11/7/2023

Therapeutic Enzymes
BIOCHEM-700

Muhammad Hamza Muneer

Therapeutic Enzymes
• Therapeutic enzymes are the biocatalysts used to treat such medical
conditions and diseases.
• 1st time used in the 19th century as therapeutic agents.
• several types of therapeutic enzymes used in the pharmaceutical
industry such as
• L-asparaginase, streptokinase, collagenase, ribonuclease, uricase, glucosidase,
• WHY????------Therapeutic enzymes possess the added advantage of
binding specifically to their respective targets which sets them apart
from non-enzymatic drugs

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Sources of Enzymes
• Therapeutic enzymes are used as antimicrobials, and anticoagulants
and in mucolytics, fibrinolytic, oncolytic, and metabolic storage
disorders.
• There are various wellsprings of remedial enzymes, for example,
animals, plants, and microbes (bacteria, fungi, yeast, etc.), which
have distinct advantages of being used to produce the enzymes,
majorly being economically feasible and consistent.
• Production and Purification: Several approaches like response
surface methodology, optimization of fermentation conditions, and
immunochemistry methods along with different chromatography
techniques have been used for the production and purification of the
therapeutic enzymes.

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Conc…

• Animal Source
• Trypsin obtained from OX bile
• Chymotrypsin for OX bile and pancreas (anti-inflammatory)
• Lipase from the pancreas (decrease the circulating immune complexex)
• Urokinase (activates plasminogen) from Human Urine
• Lysozyme form Hen egg
• Pepsin from Hog Pancreas
• Altepase (expensive) from human tissue culture

Conc…

• Plant Source
• Papain from Papaya
• Bromelain from Pineapple
• Amylase from Barley
• Nattokinase from Natto (cheese-like Japanese food). Produces by adding
basillus to fermented soybeans. Function as enhance plasmin production and
dissolve fibrin directly.
• Superoxide dismutase from Melon and green vegetables. Functions as reduce
wrinkles and cancer treatment and lung problems

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• Microbial Source
• Streptokinase (dissolve blood clots) form beta-hemolytic Streptococci
• Glutaminase from E.coli
• Amylase from Bacillus
• Lipase from Aspergillus

• Microbes are the preferred source because

• Cheaper to produce
• plants and animals contain more harmful material than microbes.

Four major therapeutic enzymes

• Collagenase,
• L-asparaginase,
• Streptokinase,
• Uricase

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Collagenase
• Collagenase was first discovered by Mandi, Seifter, Harper, Bond, Van Wart, and
their team in 1950.
• The first collagenase was isolated and produced from Clostridium histolyticum.
Also Present in tadpoles, bacteria, marine organisms, amphibians, and mammals
• Collagenases are widely found in the skin, teeth, bone, blood vessels, and tendons.
• collagenase is a Zn2+-dependent matrix metalloproteinase (MMP) and has 4
different types.
• Each of them has a different substrate to act on and the absence or non-
functionality of these may play a crucial role in various diseases
• Responsible for breaking the peptide bonds in collagen (a key part of the
extracellular matrix (ECM))
• It is mainly extracted under basic and acidic conditions from
• microorganisms such as Vibrio, Clostridium, Streptomyces, and Pseudomonas;
• fungi such as Penicillium, Aspergillus, Cladosporium, and Alternaria
• human gingival fibroblast and ligament fibroblast

Conc…

Therapeutic applications of collagenase

• Collagenases have been used as a therapeutic enzyme and has found
various applications in curing many diseases and medical conditions:
• Dupuytren’s disease (also known as Viking's disease)
• Glaucoma disease,
• Repair cartilage,
• Keloid disease,
• Cellulite treatment, etc.
• These diseases mostly occur either due to the overproduction or the
low production of collagenase in the body

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• Various MMPs have been long linked to cancer progression, which

majorly includes gelatinases. However, MMP-8 (collagenase-II) was
shown to have the opposite effect, that is anti-tumorigenic activity.
In 2003, it was first observed in a mouse model that the loss of MMP-
8 leads to an increase in skin cancer. Site-directed Mutagenesis
(Ile259Leu and Ile270Ser) of MMP-8 can retain its tumor-suppressive
activity.
• MMP-13 (collagenase-III) is involved in Bone metabolism. Estrogen
hormone down-regulated the expression of MMP-13 and its protein
level increased remarkedly, in the absence of it.

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Side effects
• Various side effects were observed in patients, after the collagenase
treatment. Some of this included
• Numbness,
• Itching,
• Dizziness,
• Peeling

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L-asparaginase
• L-asparaginase (ASNase) is a well-known therapeutic enzyme and an
Amidohydrolase.
• It catalyzes the hydrolysis of both the asparagine and glutamine.
• L-asparaginase possesses anti-cancer properties and is utilized in the
treatment of critical lymphoblastic leukemia.
• Various bacteria such as Pseudomonas aeruginosa, Enterobacter
aerogenes, Zymomonas mobilis, Corynebacterium glutamicum, Erwinia
chrysanthemi, etc., are known to produce L-asparaginase.
• E. coli are used on an industrial scale for the production of L-asparaginase

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Mechanism of Action
• The enzyme helps in the
deamination of L-asparagine to
L-aspartate (with the release of
ammonia) which eventually
leads to the death of the tumor
cells, due to nutritional
deprivation of the amino acid,
asparagine

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Conc…

• L-asparaginase enzyme has varied applications and is a single molecule for

the treatment of multiple diseases.
• This enzyme has been long known for its chemotherapeutic application
(treatment of cancer).
• It is used for the treatment of lymphosarcoma, acute myelomonocytic
leukemia, critical lymphoblastic leukemia, myelogenous leukemia, Hodgkin
disease, chronic lymphocytic leukemia, etc.
• Apart from this, it is known to treat infectious diseases caused by
pathogens like Streptococcus pyrogens, Staphylococcus aureus, Listeria
monocytogenes, and Clostridium botulinum.
• It is also known for its immunosuppressive and anti-inflammatory
properties as well as for its use as veterinary medicine.

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Side Effects
• The ASNase treatment is accompanied by several side effects, including
immune reactions, severe toxicity, etc.
• SDM (site-directed mutagenesis), has been used to develop ASNase with
better properties.
• SDM is an invitro-mutant development technique by causes mutation into
a target DNA sequence.
• This technique is used to alter the catalytic domains of the enzyme so that the role
of these domains on enzyme activity can be determined to find out the active site of
a therapeutic enzyme.
• SDM has also been used to alter residues (N24S) of the cleavage site of the
asparaginase enzyme, which otherwise reduced the in-vivo life of the
enzyme due to the action of the endopeptidases and proteases such as
cathepsin B.
• This cleavage-resistant form of the enzyme hence has a higher potential of
increased in-vivo half-life and can be used as a potent treatment option.

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Conc…

Recent Discoveries
• Novel L-asparaginase obtained from Bacillus licheniformis
• Possesses the best biochemical and biophysical properties. It also proved
to have high potential as an anti-cancer drug against various cancer cell
lines
• Studies are being carried out on two novel forms of this enzyme:
Calaspargase Pegol. And PEG-crisantaspase (Asparec). These act as 2nd
generation drugs.
• A year later, the second and third phase clinical trials of Asparec had also
begun. In 2018, Calaspargase Pegol was approved by the FDA and was
studied to provide advantages over the other pegaspargase products
because of the longer interval between doses of administration

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Streptokinase (SK)
• Streptokinase (SK) is a therapeutic enzyme, which in simple terms, is essential to
break down blood clots. It is a thrombolytic enzyme with a molar mass of 47 kDa
(415 amino acid residues) which shows a maximum activity at a pH of 7.5
(isoelectric pH is 4.7)
• It is a single-chain polypeptide, which forms a complex with the plasminogen
activator, which in turn converts the precursor (plasminogen) to plasmin. Plasmin
further degrades the fibrin (fibrinolysis)
• Streptokinase is preferred over other thrombolytic enzymes like tissue
plasminogen activator and urokinase, due to its low cost and longer in-vivo life.
• It is used in the treatment of various cardiac and thrombolytic-associated
diseases.
• It is isolated using various bacterial, yeast, and mammalian (CHO cells) systems

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Conc…

Application
• Streptokinase has various applications in therapeutics.
• Some of these are the management and medication for medical
conditions like acute myocardial infarction in adult patients, deep
vein, and arterial thrombosis, and occluded arteriovenous cannulas.
• It is majorly a part of a group of medications known as fibrinolytic
(blood clot lysis).
• Streptokinase as a fibrinolytic agent for the treatment of acute
myocardial infarction.

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Problems with SK
• One of the major problems with the enzyme, was the short in vivo half-
life, due to the action of plasmin on it, thus, breaking it into smaller
intermediates.
• Plasmin is a serine protease, which cleaves the peptide bond after specific amino
residues such as lysine or arginine
• When PEG (Polyethylene Glycol) was attached to the enzyme, there was no
evidence of any catabolic activity of the plasmin on the PEG-streptokinase
complex, thus, increasing the half-life of the enzyme and decreasing
immunogenicity.
• Also, plasmin-resistant streptokinase was generated by mutating the
amino acid residues (Lys 386 and Lys59), using the SDM technique.
• The obtained SK variant retained its activity, with the half-life increased to
21-fold.

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Conc…

Uricase
• Urate oxidase (UOX), also known as Uricase is a therapeutic enzyme that is
not endogenously present in humans and was lost because of the
evolutionary process.
• However, the cDNA sequence obtained using the mRNA from the human
liver cells traced back to the presence of the uricase enzyme in humans
• Uricase projects an important form of the therapeutic enzyme since it is
not present or produced in humans
• Utmost importance in the treatment of diseases like Gout, Hyperuricemia,
and Osteoporosis.
• It is isolated from various sources such as Pseudomonas aeruginosa,
Bacillus thermocatenulatus, Bacillus subtilis, Streptomyces exfoliates, E.
coli, etc

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• The UOX has four identical subunits containing four identical active
sites, each containing a type 2 copper-binding site, situated at the
interfaces of the four subunits.
• It metabolizes uric acid to form allantoin and hydrogen peroxide.
(Uric acid (UA) is formed as a by-product of the Protein
metabolic reaction that leads to the breakdown of the purine
nucleotides (Adenine and Guanine).

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Conc…

Applications
• Accumulation of the uric acid crystals (monosodium urate crystals) within joints
often leads to a painful inflammatory condition commonly known as Gout.
• Hyperuricemia and Osteoporosis lead to increased UA in the bloodstream
• The increase in the level of UA in the blood is associated with other disorders like
kidney stones, hypertension, type 2 diabetes, cardiometabolic diseases, Lesch–
Nyhan syndrome.
• Thus, the use of uricase enzyme is considered the most proficient treatment
option for such disorders. Diuretics and hypouricemic agents (Allopurinol, urate
oxidase, etc.) also play a significant role in the treatment of Tumor lysis
syndrome (TLS) which is a life-threatening oncological condition

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Problems
• Various attempts have been made to improve the stability, activity,
solubility, and also reduce the immunogenic action caused by the
treatment associated with the urate oxidase enzyme.
• One of the major engineering strategies used in this process was SDM.
Multiple mutagenesis and screening rounds finally led to the selection of
two mutants that showed higher catalytic activities, lower optimal
reaction temperatures, and higher thermostability
• To develop a more humanized variant of the urate oxidase enzyme, SDM,
and exon replacement/restoration methodologies were used.
• when SDM was used to create the desired mutations (E24D and E83G)
within the chimeric enzyme, A chimeric uricase (porcine-human uricase)
was hence obtained. This chimeric enzyme had more homology to the
human uricase (91.45%) and showed increased UA degradation activity
(141% of porcine-uricase).

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Conc…

Recent advancements
• The use of recombinant PEGylated uricase (sold under the name
Pegloticase and Pegadricase) is considered an efficient treatment
option for the removal of the urate crystal deposits. BUT enzyme was
highly immunogenic and induced an anti-drug antibody (ADA)
production in most of the patients.
• Further, the SEL-212 (Pegadricase + ImmTOR {immunological
tolerance}), in a clinical phase 2 trial, was also observed to diminish
immunogenicity and sustained uric acid levels in Gout patients.

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Limitations
• Many limitations such as
• high cost of production,
• purification
• targeted enzyme delivery,
• elicitation of the immune response against it,
• shorter in-vivo half-life,
• Rapid clearance from Blood
• Should be non-immmunogenic
• Also, industrial production of therapeutic enzymes presents several challenges in terms
of
• search of a new plant,
• mammalian systems, and
• microorganisms
that can be used to produce enzymes in a cost-effective manner and with fewer
adverse effects
• The real challenges on the contrary are better enzyme formulations for suitable
administration.

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Abzymes, also known as catalytic antibodies

• The possibility of catalyzing a reaction by
means of an antibody that binds the
transition state was first suggested by
William P. Jencks.
• Dr. Paul published the first example of
hydrolysis of HIV protein coat by a
catalytic antibody.
• It catalytically destroys the site, rendering
the virus inert, and then can attack other
HIV viruses. A single abzyme molecule
can destroy thousands of HIV viruses.
• This Abzyme degrades the super-antigenic antibodies that possess enzymatic activity. They are
region of the gp120 CD4 binding site. capable of catalyzing chemical reactions, similar to
enzymes.

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