THE CONTRIBUTIONS OF HAMAO UMEZAWA TO SCIENCE AND MEDICINE

Dr. Hamao Umezawa's scientific life has concentrated on the secondary
metabolites produced by microorganisms. His prime concern has been to detect
such metabolites through their biological activities, isolate them, determine
their structure, study their mechanism of action and encourage clinical trials
of those having medical significance. This work was started in 1944 and
continues today. In the early stages of the work methodology was relatively
crude. Dr. Umezawa and associates have played an extremely important role in
refining the methodology so that today very sophisticated techniques permit
studies thought impossible just a few years ago. In the course of his work,
Dr. Umezawa has had associated with him a number of young researchers who
received the benefit of his thinking, philosophy and training. Taese men in
turn have made numerous contributions both in academic and industrial research.
Thus, Dr. Umezawa has had a profound effect in the development of microbial
chemistry in Japan and has played a very important role in setting the high
standard of such studies in his native land.

Japan, of course, has had a long tradition in using fermentation processes
for various aspects of its food and drink consumption. In a’sense Dr. Ume zawa
has followed this tradition but his emphasis was to make fermentation of use
in medicine. He started his work with the study of antimicrobial agents produced
by microorganisms. In the course of this work he has discovered almost one
hundred such agents. One of these agents, kanamycin, was found to be useful
in treating human infections caused by a variety of microorganisms and is in

¢

worldwide use today.
As an indication of Dr. Umezawa's degree of penetration into scientific
problems, he studied the very interesting question as to why certain pathogens
‘became resistant to this and other aminoglycoside antibiotics. He and his
group studied in detail the mechanism of such resistance and found that there
were specific inactivating enzymes produced by various organisms which reacted
at certain sites of the molecule. With this information available Dr. Umezawa
studied how such antibiotics could be modified so that they would be resistant
to the inactivating enzyme but still retain activity. This type of work could
be carried out only by a person who had great capability in several fields,
since success in this area involved intricate studies in microbiology, enzymology,
structure determination and organic synthesis. It is indeed rare to find a
person with great competence in such a variety of scientific fields. This
integration of severat disciplines permitted Dr. Umezawa to rationally plan.
the synthesis of an antibiotic which would be active against resistant micro-
organisms. This led to the discovery of dideoxykanamycin B which is now
being used in Japan and is under clinical study in many countries of the
world. This new drug probably is the first example of a useful drug developed
rationally as a result of understanding fundamental biochemical mechanisms.
Dr. Umezawa's interest broadened into the antitumor area and again he
brought to bear his penetrating approach to the problem. Recognizing
specificity with respect to the pharmacological distribution of a potentially
toxic compound, Dr. Umezawa concentrated much work on an agent ,bleomycin,
which showed a differential distribution in certain tissues as well as tumors.
He showed that this distribution resulted in part, at least, from the presence
of inactivating enzymes in certain tissues. The fact that there was accumula-
tion in various tissues led to the hope that such an agent would not show
the typical blood dyscrasias associated with such agents. Clinical studies

of this agent did indeed indicate utility against certain types of human
cancers and the agent is used worldwide today in helping control this disease.

Dr. Umezawa's studies also led to the discovery of an agent very useful
in controlling a disease of rice caused by the organism, Pyricularia oryzae.
This drug is used widely in Asian countries in controlling the disease and
has led to increased productivity of rice. In today's food short world this
contribution takes on increasing importance.

Much of Dr. Umezawa's recent work has concentrated in finding unusual
metabolites which had the properties of inhibiting specific enzyme systems.
Again seeking a rational way to develop new drugs, Dr. Umezawa delved into
the biochemical mechanisms of various disorders and focused his attention on
enzyme systems that may be key steps. He then set up refined methods to
detect such activities and isolate and determine the structure of metabolites
having the activity. As a result of these studies a variety of specific
enzyme inhibitors have been found. A number of these are under current study
clinically and such substances are also extremely useful probes into studying
mechanisms of enzyme action. They also provide useful tools in determining
whether postulated mechanisms of certain disease states are indeed the basic
responsible events.

Dr. Umezawa's work thus represents an attempt to find through a rational
approach agents of clinical importance. This has been the hope of many
scientists working in this area and Dr. Umezawa's findings are a stimulus
for workers to do the same. His success in discovering new useful drugs
sets an example for such an approach which will have a profound and long
lasting effect on future research in the medical area.

A more detailed description of some of Dr. Umezawa's more {important
findings are presented below along with a curriculam vitae, a biography of
Dr. Umezawa's most important publications, a list of new antibiotics discribed
by Dr. Umezawa and a list of structures of enzyme inhibitors discovered by

Dr. Umezawa.
Discovery of Bleomycin and Its Action

*1 . nee
In 1956, H. Umezawa discovered phleomycin ~ which exhibits inhibition
, *
against Ehrlich carcinoma with a high therapeutic index - However, this
*
antibiotic produced irreversible renal toxicity in dogs 3, Therefore, H.

Umezawa continued the study of antibiotics of similar types and discovered

(1) (2)

bleomycin which was separated into individual components by chromatography .

The bleomycins caused hepatotoxicity in dogs but not renal toxicity‘ and

were differentiated from phleomycin by paper chromatography and by stability

in acid and alkaline solutions. Umezawa ‘4?

studied the distribution of
3u-bleomycin in organs of mice, measuring the radioactivity and the antibacterial
activity in organ extracts, and proved that bleomycin is inactivated in organs
and tissues. This inactivation is significantly slower in skin and lung than

in other organs, especially in old mice“) , This inactivation proved to be

due to an enzyme which hydrolyzed the carboxamide bond in the bleomycin

molecule 4») , Bleomycin is effective against squamous cell carcinoma induced

in mouse skin by 20-methylcholanthrene, but not against sarcoma of mouse skin

(4) (4)

induced by the same agent . Umezawa confirmed that the content of the
bleomycin-inactivating enzyme is significantly lower in the former than in

the latter. He purified this enzyme, which hydrolyzes not only the carboxamide

of f ~aminoalanine moiety of bleomycin molecule but also lysinamide,

 

*] Maeda, K.; H. Kosaka, K. Yagishita.& H. Umezawa: A new antibiotic, phleomycin.
J. Antibiotics, 9A, 82 (1956). ¥*2 Umezawa, H.; M. Hori, M. Ishizuka & T.
Takeuchi: Studies on antitumor effect of phleomycin. J. Antibiotics, 15A,

274 (1962). *3 Ishizuka, M.; H. Takayama, T. Takeuchi & H. Umezawa: Studies
on antitumor activity, antimicrobial activity and toxicity of phleomycin.

J. Antibiotics, 19A, 260 (1966).
lysy1-$-naphthylamide and argininyl~{-naphthylamide and which was found to
(6)

be a new aminopeptidase Moreover, 34-bleomycin is distributed in squamous
cell carcinoma in a significantly higher concentration than in sarcoma. Thus,
the selective effect on squamous cell carcinoma was shown to be due to the
low content of the bleomycin-inactivating enzyme and a high concentration of

the antibiotic in this tumor. The data also indicates that initial toxicity

should appear in the skin and lung because of weaker inactivation of bleomycin

(4)
(7)

by these organs, especially in old animais

As described in a review on bleomycin » Umezawa clarified the structural

*
feature of bleomycin molecule 1 and found that bleomycin reacts with DNA

(7) 2

*
and causes single strand scission This reaction occurs even at 0°C “,

| 2
“ N~ aA NH,
I ; 0
OP ty on
tH
“ ON, wos ’ S Jt
Mig SO HOS ee nh mS
CH, N at “CH A» oo Us ‘|
07 ™_ N H
HO © o40/ Ay | A, :  R=NHCHACHACH,s~o43
| ue en ae 2 3 genet? CH
OH L (9) Yo“ OH . - 2NH
| AG By: R NHCHCHCHCHNHC® yy
' 9
OH * At-b: R=NHCH,CH,CH.NH
07" NH 2 ab hotiy Ni,

Bleomycinic acid: R=QH

A,-b

Fig. 1. Structural Features of Bleomycins Ags Bos D

and Bleomycinic Acid

 

*] Takita, T.; Y¥Y. Muraoka, T. Yoshioka, A. Fujii, K. Maeda & H. Umezawa: The

chemistry of bleomycin. IX. The structure of bleomycin and phleomycin. J.
Antibiotics, 25, 755 (1972). *2 Umezawa, H.; H. Asakura, K. Oda & M.
Hori: Characteristics of bleomycin action which produces a single

scission in a superhelical form of SV40 DNA. J. Antibiotics, 26, 521 (1973).

 

‘

Various bleomycins are different from one another in the terminal amine

y)

moiety (R in the ‘structure . The addition of an amine to the fermentation

medium suppresses the production of natural bleomycins and causes the production
of bleomycin containing the added amine 6977), Bleomycinic acid can be obtained
by an enzymatic hydrolysis (a new enzyme, agmatine amidohydrolase -) of bleomycin
Bo and is used for chemical synthesis of new bleomycins‘”). |

Bleomycin is now widely used for the treatment of squamous cell carcinoma.
It is especially effective on the well-differentiated type. There are patients
who were treated with bleomycin who survived for more than 5 years -. Bleomycin

has also been ‘found‘to be an effective agent in the treatment of malignant

*2
lymphoma ~.

 

*1 Umezawa, H.; Y. Takahashi, A. Fujii, T. Saino, T. Shirai & T. Takita:
Preparation of bleomycinic acid: Hydrolysis of bleomycin Bo by agmatine
aminohydrolase of a Fusarium sp. J. Antibiotics, 26, 117 (1973). *2 New
Drug Seminar on Bleomycin. National Cancer Institute, Division of Cancer

Treatment, Bethesda, Maryland, (1974).
Enzymatic Mechanism of Resistance and Synthesis of Useful Agents
Against Resistant Infections

The enzymatic mechanism of the selective effect of an antibiotic agent
on different microbes can be applied to the development of active agents
against resistant infections. Umezawa clarified the enzymatic mechanism
of resistance, predicted the active structure and succeeded in the synthesis
of such a compound with practical usefulness. This is very important, because
the probability of finding new useful antibacterial antibiotics has become
very small.

In the study of water-soluble basic antibiotics, Umezawa discovered
kanamycin in 1957, which was evaluated for its effect against infection of
staphylococci resistant to drugs at that time and against tuberculosis resistant
to streptomycin, as shown in papers reported at the New York Academy Symposium

1

Ie .
in 1958 ~. Later, when resistant Gram negative organisms appeared and

synthetic penicillins were introduced, the usefulness of kanamycin in treating
infections caused by resistant Gram negative bacteria became especially important,
as described in papers at the New York Academy Symposium in 1965", Kanamycin
resistant strains appeared in 1965. In 1967, Umezawa © extracted enzymes

from disrupted cells of E. coli K12 ML1629 which he obtained from 5. Mitsuhashi
(Medical School, Gumma University) and E. coli K12 R5 which he obtained from

H. Okamoto (National Institute of Health, Tokyo). ne 8)

isolated and purified
the reaction products of these enzymes and elucidated their structures, clarifying

the nature of the enzyme reactions. Thus, the enzyme which was obtained from

most common resistant strains was shown to transfer phosphate from ATP to the

 

*1 The basic and clinical research of the new antibiotic kanamycin. Ann. N.
Y. Acad. Sci., 76, Art. 2, 19-408 (1958). .*2 Kanamycin: Appraisal after

8 years. Ann. N. Y. Acad. Sci., 132, Art. 2, 773-1090 (1966).
3'-hydroxyl group of kanamycins.

Based on these findings, 3',4'-dideoxy-

kanamycin B, which does not contain the 3'-hydroxyl and does not undergo

the reaction of the phosphotransferase, was synthesized .

(9)

It was shown that

this compound inhibits most resistant staphylococci, resistant Gram negative

organisms and Pseudomonas aeruginosa.

The usefulness of 3',4'-dideoxykanamycin

B in the treatment of resistant infections was confirmed by large-scale clinical

studies in Japan in 1970-1971.

In another aspect, the confirmation of the ability of 3',4'-dideoxykanamycin

B to unhibit resistant organisms was proof of the involvement of the enzyme

. in the mechanism of resistance.

6" 2"
CHAOH “8
1 2 Ry r 3'
, “gn HO 1'
rae Oy
/ 4 a7 5 4\ 0
a \ ve
HO os 3 1 2 3 2
NH,

J
OH is phosphorylated by

CHAR

OH 4°

22

Ro=NH,

2

kanamycin A: R,=0H,

]

kanamycin B: R,=NH R,=NH

2° 2
kanamycin C: Ry =NH . R,=0H

kanamycin-neomycin phosphotransferase.

Besides 3',4'-dideoxykanamycin B, other interesing derivatives have been

synthesized.

1-N- (4-amino-2-hydroxybutyryl)-~6'-N-methyl-3' ,4'-dideoxykanamycin

B is active against resistant organisms producing kanamycin-neomycin phospho-

transferases I and II, kanamycin-gentamicin nucleotidyltransferase, gentamicin

acetylase, and kanamycin acetylase.

Umezawa and his coworkers also elucidated the mechanism of resistance
to the other aminoglycosidic antibiotics (streptomycin, lividomycin, etc.)
and determined the structures of most of the products of enzymes which are

(10)

involved in the mechanism of resistance to aminoglycosidic antibiotics .
The Study on Enzyme Inhibitors of Microbial origin t))

H. Umezawa extended his antibiotic studies to include enzyme inhibitors
produced by microorganisms =. Compared with inhibitors obtained from animal.
and plant tissues which generally are of macromolecular nature, the inhibitors
that he found in microbial culture filtrates are small molecules of unique
structural types which no one had detected before. He found leupeptin
inhibiting plasmin, trypsin, papain and cathepsin A, antipain inhibiting
trypsin, papain and cathepsins A and B, chymostatin ” inhibiting chymotrypsin,
and elastatinal > inhibiting elastase. These inhibitors contain a C-terminal
aldehyde group and he showed that argininal in leupeptin, phenylalaninal in
chymotrypsin, alaninal in elastatinal werenecessary for inhibition of the
enzymatic cleavage at the carboxyl side of arginine, phenylalanine, and alanine
(or glycine). He found pepstatin inhibiting pepsin, cathepsin D and renin.

Pepstatin is the first small molecule found to inhibit these enzymes.

 

*] Umezawa, H.: Enzyme inhibitors of microbial origin. University of Tokyo
Press, Bunkyo-ku, Tokyo, 1973. Umezawa, H.: Chemistry of enzyme inhibitors
of microbial origin. Pure and Applied Chemistry, 33, 129 (1973). ¥*2 Tatsuta,
K.; N. Mikami, K. Fujimoto, S. Umezawa, H. Umezawa & T. Aoyagi: The
structure of chymostatin, a chymotrypsin inhibitor. J. Antibiotics, 26,

625 (1973). *3 Umezawa, H.; T. Aoyagi, A. Okura, H. Morishima, T. Takeuchi
& Y. Okami: Elastatinal, a new elastase inhibitor produced by actinomycetes.

J. Antibiotics, 26, 787 (1973).
-10-

Phosphoramidon - inhibiting thermolysin was found to be a new inhibitor against
a metal protease which cleaves the amino side of a hydrophobic amino acid.

He and his coworkers ‘1? also isolated inhibitors of enzymes which are
involved in norepinephrine biosynthesis and elucidated their structures:
oudenone and aquayamycin inhibiting tyrosine hydroxylase; fusaric acid and
dopastin inhibiting dopamine p-hydroxylase, methylspinazarin and dihydro-
methylspinazarin inhibiting cathecol-O-methyl transferase. Inhibitors of
dopamine (} hydroxylase and tyrosine hydroxylase showed hypotensive effects.
Structures of enzyme inhibitors are shown in an attached paper. Thus, H.
Umezawa has demonstrated that microorganisms are rich sources of enzyme
inhibitors of small molecular nature which exhibits pharmacological activities.

Pepstatin has been shown to be effective in treatment of gastric ulcer.
Leupeptin ointment applied immediately after burn suppresses pain and
blister-formation. The hypotensive effect of fusaric acid has been confirmed
clinically. The study of enzyme inhibitors, thus, has been shown to be a

rational approach to the discovery of useful drugs.

,

 

*1 Suda, H.; T. Aoyagi, T. Takeuchi & H. Umezawa: A thermolysin inhibitor
produced by actinomycetes: phosphoramidon. J. Antibiotics, 26, 621

(1973).
A Short Curriculum Vitae of H. Umezawa

Date of Birth: October 1, 1914
Address: 23, Toyotama~kita 4-chome, Nerima-ku, Tokyo, Japan
Present positions

Member, The Japan Academy

Director, The Institute of Microbial Chemistry (Kamiosaki,
Shinagawa-ku, Tokyo)

Director, The Department of Antibiotics, The National Institute
of Health, Japan (Kamiosaki, Shinagawa-ku, Tokyo)

Curriculum vitae

'

March, 1937 Graduated from Medical School, University

of Tokyo

June, 1944-July, 1947: Associate Professor, University of Tokyo
, and Member, The Institute for Infectious

Diseases, University of Tokyo

April, 1945 — > Doctorate of Medical Science, University
of Tokyo
July, 1947 - : Director, Department of Antibiotics, The

National. Institute of Health, Japan

August, 1954 - : Professor, University of Tokyo, Institute
March, 1975 of Applied Microblology

May, 1962 -
April, 1963 -

Director, Institute of Microbial Chemistry

Honorary Member, Japanese Association of
Medical Science

November, 1969 - : Member, The Japan Academy

August, 1973 - : Member, Deutsche Akademie der Naturforscher
Leopoldina

April, 1974 - : Honorary Member, Pharmaceutical Society
of Japan

Prizes

Culture Medal (Bunka~-Kunsho) conferred by the Emperor, November, 1962
Japan Academy Prize, May, 1962 4
Fujiwara Prize, June, 1971
Asani Prize, January, 1959

Commandeur de l'Ordre de la Santé Publique (France), December, 1960
List of New Antibiotics and Their Derivatives Discovered

by H. Umezawa

Antitumor Antibiotics:

No. 289 Substance (1953), Sarkomycin (1953), Actinoleukin (1954),
Ractinomycins A and B (1955), Pluramycins A and B (1956), Phleomycin
(1957), Raromycin (1957), No. 418 Substance (1959), Peptimycin (1961),
Enomycin (1963), Labilomycin (1963), Hilamycin (1963), Formycin A (1964),
Bleomycins (1965), Plurallin (1966), Phenomycin (1967), Macromomycin (1968),
Acrylamidine (1968), Coriolins (1969), Neopluramycin (1970), Diketocoriolin

B (1971).

Antimicrobial Antibiotics:

Two Streptothricin B of S. fradiae (neomycins) (1948), Aureothricin
(1949), Griseolutein A and B (1950), Nitrosporin (1951), Actinomycin J
(1951), Abikoviromycin (1952), Exfoliatin (1952), Moldin (1952), Phaeofacin
(1952), Sarcidin (1953), Achromoviromycin (1953), Thiazolidone antibiotic
(1953), Pyridomycin (1953), Azomycin (1953), Phthiomycin (1953), Seligocidin
(1954), Aureothin (1954), Mediocidin (1954), Tertiomycin A and B (1955),
Antitoxoplasmic Substance No. 534 (1955), Mesenterin (1955), Kanamycin A,

B and C (1957), Althiomycin (1957), Alboverticillin (1958), Mikamycin (1959),
Blastmycin (1957), Niromycin A and B (1960), Unamycin A and B (1960),
Amidinomycin (1960), Emimycin (1960), Ilamycin Ai Ays By» Bo» Cy and C,
(1961), Cytomycin (1961), Griseococcin (1962), Monazomycin (1963), Bottromycin
Ay» Ay and B (1965), Kasugamycin (1965), Spinamycin (1966), Josamycin (1967),
Leucinamycin (1967), Pepthiomycin (1968), Ablastmycin (1968), Laspartomycin
(1968), Oryzoxymycin (1968), Gougeroxymycin (1969), Deoxynybomycin (1970),
Macarbomycin (1970), Negamycin (1970), Leucylnegamycin (1971), Requinomycin

(1972), Minosaminomycin (1974), Amiclenomycin (1974).
Derivatives of Aminoglycosidic Antibiotics:

3',4"-Dideoxykanamycin B (1971), 5"-Deoxylividomycin A (1972),
5"-Amino-5"-deoxylividomycin A (1972), 5"-Deoxylividomycin B (1972),
3',4'-Dideoxy—6-N-methylkanamycin B (1972), 3'-Deoxykanamycin (1972),
3'-0-Methylkanamycin (1972), 1-N-(4-Amino-2-hydroxybutyry1) lividomycin A
(1973), 3',4'-Dideoxybutirosin B (1973), 1-N-(4-Amino-2-hydroxybutyry1)-
kanamycin B (1973), 3'-Deoxybutirosin B (1973), 1-N-(4-Amino-2-hydroxy-
butyry1)~6-N-methy1-3' ,4'-dideoxykanamycin B (1973), 6'-Amino-6'~deoxy-
lividomycin B (1973), 6'-Deoxy-6'-methylaminolividomycin B (1973), 6'-
Deoxy-6'-hydroxyethylaminolividomycin B (1973), 1-N-Isoserylkanamycins

(1974), 4'-Deoxykanamycin (1974).

Enzyme Inhibitors:

Leupeptin (1969) inhibiting trypsin, plasmin, papain, thrombokinase
and cathepsin B, Antipain (1972) inhibiting trypsin, papain, thrombokinase,
cathepsin A and cathepsin B, Chymostatin (1970) inhibiting chymotrypsins,
papain and cathepsin B, Elastatinal (1973) inhibiting elastase, Pepstatin
(1970) inhibiting pepsin, gastricsin, cathepsin D and renin, Hydroxy pepstatin
(1973) inhibiting pepsin, gastricsin, cathepsin D and renin, Pepstanone
(1972) inhibiting pepsin, gastricsin, cathepsin D and renin, Phosphoramidon
(1973) inhibiting thermolysin, Panosialin (1971) inhibiting trypsin, . plasmin,
pepsin, sialidase, acid phosphatase and polygalacturonase, Aquayamycin (1968)
inhibiting tyrosine hydroxylase, Oudenone (1970) inhibiting tyrosine hydroxylase,
Fusaric acid (1969) inhibiting dopamine 8-hydroxylase, Dopastin (1972) inhibiting
dopamine $-hydroxylase, Oosponol (1972) inhibiting dopamine B-hydroxylase,
Pimprinine (1973) inhibiting monoamine oxidase, Cinnamic acid amide (1973)
inhibiting monoamine oxidase, Methylspinazarin (1973) inhibiting catechol-
O-methyltransferase, 7-O-methylspinochrome B (1973) inhibiting catechol-—0-

methyltransferase, Dihydromethylspinazarin (1973) inhibiting catechol-0-
methyltransferase, 6~-(3-hydroxy-n—buty1)-7-0-methylspinochrome B (1973)
inhibiting catechol-O-methyltransferase, 1-[2-(3,4,5,6-tetrahydropyridyl) ]-
1,3-pentadiene (1974) inhibiting N-methyltransferase, Lecanoric acid (1974)
inhibiting histidine decarboxylase, 5-Formyl uracil (1972) inhibiting
xanthine oxidase, Coformycin (1967) inhibiting adenosine deaminase, 8-

Lactamase inhibitor (1974).
1)

2)

3)

4)

A Bibliography of H. Umezawa's Most Important Publications

Umezawa, H.; K. Maeda, T. Takeuchi and Y. Okami: New antibiotics bleomycins
A and B. J. Antibiotics, 19A, 200 (1966).

In the study of antibiotics resembling phleomycin, new antibiotics
which were differentiated from phleomycin by their stability in acid and
their behaviour in paper chromatography were isolated by cation exchange
resin chromatography, and named bleomycins A and B.

Umezawa, H.; Y. Suhara, T. Takita and K. Maeda; Purification of bleomycins.
J. Antibiotics, 19A, 210 (1966)

Bleomycins A and B were further separated into Ay - Ae and B, - Bos
and each of them completely purified by chromatography using a gradient
of ammonium formate.

Ishizuka, M.; H. Takayama, T. Takeuchi and H. Umezawa: Activity and
toxicity of bleomycin. J. Antibiotics, 20A, 15 (1967)

Bleomycin A and B exhibited inhibition of Ehrlich carcinoma with a
high therapeutic index. They also inhibited microorganisms. Bleomycin
showed no irreversible renal toxicity in dogs and exhibited a therapeutic
effect against round cell carcinoma near the vagina in dogs.

Umezawa, H.; T. Takeuchi, S. Hori, T. Sawa, M. Ishizuka, T. Ichikawa and
T. Komai: Studies on the mechanism of antitumor effect of bleomycin on
squamous cell carcinoma. J. Antibiotics, 25, 409 (1972)

34-bleomycin was prepared and after subcutaneous injection in mice,
all organs were homogenized and concentrations of bleomycin in each organ
homogenate were determined by radioactivity and antibacterial measurements.
The results indicated that all tissues contain a bleomycin-inactivating
enzyme. The inactivation was significantly slower in skin and lung,

especially in old mice. Squamous cell carcinoma and sarcoma in the skin
5)

6)

of mouse was induced by methylcholanthrene. These tumors were homogenized,
and the activity in inactivating bleomycin was shown to be significantly
higher in the carcinoma than in the sarcoma. The bleomycin-inactivating
enzyme was extracted from mouse liver homogenate by successive application
of protamine treatment, precipitation with 60% saturated ammonium sulfate
and Sephadex 100 chromatography. The enzyme was shown to hydrolyze the
carboxamide group of bleomycin. The inactivated bleomycin was isolated
and was shown to have a carboxylic acid group. During inactivation, one
mole of ammonia was released.

Umezawa, H.: Natural and artificial bleomycins: Chemistry and antitumor
activity. Pure and Applied Chemistry, 28, 665 (1971)

Sequential hydrolysis of bleomycin resulted in each amino acid
component and indicated that various bleomycins are different from one
another in the terminal amine moiety. Addition of an amine to the culture
suppressed the production of natural bleomycins and caused the production
of bleomycin containing the added amine. This technique was useful in
providing a bleomycin mixture containing various bleomycins in a constant
ratio. This paper also describes the isolation of the inactivated bleomycin.
Umezawa, H.; S. Hori, T. Sawa, T. Yoshioka and T. Takeuchi: A bleomycin-
inactivating enzyme in mouse liver. J. Antibiotics, 27, 419 (1974)

The enzyme which hydrolyzes the carboxamide group of bleomycin was
purified form mouse liver by affinity chromatography using Sepharose 4B-
lysinamide. The enzyme thus purified also hydrolyzed L-lysinamide, L-
lysyl-j-naphthylamide and L-arginyl-(-naphthylamide, but not leucyl-{-
naphthylamide. Thus, this enzyme was shown to be aminopeptidase B. However,
in mice this bleomycin-inactivating enzyme was very unstable. Using rat
liver it was differentiated from known aminopeptidase B by chromatographic

\

separation.
7)

8)

9)

10)

Umezawa, H.: Studies on bleomycin: Chemistry and biological action.
Biomedicine, 28, 459 (1973)

H. Umezawa's study on the discovery, isolation, chemistry, mechanism
of action, mechanism of the effect on squamous cell carcinoma, and
production of artificial bleomycins are reviewed in this paper. H. Umezawa
first found that bleomycin binds with DNA and causes single strand scission.
Umezawa, H.; M. Okanishi, S. Kondo, K. Hamana, R. Utahara, K. Maeda and
S. Mitsuhashi: Phosphorylative inactivation of aminoglycosidic antibiotics
by E. coli carrying R factor. Science, 157 (3796), 1559 (1967)

Resistant E. coli carrying R factor were shown to contain intracellular
enzymes inactivating kanamycins, streptomycin, and neomycins. These enzymes
were present in the supernatant of disrupted cells, and the products of
the enzyme reactions were isolated. Thus, kanamycin resistance was shown
to be due to a phosphotransferase which phosphorylates 3'-OH of kanamycins.
Umezawa, H.; S. Umezawa, T. Tsuchiya and Y. Okazaki: 3',4'-Dideoxykanamycin
B active against kanamycin-resistant E. coli and Ps. aeruginosa. J.
Antibiotics, 24, 485 (1971)

3'-OH kanamycin B underwent phosphorylation by the enzyme in resistant
organisms. Therefore, 3'-OH and 4'-OH were removed chemically from
kanamycin B. 3',4'-Dideoxykanamycin B thus prepared inhibited the resistant
organisms. This proves the involvement of enzymatic inactivation in
resistance. The compound represents the first successful application of
knowledge of the biochemical basis of resistance to the development of
an active agents useful in the treatment of resistant infections.

Umezawa, H.: Biochemical mechanism of resistance to aminoglycosidic
antibiotics and development of active derivatives. Advances in Carbohydrate

Chemistry and Biochemistry. Academic Press, New York and London, 1974.
11) Umezawa, H.: Enzyme Inhibitors of Microbial Origin. University of

Tokyo Press, Bunkyo-ku, Tokyo, 1973. Umezawa, H.: Chemistry of enzyme

inhibitors of microbial origin. Pure and Applied Chemistry, 33, 129
(1973).
List of Structures of Enzyme Inhibitors Discovered by H. Umezawa

tHe
CH, CH, C=NH
CH-CH, CH-CHg NH
CHy CH (CH)
R- CO-NH- CH CO-NH-CH-CO-NH-CH-CHO
(S) (S)
R=CH2 or
Leupeptin Cols

inhibiting trypsin, plasmin,
papain, thrombokinase, |
cathepsin B

NH, " Nip
CNH C=NH
NH GH, i
CH (CH) 3 GH-CH, (CH) 3
HOOC-CH-NH-CO-NH-CH-CO-NH-CH-CO-NH-CH-CHO
(Ss) (S) (S)
Antipain

inhibiting trypsin, papain, thrombokinase,
' cathepsin A and B

| H CH, CH, | (OMe
Cie CH-CH, CH-CH en CH
CHy (s) CHa CH CH (8) CH CH,
HOOC- dye ve -CO-NH-CH-CO-NH-CH-CO- NH- CH=CHO - HOOC-CH-NH-CO-NH- i CO-NH- ie CO-NH- CH- CHO
(S) (S) (S) (S) (S) (S) (S)
Chymostatin | Elastatinal - |
inhibiting chymotrypsins, papain, inhibiting elastase
cathepsin B
CH, - CH,
CH, CH, CH-CH, . tH ~CH,
(- -CH CH ~CH, CH OF CHy CH OF
R-CO-NH- CH CO-NH- CH CO-NH-CH- cH -CHo~ ~CO-NH- de CO-NH- du- tHe -CH,- -COO0H
— (Ss) (8) (S)'(S) (S) (S)(S)
Pepstatin R°CH3, CoHe» C3H7» CyHg»
CoH, 4 weeee etc,
inhibiting pepsin, gastricsin, cathepsin D, renin
CH3 oo CH |
CH CH3 CH-CH3 CH-CH3
CH- -CH, CH- -CH CH, OH CH0H CHOOH
R-CQ~NH- cH CO-NH- due CO-NH- be H- -CH,-CO- ~NH- cH CO-NH- dy- -CHI-CHy- -COOH
(S) (S) (S)(S) (S) (S)(S)

R=CHy, Coz» C37. Cqlg

Hydroxy pepstatin
. . CoH eeeeve etc,

inhibiting pepsin, gastricsin, cathepsin D, renin
CH CH
| 3 3
CH 7 CH-CH cH CH,

3 3 3
cH -CH, CH -CH, CH, OW CH, CH,

R-CO-NH-CH-CO-NH-CH-CO-NH-CH-CH- -CH,-CO-NH- cH-CO-NH- bye C ~CH,
(S) (S) (S)(S) (S) (S)

R=CH,, C Hes CaH5, C Hay we. etc

Pepstanone 3° “2 37° “5

inhibiting pepsin, gastricsin, cathepsin D, renin

  

N K0,S0
CH, CH, H
‘NG \ R
i
OH CH
L. { 2 K0,S0
Oe H—COOH R: -(CHy)49-CH(CH3) 05
~(CHy)44-CHa ,
Phosphoramidon
H3¢ 0 | ~(CHy)13-CH(CH3) 9
inhibiting thermolysin Panosialin
HO OH inhibiting trypsin, plasmin,
OH pepsin, sialidase,
acid phosphatase,
pol ygalacturonase
“CH-CH5-CHy
Qudenone
Aquayamycin inhibiting tyrosine hydroxylase
inhibiting tyrosine hydroxylase
. OH 0
CHa(CH) | v0 |
3(CH,) 5 | CH3. YH HOA CF a | 0
~ ae 13 ~
N~ ~cooH H C-NH- -CH,-Cl- -CH-CH, LZ
. 07 (S) C=0
Fusaric acid y
inhibiting dopamine Dopastin CH,0H
-hydroxylase . inhibiting dopamine
6 é-hydroxylase Cosponol
Nw cH inhibiting dopamine
\ } 3 B-hydroxylase

N
H

Pimprinine

sl lgntas

Cinnamic acid amide

inhibiting monoamine oxidase

. *
PECP ae hs ere Tara fyye t den
OH 0
CH OH
OH 0

Methyl spinazarin

inhibiting catechol-0-methy1-

transferase
0
CH-CH-CH)~CH,
OH OH 860

6-(3-hydroxy-n-butyl )-7-0-
methyl spinochrome B

inhibiting catechol-0-
methyl transferase .

OH
fi CHO
C—0— CHy HN |
A
HO CH, | ‘COOH 0
OH

Lecanoric acid
inhibiting histidine
decarboxylase

Q
CH,0 ee OH
OH

OH 0

7-O-methylspinochrome B

inhibiting catechol-0-
methyltransferase

OH f
iP ~~ OH
CH2
H L OH
OH 0

Dihydromethyl spinazarin
inhibiting catechol -0-
methyl transferase

N
CY -oiecieorech-

}-[2-(3,4,5,6-tetrahydropyridyl )]-
1,3-pentadiene

inhibiting N-methyl transferase

0

N
H
5-Formy] uracil

inhibiting xanthine
oxidase

OH
« TS
\e yn
HO 0

OH OH

Coformycin

inhibiting adenosine
deaminase