MEMORANDUM

BRISTOL LABORATORIES

UNIT OF BRISTOL-MYERS COMPANY

J, Lederberg Lecember 22, 1955

 

 

 

 

FROM. DATE.
TO J. Leia SUBJECT Bew ooeeeeosiee Sereening Program «
CL.

pear Joet

It wae very good to heer your recent word on the preductivity ef the orogras for
eereening for inducing agents. If nothing elee, the procedures is at least turning

ap cempoundes of se~far unknowa possibilities that would have Been overlecked by the
other procedures. I¢ happens that I was preparing « more general verspective eusmary
of my consaltation work at the tine that your last record arrived, I will defer that
te other records and teke this eccasion to anawer some of your eveeific questions
about the induction vrogran.

The instability of your active culture is certainly curious but the first question

I would ask is whether it has areoyped up agsin after repurifiestion of the isolate.
It ie perfeetly possible that it has no particular significance at sll. If it
really proves however that the ability to form this agent in an unstable property I
doubt if the most likely explanation is to be found in teres of an umueually high
wmatation rate. It would appear more likely that the agent haa a strong selective
effect against ite own parental type of cell. In that case onee you have obtained a
moderate amount of the purified material 1¢ may be feasible for you to sslect with
thie substance for colonies that would be more resistant to it and yet continue to be
able to form it. I¢ is ef course quite possible that mutagenic action of the agent
playe some rele in the higher variability of the eulturs.

I suppose the next question that is going to come up ie what to do with this tyne of
agent. [I have not heard any details at all from you about the technique of your
anti-tusor screening orogram and I wonder if you have developed this to the point
where I could have some rnowledge of it. It sveme to me rather important that «
possible anti-tumor agent be teated on more than one systes, although of course the
Ancites tumore are the most likely candidates for a soreening system. It is
vartioularly encouraging that so small a proportion of your broths are proving to have
indueing activity as this does make it likely that you are running into something a
Little bit new in the screening crogras. If your compound really has little or no
entibecterial activity then perhate it is going to prove to be of some considerable
imvortance for theoretical investigations whether or not it is something thet will
earn Briste] any profits right away.

Avrevcs my earlier croposnls, of course what I had in mind as the justification for
leoking for inhibition of inducing activity was the possibility of using this as «
system for screening for antiviral activity. There is really not such that I can say
about the poesible superiority ef this system over the more thoroughly worked over
setups that Asheshov and others have been looking into with regard te the inhibition
of phage lysis of bacteria. If after a brief introduction you should in fact find that
a large fraction ef your brothe were showing thie type of inhibition there would not
be a great deal of point in continuing with the program. As far as I know, however,
2

Tot J. Lein secenber 22, 1955 New Antibiotics Sereentng *regran «
Conwaltantship Arrencenent

nothing whatsoever i¢ *nown about the mechenian of action of the inhibitors thet the
Fetanical Sarden group had uncovered and for that reason there is scareely any
theoretical basie on which te dudge the veseible advantages of another kind of
screening progran. I would be rather astent shed myeeli if any general antiviral agent
were to be discovered and in particular it weuld surprise me if quch agents vere
picked up by the use of wacteriovkage systems, but I really could not give you any
sound theoretical reasons for that peesinian siwply beenuss we know eo little about
the poasibdle mechanisus which may be involved. On this surely empiriee] basis
therefore it would seen te mo that it would be worth giving © summary sort of trial
of » couple hundred broths fer this rind of inhibition. I¢ would be of Particular
interest to focus on those broths which had inhibitory activity on vhage deovelonment
without any great antibectertal action.

There are two possible pointe at whieh such aa inhibitor aight be expected te act in
the development of lambda. Ons would be with regard to the induction recess itesif
thet the vrovhage might somehow be desensitized to the intmetive effect. Thies
think would be waique for the lysogenic system and represent something that could aot
be picked up by leoking at the ordinary infective systens. Whether this veuld in
ture give you compounds that vould be of interest in antiviral theravy in quits
predlematicn] but we are working so much in the dark that it seens to me even « fey
ttrawe would be worth leoking at.

The second point where an inhibitor might be acting would be in the grevth of the
aotivnted phage iteelf and in this respect one would anticipate there would bo no
areat difference between the lysogenic eyetem and the lytic syateme which asheshor
hae examined. fRowever, just Because lambda ie a temperate vhage, not te mention the
fact that {t is simply a different phage from the syatems which Asheshov had been
uaing, I am sot eure how far ons should apricri axtrapolate from hie results. If ny
Understanding is correct, very few antibiotic broths have been found te have any
detectable activity agninet saimal virus aysteme. In the cireunstances {t would seen
to me worthwhile to make at least s trial to ses whether anti~lambda induction aetivity
is as crevalent ae the antie-phaze activity cf the Betgnical Garden greup or whether on
& purely empirical basis thie ia not an equally unique syaten. It seame to me
iapertant however that any follew-ups on the therapeutic poesibilities of anti-vhae
or anti~inductive compounds ought to be sade on systems which have at least a chemical
eistlarity to the bacteriophages, that is to aay, they should be tried on IW; ne vell
as WWA-containing animal virases. I know tee Ulttle ebout the kind of work that you
may have already deen doing or aaticipating doing ta thie field to dudge the efficacy
of your possible follow-up trials. I realise that the screening that I am avg¢esting
is one of the chancier things thet I would have any oceasion te sropoes te you «and I
certainly would net press the matter if you consider the preliminary routine sore
expensive of time or money than any possible benefits that You ean envisages. Theres

ie only the long-chance that the lanbde eyetem may vrove to be rather different from
the other vhage ayetems that have been examined.

Thies is the kind of thing of course that {t would be very much mere apvrepriets to

diseuse personally and I am sorry to heave te be in a position of mutting you off se
frequently about a yessidble visit. My work here is piled so high just at the sonent
that I can't see iweediately when 16 will be possible for me te come wat I will rake
every effort to find a date when I can do it, verhaps ta mid-January or thereaboute.
I would recommend thet we not count on thie as far at the exchange of information or
consultation is concerned until I tell you just when such a tris anotually would be

possible. If it would be feasible to manage this eo that we could soneult, say on «4
3

fo: J, Lein Lecember 22, 1955 . Wew Antibloties Seresning *regran ~
Consultantehip Arrangement

Saturday, that would give me much greater leeway in trying te plan the triv.
Unfortunately I have had some very preselng personal odiigations which have taken «
little of the time that I might otherwise have been able to extract from ay labors tory
duties, Ie there any chanee that you or Feliz or beth of you would be iaterested to
travel yourself and visit us im Madison? Ae leng as I can keep wo ay lab routine with
an hour or two's attention I would be able tc disense 211 of these eatters with you at
great length if thera vould be any poseibility of your coming here. I won't extol for
you the Yieconsin climate in the wintertine although I suspect 14 is not greatly
different from what you are experiencing yourself at the present tine.

On re~liatening te thie record, I realise that I had left out one point in poesible
mechaniens of inhibition and that is that an agent might prevent the lyets of 2
bacterium even though it had grown a fall quota of phage. If this were the ease, the
infective cemter ought st111 te be detectable as euch after the agent is diluted away
amd this would be a somewhat simpler thing to set up for the lysogenie responses than it
would be for ordinary vchage infective bacteria, I will give the whele question of
antivire) possibilities more thought and tell you whet I ean find out in due course,

Before I forget, I should caution yeu against the possibility of picking up hydrogen
peroxide ag an induging agent. Since this is possibly going to Be extracted by orennic
eolvents and since hydrogen peroxide iteelf has been reputed to have atrong inducing
activity, it is something te leek out for. If you have any reason te suspect caroxide,
I suppose the easiest way of ruling 1t out vould be ite inactivation by catalase.

t would like te go on now to a more general set ef topics. My understanding of my
position with you, Joe, is thet I am supposed to try to keep as much of a general
peravective as I ean and help to remind you of general {eseuss at some intervale in order
to help you and myself from becoming too much preoccupied with imediate details. For
thet renaen I have gone ever notes of our previous discussione and there are 4 list of

topics thet I think cought to be kept alive wrether or mot any immediate consideration
ie given of them.

le Want are eatidloties? I won't co ever any detailé of vrevious discussione which
would be somewhat obvious. The queetion I find myself left with is on vhat Basis
should Actinomycetes be unusualiy favorable sources for new antibiotics and as s
corollary is this preeumstion really true er should more attention be ziven to
ether erganians ae sources of new materiale?

2. ‘het is the statue of the operational evaluation of the screaning program? At what
stages are different percentages of cultures eliminated in terms of the floy sheet?
These are the Find of data that I would find very useful in trying to verfect
further eriticlems of the existing procedures. ‘that are the means of clasvifying
antibiotics te be sure of their identity excluding duplications ete. or is this 2
vreblem which ie outeide the Microbiology greupt Is the renewed emrhasie on low
Yielding eultures working cut im any reasonable fashion? 4nd in order to evaluate
any part of the program at least some arbitrary statenent has to be nade coneerning
the relative cost of each stage of sereeniag. Without an estimate, even an

arbitrary ons, it seems te me impossible to try to reconstruct the progres on any
rational desis.

3. We have had some mention of sersening compounds for usefulnese as adjuvante rather
than as primary antibictics. Has anything more come of thie?
pe

Ter

Be,

ub

6.

&

J. Leia Becember 22, 1955 New Antibiotics Sereening
Pregrem ~- Gonaultantehip
&rrangenent

IT am etill semewhat concerned about the prepertion ef celenies which are stated

to fail toe grow on transfer fren the original sei] inoeula. Can you remind me
again what the plating media are fer these initis] iselations? I an concerned
with the question es te whether these sedis would allow the development of
auxotrophic speeies of Actinomyestes. I redise that most Actinemyestes grow well
on o sinimal medium without supplesestation but aay saphaeia on finding new
wnterials eught te pay seme attention te the possibility ef Actinomyestes with
unusual autritional requirements. It sight even be worthwhile considering whether
te aelect again vigereus crototrophic forma by some nodifiention of the venieillin
seleetion method for exazple ae a means of looking for unicue auxotrovhie fores.

T realize the cemplications that you will have by way of developing seleetive media
with such «a proceedure. In our lab we have found Mycostatin to be « very useful
adjunct in preventing the growth of weld contaminants {in our Actinomycetes plates.
Of course you run the risk of inhibiting some Actinomycetes but thie is sousthing
werth considering.

Leeking over the flow sheet, I see there is some reference to contaminated breths
which I gather are disterted sven though they any show considerable sctivi ty.

Would it Be worthwhile to take such broths toe find out what the soures of antibiotic
activity ief You may either aise an antibiotic that could be chtained by
reivolating the Actinouycete or even conceivably this would de one way of stumbling
eato an antiblotic produced by s bacterium. At any rate I would think thet if a
broth hee substantial sctivity it ought net to be simply discarded because it
aypenre to be contaminated. Dose contaminated mean turbid? [ff se, I think there

ia the question whether in fact thees are contaminated broths or whether you my
not be rumuing inte seme peculiar Actinemycete forae which are capable of giving

a certain anount of turbid growth. In any event I weald be interested te hear

Jast what these oontacinated broths are and why they are Being discarded. Also,

ae concerns the flew aheet, the main critiaien that I think I might sake of the
precedure is the failure to subject every farment: tion broth that shewe a trace of
activity te a time series. It seens to me that the adiitional effort recuired te
test vhether substantial activity might aot be produced over different ranges of
Sime would be well-werth act drepzing any culture thet shows even a trace of activity
tn the firet sereening en a varicty cf medin. I am else concerned abont the

fallere of replication, that is te say, the indicated eccurrense of cultures which
geve satisfactory broths in the firet screening and which *fatied te hold up” on

the second sereening. Have you any ideas for the reasons behind thease discrepancies?
t emphasise minutine of this Kind beeeuse it seene to we thet the amall lsbdoratory
ia netually in a better position to trace down thia kind ef distraction than is the
large one which fe set up on an even more invalved routine basis and ae you have
reveatedly enshagized to me you have to find the ovtimal basis for your competitive
position as aginst the larger setups.

The flow sheets refer to a matation progras but I have heard oraetically acthing
from you ebeut what is actually done here and would like te knew {f you are wareuiag
thie avenue to speak of. It sseme to me that 2 more routine arcliieation could be
made of the mutation srogras to try to impreve tha activities of some of ths
marginal oredusers that you doen't quite want to diseard during the intersedtiate
sereening stazes. That in te say, on your flow sheet #1 near the vottom center
where 1% says further fermentations until active or disenrded, it seeme te me that
these would be umnsually ept candidates to put inte the matation program. Ones
again i¢ would seem to me thet 1% le in this group that you are most likely te find
Fart

6.
7.

&.

J. Leia December 22, 1995 Yew Antibleties Sersening frogranm -
Consultantehin Arrangement

agents which would be overleeked by other workers,

Since the purposes of a screening program is to start with e maxiaws of genetic
heteregeneity in the first place I wander if 1% would sot be worthwhile trying

te increase that initial heteregeneity by « rather gross treatment of your

sanplee before they are even plated with mutagenic agente. For exemnie, it

might met be completely unvwerthwhile to include euch an active mutagen as eraserine
in the medium in which the samples are held Just prier te plating or even ia the
plating media themselves. This ie kind of a wild abot but it would de one meane
of increasing the diveraity of types on which your reutine Weresning is going to

be exercised.

AS anether possible apclication of wutagenle or inducing agents, I am reminded
ef the finding that the vactoriocina, like nyocinaas, are released from daeterin
under conditions which are closely analagoue te the indugtion of bacteriovhace.
Thie might have some Bearing on the improvement of yislds ef antibiotics.

Thie wae juet a small taterraption now, Joe. There wae a little fire in the roem next
deer but after 10 or 15 minutes ef scurrying around the thing wee out under contre}
eo I'll try to get back to order new.

9.

Perhaps the indusing antibiotie that you've just picked ur would be worth ueiag

in a triel of this princi, firet decause it has at least a low antibacterial
activity asd would net confuse the test and escond because of the presumption

that it does have some effect on the Actinomyoots. At least if you are constructing
a wearisty of media with various inhibitors destgned to help croveke the

production of antiblotios, I would suggest including in such a series compounds

with Enown induelug activity.

One of the most familiar facts of screening for antibiotics is the very high aninal
toxieity which most of these compounds demonstrate and which ef course makes then
selese for therapeutic purpeses. Save you any notion why se many of these agents
should be so texic? Ferhave this reflects « general protoplasmic toxicity fer
whish the screening crogream would be of course selective bat it's a little
surprising in a way then that the Actinomycetes themselves should fail te be
muscentible te agente whose common bond of toxicity will then be seme bacteria

and some samzals. Sven if thie is true it suggests the possibility that there may
be seme evecies specificity in the toxic action of seme of theses therapeutically
useless materiale which raises the question of whether you thought there sight be
other croduct applications fer these texic agents. I had in mind the nossibdility
of one use as pesticides of some Kind for example in the slisination of rodenta,

a use which provebly would require denenttration of a certaia amount of selectivity
or apolications such as that of velsoming of lakes in order te remove fish. ould
it be toe far fetched to propoes some consideration of toxicity tests on, say for
example, gold fish, as part of the pregranm becanes I atepeet that thers might be a
market for any extremely toxic ageate for such diverse purposes, «nd you wight eat
a lead on some activity of toxic effects. Wow I appreciate that antibiotics may
turn out to be tee expensive in terms of coet of production to compete with other
agente but thie is « vosaibility at least to be kept in the back of one's mind and
the particular question where there aay be some scecies svecificity and toxieity
which would meen of course that using, say the mouse, 26 2 screening sgent may not
tell you all that you want te kmow about man. Jomewhat amusedly I vould alea be led
Ter

9.

10,

ii.

12.

6

d. Lein tecenber 22, 1955 New Antiblotios Sersening Progran -
Consultastehio Arrangeseat

to inquire whether some of your products aight ast have other kinde of toxic
activity and might for exanple furaich a cheaper and atf1l marketable eubdsti tute
for colchiein. 1 don't knew how large the market for a drug of this kind ie wut
{f you leek at the price which ia being oherged for it, I think you can see that
1% would not take a tremendous market to wake it worthwhile te preduece an
effeetive eubstitute. I'm sures I don't have to orepese how one sight go about
serevning for thia type of setivity. it would tavolvwe I imagine fer the roughest
tests looking for the formation of © tumors in onion roots. 1 would keep in eind
that compeunde with C-mitotie activity have Been proposed also as sati-tumor
agente and if yeu are disposed te wild flings 1¢ wight act be completely worthless
to give seme consideration te sereeniag for thet sort of sotivity in relation te
tumor therecy a6 well as fer a polypleidising agent.

A deoieive atage in your sereening program 19 the initial oleking of the colonies
which are going to be follewad in mere datailed study. Yerhars the tine thet I
epent during my last vielt wae not entirely typical ut I nave the Sealing thet
this particular phase of the work was perhapa not es cleaely tied in with the rest
of the program ae cerhapea it sould be, and that 14 would be worthwhile considering
whethsr a great deal of professional attention should be concentrated on this
specific vhave of the sereening program.

fe you happen to know whether chiertetracyeline is the yellow pigment which is
produced ty ot ? &If this is so, then 16 seems to me that

it would be useful ae an agceet of the mutation progran te leek inte the
pigmentary variation of this Actinomycetes. I Rad in mind that the cceurrence of
mutants whieh still had apurectabdle antibiotic activity, sithowgh lneking the
original yellow pigmentation, might furnish a technique towards the discovery of
etill further antibiotics related to tetracyeline. I will be aildly surprised
should this be a feasidle cropesal you have not already explered from a womber of
angles. If this is the ones, I would appreeiate for the ourpese of my @wn general
background being referred te any literature or other informetion on thet partionler
saint.

4o have had some diseussion about sereening prograne for the detection of Plolorical
activity other than dDecterial iahibition, These have included virus induetion in
the F-12 eyeten whieh seems to be very well wader contrel, the inhibition of virus
develepnent which ts diseuesed in more detail on the other side of this record end

a orepees] that you brought up on testing for the produstion ef smell colony variants
ig yeast. Gm this last poiat we have been im some contact with Evhrasei for our

awn purveses and I an eti1l net very hapoy about the aprlicabdility of the red
adenine-requiring mutant fer a simplified vrecedure. HEvhrused hee sent ue cultures
in which thie affeat is suppcesd to pertsin but evidently « certain amount of
maniculation of the medium is racuired in order to insure thet the red adenine

stook in allowed differentiation of normal and vetit. Enhrovel writes thas it ts
necessary to use a very low sugar vedium in order to nrevent the vetit tyres from
showing the red color ss well. In faet, sinee we are trying to adapt alnort
preaiaely the same eyeten for cur awn parsoses it seams te me that i¢ would be xoet
practical for you te walt on thie until ve heave leoked inte thie curselves unlese

of course you heve euch mere encouraging and detailed information from other sources.
Tam willing to predict that you will find 2 rather close tut act «n absolute
correspondence between the agents which are detected ae inducing agentes and these
which will ehew up by thie varticular syeten whitch {s not to say that it would not
be worthwhile to go shead with the yeast orovided thet it does not reorséent too
?

fot J, Lein Decenber 22, 1955 Hew antibiotics Sereesing rogram -
Con@ultantehio Arrangement

12, much of a distraction to your ether efforts. The main question is vhat to de
with the further evaluation ef agente which are picked up by these biological
activities. Any possible virus inhibitors would of cource be worth studying
fer their chemotherapeutic effects in egge, in mice etc. and I think, unless
too many of them tura up, they should de given a fairly comprehensive
oxemiantion with a variety of animal viraces. I have already asked you the
question ae te the statue of the tumor agent soreening progran.

13. A little while age you seked me if I had any notione about properdin. I have
asked seme of my iwmanclogies)] colleagues about this. There evidently is aot
very much exagt information on whet properdia is or what it ean de. It can be
best summarised by calling it a component ef complement and in that reepect
nothing very remarkably sew. I could act find out anything that would suggest
the desirability of geing inte thie kind of vrogras unleaw. you meant te de it in
suite a large way at a sajer eter.

14, Apropos of cur earlier discussione on beeterinl replacement therany to mitigate
complication of the use of bread epectrum antibiotics, I sav an séverti cement
in a Jew York paver last week which advertised the uae of acidovhiluese atlk ‘te
counteract the dangerous side-effects of antibioties". I am not slear whether
thie is intended to be used in conjunction with antibiotic therapy or as « means
of hastening recovery from it. Have you heard anything about this? I have aot
agen any techaical medionl literature thst would helo to justify the uee of this
particular prevaration. It does however suggest an anele, nawely, inatead of
feeding something called Serratia ae 2 separate sedical article the development
of approvriately antibietia-reei stant strains of organinws used in the aenufacture
of milk, sour creas, cheese ete, which might at least carry net «o bad an oder
to the median] srofeseion in their use ae Biclegieal therareuties, I am still
concerned ghout the possible dangers of introdueing any new erganiame inte tha
eituation until there has been a very thorough etudy of the noasible cerile. Te
feed aciieochilue of a standard variety to a person reseiving antibiotic theracy
ie ome thing and if the Lactobacilli are still eneseptible to the antibiotic it
may be expected to do very little good at all. ‘If we really wanted to secoamlich
something by using antiblotie=-resistant Lactobacilli. to start with, we rum the
risks that awe aseociated with their introduction in sesentially oure culture inte
the human cut. I will be very euch interested to hear the Llamediatea quteome of
the experiments that you cxzid were being started with things like the Serratia
organine that I had sent te you. In any event there does sean to be some
poasidility that should be lecked into of modifying that crogram to the development
of antiblotico-resistant strains of micreerganieme which are alrendy customarily
weed in the food industries, Of course from the point of view of ths Bristel
Cewpany one difficulty with thet is that te does net directly lead to further
produet develonpaent wat it cerhaps fe something that you coulé work an together
with some of the dairy industries or some veeple of that rind. In any evant I
am mare that you vould have some interest in any vrocedure which «ould helv te
ereve the suitability ef antibiotics and chemotheravy.

Thies is about al] I have to say ty way of Lemediate summary. The only thing I would
repeat fer vurpove of emphasio and just because it is a rather conerste thing concerse
thie detail on flow sheet #1 in the sereening oregran. In ene eample of data that you
geve me there wan as I reeall a enlit of so-called 75 non-active to 9] active breths in
the first scresning. Of these 91 netive broths in this partienler serier, 14 were
rejected for one reason or another and 75 were given a time study. That ie te sey,
fo: J. Lein Lesember 22, 1955 Hew Antibiotics Yereening Frograa -

Consultantship Arrangement

about an equal nusber were discarded as being “non-active” or being active. Hovever,

ag best I can wake out the se-called non-active organisms did include types which had
ehovn very snail degrees of setivity. I am sure thet you're aot doing a mtation
program on all ef them. My immediate suggestion is thet every culture which hes

shown any activity whatsoever in any broth ought te be given a time study and in fact

it might be worthwhile giving tines studies even te the arvareat negative cultures. Few
that meane of course thet you'll Reve to cheese your media semewhat Dlindly. The

ether situation wae that the main scuree for cultures to be used in the sutction crogrem
or perhaps not the ones which are completely inactive but the ones whieh wight otherwise
be discarded for lack of sufficient aetivity to be rundown biccuenteally.

A& per custom, vleaee let me knew if I have been clear enough about any particular
peiat. The reason that I wrete to br. Gourevitch rather than use the VYelecewriter was

sizmply that I hed the typewriter handy and net the department machine at that partienler
time.

Beet wishes to you and Pat.
Youre sincerely,

Teshua
fii fig