Okafor N. Modern Industrial Microbiology and Biotechnology

Подождите немного. Документ загружается.

"# Modern Industrial Microbiology and Biotechnology

agents it is usually possible to isolate the pathogen from the diseased animal and test the

isolate against a wide range of possible antibiotics in vitro and in vivo in experimental

animals. In general an antibiotic successfully tested in vivo in experimental animals will

be expected to be reasonably efficacious in treating human disease. In the case of anti-

tumor agents the relationship is not so straight-forward.

As is the case with anti-microbial antibiotic no order of procedure can be prescribed.

The description that follows is built up from published materials from several groups,

especially at the National Cancer Institute, and the Cancer Research Laboratories,

Kalamazoo, both in the USA. The stages involved in the search and initial development of

anti-tumor antibiotics may be enumerated as follows.

24.5.3.1 Screening of potential antibiotic producing organism

A wide variety of microorganisms is obtained from all over the world and their

fermentation broth is evaluated for the presence of anti-tumor drugs. Cultures whose

identities are established as well as those still to be established are obtained from culture

collections and individual scientists around the world. Fresh isolations are also made

from natural habitats including soil, aquatic and other environments.

Adriamycin OCH

OHHHHOH

Daunomycin OCH

HHHOH

Carminomycin OH CH

HHHOH

Fig. 24.7 General Formula of Anthracyclines

Production of Antibiotics and Anti-Tumor Agents "#

For the isolation (and often the maintenance of the organisms, a wide variety of carbon

sources is used. Especially in well-studied groups, such as actinomycetes, novel carbon

sources are used in the hope that fermentation broth may contain some anti-tumor

antibiotics, as a result of the blockage of certain pathways or the enhancement of others.

Such carbon sources include monosaccharides, glycosides, substituted sugars,

polyhydric alcohols, oligosaccharides, terpens, and hydrocarbons.

Isolations of microorganisms are done by sprinkling soil on plates containing the

above carbon sources. Perfusion technique in which soil is bathed constantly with a

solution containing the chosen carbon source may also be used. Isolates are grown in

shake flasks using the various carbon sources and a variety of environmental conditions

including pH, minerals, temperature, nitrogen sources, and aeration.

24.5.3.1.1 In vitro prescreening

Since large numbers of samples are generated, it would be extremely expensive to test

them directly in tumor-bearing animals. Prescreens are therefore used. Such prescreens

should ideally select the broths successfully containing potentially in vivo active

components, should be relatively inexpensive in terms of money and time and should

require only small quantities of the test broth. In vitro screens are also used to follow the

course of fermentation. In vitro methods are particularly essential because the frequency

of occurrence of active anti-tumor components in fermentation beers is low, and when

present at all, the concentration especially initially is also low. Some of the in vitro

screening methods which are currently in use are as follows:

(i) Use of anti-microbial activity as prescreens: As most anti-tumor agents will also inhibit

micro-organisms, the latter came to be used during the early stages of the search for these

drugs. Indeed a number of drugs possessing carcinostatic properties were first isolated

as anti-microbial agents. These include actinobolin, cycloheximide, and actinomycin.

Azaserine was the first anti-tumor drug isolated following activity against a bacterium,

which in this case was E. coli. A wide range of microorganisms can and indeed have been

used in prescreens. However, many groups favor using a set containing a few

microorganisms to facilitate the identification by ‘finger printing’ of already discovered

antibiotics. Finger-printing will be discussed more fully below. A set of microorganisms

which has been used include Bacillus subtilis ATCC 6633, Sarcina lutea ATCC 9341,

Torulopsis albida NRRL Y1400 and Escherichia coli M 1262.

The beer to be tested is spotted on filter paper disks placed on freshly made pour plates

of these organisms or in agar wells dug from such plates. The extent of the inhibition is

determined by the diameter of the zone of clearing.

(ii) Anti-metabolite activity: The use of anti-metabolites as a prescreen was based on the

observation that some drugs or broths which did not inhibit microorganisms in complex

media such as nutrient agar did so on synthetic minimal media. It was soon shown that

by adding various compounds to the minimal medium it was possible to determine the

missing metabolite. Broth samples are incorporated into rich agar and minimal agar

respectively. Those broths which are more active against susceptible microorganisms in

synthetic minimal medium are regarded as potential leads. This methods led to the

discovery of 5-Azacytidine and its principal advantage is that it gives an idea at an early

stage of the mode of action of the active component of the broth.

"# Modern Industrial Microbiology and Biotechnology

(iii) Inhibition of tumor cells in culture: The ability of the broth to inhibit animal tumor cells

in tissue culture is tested. Among cells which have been used are L1240 (mouse leukemia

cells), KB (human carcinoma cells of the nasopharynx) and p388 mouse leukemia cells.

L1240 appears to be most widely used. The tumor cells are grown in liquid culture with

and without the broth over a period of about three days. They are then counted in a

coulter counter. The potency of the same is given as ID

or ID

or the dilution that will

cause 50% or 90% inhibition of growth as compared with control.

(iv) Nuclear cytotoxicity: Instead of using cell cultures, nuclei from tumor cells can be

isolated and the broth tested against these. Isolation may be achieved using citric acid,

detergents, organic solvents, and glycerol. The limitation of the system is that it can be

used to detect only those agents which in some way affect nuclear synthesis. It however

lends some insight into the mode of action of the agent.

24.5.3.2 Finger printing of anti-tumor antibiotics in culture

Broths showing some activity by any of the above prescreening methods are subjected to

‘finger-printing’ or ‘dereplication’ in order to avoid the isolation of already known

antibiotics. The data used include the following:

(i) The anti-microbial spectrum of the active components of the broth is determined

using a set of microorganisms. Besides bacteria and yeasts some workers use

protozoa and algae.

(ii) The characteristics of the culture used in the fermentation.

(iii) Chromatographic analyses of the broth using paper, thin layer, and high

performance liquid chromatography.

(iv) Ultraviolet absorption.

24.5.3.3 In vivo assessment

If the results of the finger-printing indicate that the active components are new then in

vitro testing is done. The one common characteristic of tumors or neoplasms is the

uncontrolled growth of cells. Outside this property they are in fact biologically

heterogeneous in terms of site of origin, cell type involved, the course of the disease, or

response to curative procedures. In assessing active components in vivo therefore this

diversity is acknowledged by testing various types of artificially induced tumors usually

in mice. These include mouse tumors of the colon, breast, lungs, and white blood cells

(leukemia). Human cancer from the colon, breast and lungs, are also grafted on these

regions of mice whose thymus glands have been removed to avoid rejection of the grafts.

Often it is necessary to have an in vivo prescreen before subjecting the broth to the

above tests. Some workers have found that mouse leukemia P388 is more sensitive as an

in vivo prescreen than L1240.

The parameters for in vivo tests are increased lifespan of the animal or tumor growth

inhibition as measured by tumor weight inhibition over the control.

The in vivo test is expensive in time and money. It takes three to four weeks to perform

whereas cell or microbial cultures take about three days or less.

Production of Antibiotics and Anti-Tumor Agents "#!

24.5.3.4 Extraction and manipulation of the pure drug

Since the active component may often be present in very low concentration it is necessary

to obtain the active component in the broth in a reasonably pure form. Subsequent to this

it is characterized chemically and then subjected to further animal tests before being

assessed clinically. Since many anti-tumor antibiotics have serious side effects,

analogues of the drugs are produced and modifications to the molecule are then carried

out. Other procedures, e.g. improvement in the environmental conditions of the broth,

development of optimum isolation procedures, scale up, sales, etc. are carried out as for

anti-microbial antibiotics.

24.5.3.5 Towards a new definition of ‘antibiotic’

The current definition of the term ‘antibiotic’ which restricts them to chemicals produced

by microorganisms is credited to Waksman who had won the Nobel Prize for discovering

streptomycin. However, when screenings have been done outside microorganisms, the

higher organisms so screened have been shown to produce anti-microbial substances.

Such substances are low molecular weight secondary metabolites in the same way as

regular antibiotics are. Due to this, there is now a tendency to extend the term antibiotic to

all secondary metabolites, irrespective of their origin, which are able to inhibit various

growth processes at low concentration. Not only that, even wholly synthetic

antimicrobials such as ciprofloxacin are now legitimately termed antibiotics. It is not an

altogether unreasonable redefinition. After all, the word antibiotic derives from two

origins, anti (against) and bios (life). Nothing in the word itself restricts antibiotics both

in origin or in use to microbial life

24.6 NEWER METHODS FOR SEARCHING FOR

ANTIBIOTIC AND ANTI-TUMOR DRUGS

In recent times newer method have been developed for searching for new antibiotics, anti-

tumor agents and other drugs. These methods include computer-aided drug designing,

synthesis of new drugs by combinatorial chemistry and genome-based methods. These

are further discussed in Chapter 28, where drug discovery is examined.

SUGGESTED READINGS

Allsop, A., Illingworth, R. 2002. The impact of genomics and related technologies on the search

for new antibiotics. Journal of Applied Microbiology, 92, 7-12.

Anon, 1993. Congress of the United States, Office of Technology Assessment. Pharmaceutical

R&D: Costs, Risks and Rewards: 1993; pp. 4-5.Washington, DC, USA.

Anon, 1999. From Test Tube to Patient: Improving Health Through Human Drugs. Special

Report, Center Drug Evaluation and Research. Food and Drug Administration. Rockville,

MD, USA.

Austin, C. 2004. The Impact of the Completed Human Genome Sequence on the Development of

Novel Therapeutics for Human Disease. Annual Review of Medicine, 55, 1-13.

Bansal, A.K. 2005. Bioinformatics in the microbial biotechnology – a mini review. Microbial Cell

Factories, 4, 4-19.

"#" Modern Industrial Microbiology and Biotechnology

Beamer, L. 2002. Human BPI: One protein’s journey from laboratory to clinical trials. ASM News.

68, 543-548.

Behal, V. 2000. Bioactive Products from Streptomyces. Advances in Applied Microbiology. 47, 113-

156.

Bull, A.T., Ward, A.C., Goodfellow, M. 2000. Search and Discovery Strategies For Biotechnology:.

The Paradigm Shift. Microbiology and Molecular Biology Reviews, 64, 573-548.

Dale, E., Wierenga, D.E., Eaton, C.R. 2001. Processes of Product Develpoment. http://

www.allpcom/drug-dev.htm. Accessed on September 28, 2005 at 12.05 pm GMT.

Debouck, C., Metcalf, B. 2000. The Impact of Genomics on Drug Discovery. Annual Review of

Pharmacology and Toxicology, 40, 193–208.

Fan, F., McDevitt, D. 2002. Microbial Genomics for Antibiotic Target Discovery. In: Methods in

Microbiology. Vol 33, Academic Press. Amsterdam: The Netherlands. pp. 272–288.

Feling, R.H., Buchanan, G.O., Mincer, T.J., Kauffman, C.A., Jensen, P.R., Fenical, W. 2003.

Salinosporamide A: a highly cytotoxic proteasome inhibitor from a novel microbial source, a

marine bacterium of the new genus Salinospora. Angewandte Chemie International Edition,

42, 355-357.

Fraser, C.M., Rappuoli, R. 2005. application of microbial Genomic Science To Advanced

Therapeutics. Annual Review of Medicine, 56, 459–74.

Handelsman, J., Rondon, M.R., Brady, S.F., Clardy, J., Goodman. R.M. 1998. Molecular biological

access to the chemistry of unknown soil microbes: a new frontier for natural products.

Chemistry and Biology, 5, 245-249.

Hill, D.C., Wrigley, S.K., Nisbet, L.J. 1998. Novel screen methodologies for identification of new

microbial metabolites with pharmacological activity. Advances in Biochemical Engineering

and Biotechnology, 59, 75–124.

Maxwell, S., Ehrlich, H., Speer, L., Chandler, W. 2005. Medicines from the Deep Sea. Washington,

DC, USA.

Rosamond, J., Allsop, A. 2000. Harnessing the Power of the Genome in the Search for New

Antibiotics. Science, 287, 1972-1976.

Wlodawer, A., Vondrasek, J. 1998. Inhibitors of HIV-1 Protease: A Major Success Of Structure-

Assisted Drug Design. Annual Review of Biophysics and Biomoecular Structurrs, 27, 249–84.

25.1 NATURE OF ERGOT ALKALOIDS

Alkaloids are laevorotatory basic naturally occurring hetereocyclic organic nitrogen

containing compounds which are biosynthesized from amino acids by plants,

microorganisms and some animals. Many of them are pharmacologically active and are

consequently used as drugs. The main precursors for alkaloid biosynthesis are ornithine,

lysine, aspartic acid, phenylalanine, tyrosine and tryptophan. For example the alkaloid

in tobacco, nicotine, is derived from ornithine while phenylalanine and tyrosine give rise

to simple alkaloids such as ephedrine or more complex ones such as morphine.

The name alkaloid (literally alkali like) derives from their basic nature, because of

which they readily form salts with acids present in the natural sources from which they

are derived. Their chemical classification is based on the carbon-nitrogen skeletons.

Ergot alkaloids, the subject of this chapter are of the indole type and are derived from

tryptophan.

The ergot alkaloids are so called because they were originally derived from ergot, a

sclerotium (twisted mat of fungal hyphae) formed as a disease on the grain of rye (Secale

cereale L.) a temperate cereal. The dried ergot is known among pharmacists as secale

cornutum. The cause of the rye disease is a fungus, an ascomycete, Claviceps purpurea. As

will be seen later, ergot contains several (more than 40) highly potent alkaloids and the

unwitting consumption of grain attacked by fungi producing ergot alkaloid has led to

‘ergotism’, (previously known as ‘Holy fire’ or ‘St. Anthony’s fire’ when it was not

understood) a disease characterized by among other symptoms, convulsions. . Ingestion

of contaminated grain, most often after the grain has been made into bread, causes

ergotism, also known as the ‘Devil’s curse’ or ‘St. Anthony’s fire,’ and has been a problem

for centuries. It has been noted in writings from China as early as 1100 B.C. and in

Assyria in 600 B.C., and Julius Caesar’s legions suffered an epidemic of ergotism during

one of their campaigns in Gaul (France). In 994 A.D., an epidemic in France killed

between 20,000 and 50,000 people, and in 1926, at least 11,000 cases of ergotism occurred

in Russia.

Ergotism can cause convulsions, nausea, and diarrhea in mild forms, and there is

some thought that an outbreak of ergotism may have been the cause of the ‘bewitchings’

Production of

Ergot Alkaloids

+0)26-4

"#$ Modern Industrial Microbiology and Biotechnology

which led to the Salem witch trials in the United States in 1691. Ergotism may also have

caused some of the extreme destruction associated with the French Revolution. In the

Middle Ages, ergotism was described as causing victims to die “miserably, their limbs

eaten up by the holy fire that blacked like charcoal.” People turned to the church for help,

assuming that the disease was retribution for their sins. In particular, they prayed to St.

Anthony for deliverance, giving rise to the name for the disease. Ergotism takes two

forms, gangrenous ergotism, in which tingling effects were felt in fingers and toes

followed in many cases by dry gangrene of the limbs and finally loss of the limbs, and

convulsive ergotism, in which the tingling was followed by hallucinations and delerium

and epileptic-type seizures. In both cases, death was slow and painful. Ergotism has now

been recognized as a result of infection by a mycotoxin, and the ergotism plagues have

been eliminated.

About 50 ergot alkaloids are known today. While most of these alkaloids are derived

from the Claviceps sclerotium formed on the rye grain, hundreds of other cereals and

grasses can serve as hosts for the fungus. About 50 species of Claviceps itself are known.

In addition, in recent times the alkaloids have been produced by other fungi including

Aspergillus, Penicillium, and Rhizopus. Some recent fungi shown to produce alkaloids are

Balansia epichloe, B. henningsiana, B. strangulans, Myriogenospore atrementose and Epichre

typhine. Furthermore, ergot alkaloids have recently been found in the seeds of some

higher plants, Ipomea, Rivea, Agyreis which belong to Convulvulaceae the family to which

the flower morning glory belongs.

The life cycle of Claviceps is given in Fig. 25.1. The sclerotium forms in the size and

shape of the grain which it replaces. These sclerotia fall to the ground at the end of the

growing season and remain dormant till the beginning of the next growing season, when

they germinate and form ascocarps (perithecia). The ascospores are distributed by wind

to the newly formed flowers of grains. The germinated ascospores yield hyphae which

produce masses of conidia supported in a sugary liquid which attracts insects. These

insects further help distribute the conidia to other plants.

The ergot alkaloids are classified as indole alkaloids, which are derived from

tyrptophan. With one exception, chanoclavine, the ergot alkaloids possess the basic

tetracyclic (four-ringed) structure known as ergoline (Fig. 25.2).

The naturally occurring ergot alkaloids can be divided into two groups. (a) lysergic

acid derivaties (Fig. 25.2) and (b) clavine alkaloids. Although the clavine alkaloids were

the first to be prepared in fermentation broths, and were used for biosynthetic studies,

they are much less known than the lysergic (Fig. 25.3) acid ones in terms of their

fermentation and even in terms of pharmacological activity. Therefore only the lysergic

alkaloids will be handled in this discussion.

The lysergic acid derivates can be further divided into two depending on the nature of

the amide substituents. First are the simple amide substituents and second are the

peptide alkaloids in which a cyclic peptide is attached to lysergic acid. The greatest

interest appears to center on the peptide alkaloids. In this group a modified tripeptide

containing proline and an a–hydroxy - a-amino acid which has undergone cyclic

foundation with the carbonyl atom of proline substitutions at C-2 and C5 of the cyclol

peptide moeity creates variability as shown in Fig. 25.4. This group includes ergotamine.

Production of Ergot Alkaloids "#%

Fig. 25.1 Life cycle of the Ergot Fungus

Ergoline

Fig. 25.2 Structure of Ergoline

Fig. 25.3 Lysergic Acid

25.2 USES OF ERGOT ALKALOIDS AND THEIR DERIVATES

Ergot alkaloids and their derivates are powerful drugs and may be used as such or may

be the basis of semi-synthetic preparations. Almost all of them have one important

pharmacological effect or another, depending on the nature of the substituents and on the

tissue of the body concerned. Thus ergotamine will cause vessels to constrict while

ergometrine has a minimal effect on blood vessels but will cause the uterus (womb) to

constrict; LSD on the other hand will excite the brain cells in a manner not achieved by the

"#& Modern Industrial Microbiology and Biotechnology

Ergonovine (uterine contraction; treating post-partum hemorrhages). Methysergide (cranial

vasodilator; treatment of migrane headaches). Ergotamine (treatment of severe migrane head aches). 2-

Bromo-=-ergokryptine (semi-synthetic, reduction of lactation in women). Ergometrine (used for

treating post-partum hemorrhages). Lysergic Acid Diethylamide (for treating psychiatric disordrers).

Fig. 25.4 Some Therapeutically Useful Ergot Alkaloids

Production of Ergot Alkaloids "#'

other three. These various effects are exploited in using ergot alkaloids as drugs in the

manner shown below:

(i) Ergot alkaloids (e.g. ergometrine) have been used in mid-wifery to induce labor for

centuries.

(ii) Ergonovine, (the 2-aminopropanolamide derivative) is used to stop bleeding after

birth due to its stimulatory effect on the sympathetic nervous system.

(iii) Ergotamine blocks the sympathetic system and is used for treating strong

headaches such as migraines.

(iv) The diethylamide derivate of lysergic acid (known as LSD) is a powerful

hallucinogenic drug and is often utilized illegally for this purpose in many

countries, as well as for experimental psychotherapy.

(v) Most of the clavine alkaloids do not possess strong pharmaceological properties.

However a few of the didydro derivates have found use as strong stimulants of

oxytoxic (milk secreting) activity or of uterine contractions.

In recent times newer uses have been found for ergot alkaloids especially semi-

synthetic ones. These include:

(vi) ‘Nigericoline’, a derivate of lysergic acid which is a receptor-blocking agent and is

used for treating peripheral and cerebral circulation disorder.

(vii) ‘Lysenyl’ a diethyl derivate of isolysergic acid is used to treat hypertension and

migraine, since it is a serotonin antagonist.

(viii) In recent times it has been found that some ergot alkaloids affect functions

controlled by the hypothalamic pituitary system particularly the release of

prolactin (which deals with milk secretion) from the pituitary gland. Since the

prolactin level seems to play a part in the growth and development of certain breast

cancers they are used for therapy.

(ix) Some of newer ergot alkaloids have also been implicated as potential therapeutic

agents in the treatment of diseases such as Parkinson’s disease, lack of milk

production after child birth, and cancer of the prostrate.

(x) The alkaloids have been used as models for the synthesis of several potent drugs.

25.3 PRODUCTION OF ERGOT ALKALOIDS

A large market based mainly in Europe derives from ergot drugs; this market seems to be

expanding as more and more pharmacological properties are discovered in the ergot

drugs.

The methods for producing these drugs are three.

(a) Isolation from field cultivated ergot.

(b) Fermentation of the ergot fungus

Until now the bulk of the production seems to be the extraction of field inoculated

ergot. The pattern seems however to be changing. Fermentation is increasing and as with

the antibiotics wholly new drugs are being produced by semi-synthesis with substrates

derived from fermentation.