binding site and Clbinding
site
O
O
OH
OH
O
OH
O
OH OH
OH
O
OH
OH
OH
OH
n
OH
OH
O
O
OH
O
O
OHOHOH
O
OH
OH
OH
OO
OH
O
O
OH
O
OH
O
OH
OH
O
OH
300 < n < 1000amylose amylopectin fragment
cleaved bond
cleaved bond
α−(1,4)-glycoside bond cleaved by α−amylase
Tertiary structure of α-amylase with bound „pseudotetrasacharide“ inhibitor acarbose
H
NH
H
H
CH3
O
H
O
H
H
OH
O
H
O
H
H
OH
O
OH
H
OH
H
OH
H OH
H
OH
H
OH
H
OH
OH
H
OH
H
OH
H
OH
O-4,6-Dideoxy-4-(((1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)-2-cyclohexen-1-yl)amino)-α-D-
glucopyranosyl-(1-4)-O-α-D-glucopyranosyl-(1-4)-D-glucose
acarbose
Glucobay®
●
inhibitor of α-amylase – a drug for diabetes
Esterhydrolases
Triacylglycerollipase, syn. triacylglycerolhydrolase, EC 3.1.1.3
●human gastric lipase: 379 AA rests, Mr
∼ 45 000
●human pancreatic lipase: 449 AA, Mr
∼ 50 000
● Asn166 has attached an oligosaccharide chain with high occurence of mannose, or some
more complex
●interspecies variability, but high homology: murine lipase sequence is in 92 % and the rat
one in 93 % identical with the sequence of the human enzyme
●released in „ready-made“ form, not as a proenzyme
●functional at pH 4.5 – 9, optimal pH 7.0
●
the active site is not availble in a solution ⇒ hydrolysis runs on inteface water-lipid phase
●function needs binding of colipase, a protein of Mr
∼ 10 000, released from pancreas;
lipids are emulsified by bile acids salts, which avoid adsorption of the enzyme to their
surface, colipase binding facilitates adsorption to the interface water-lipids covered with
bile acids salts (released as procolipase, a peptid from 95 AA, activated by trypsin in the
intestine)
●structure: 2 basic domains: the big N-terminal (1 - 336) and the small C-terminal (337 –
449); the big one belongs among α/β-hydrolase structures and contains the active site;
superficial loop „lid“ domain covers the active site in „closed“ lipase conformation; colipase
binds to the C-terminal domain and pushes hydrophobic tips of its „fingers“ to the opposite
side of the domain, which leads to lid opening; colipase simultaneously forms a large
hydrphobic plane, which interacts with phase interfaces water-lipid
●„catalytic triad“: Glu354, His468, Ser221
●porcine: Mr
also ∼ 50 000 (50 084 exactly), 450 AA rests, glycosylation
„Closed“ (E) and „open“ (E*) lipase
conformation
Quarternary structure of the lipase. Putative NLBD = proposed domain of binding of neutral lipids.
O
O
O O
NO
H
1-[(3S,4S)-3-hexyl-2-oxooxethan-4-yl]-2-(2S)tridecyl-N-formyl-2-amino-4-
methylbutanoate
orlistat
Xenical®
, Alli®
pancreatic lipase inhibitor – peripherally acting anobesic