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The inhibition of ribonuclease by acidic polymers and their use as possible antiviral agents
ARCHIVES
OF
BIOCtI~MIS’TRY
ASI)
HIOI’IIYSICH
73, :%jti--:%j:j (1068)
The Inhibition of Ribonuclease by Acidic Polymers and Their Use as Possible Antiviral Agents Hans Heymann, Z. R. Gulick, C. J. De Boer, G. de Stevens and R. L. Mayer
Invasion of I~schcrichia coli ~11s by the T-coliphagcs in\‘olv(:s, ill general, a cytological disorganization of the host c*ell, particularly of its deoxyribonucleic acid (DNA) (1). A n increase in intracellular deoxyribonuclease (DNase) activity upon ‘I’z-bacteriophage invasion has been demonstrated, although it has been st,ressed that a necessary connection between activated DiYnse and phage multiplicat,ion htls not been established (2). T,ess-detailed biochemical illformation is available regarding viral invasion of animal cells, but an at,t,ack on, and a reorganization of the protein-synthesizing system, gencrnlly believed to be ribonwleoprotein, has heel1 recognized (3, 4) for, c.g., vwcinia virus grown in cmhryonated (‘ggs. On the hypothesis that interference with trwleic: acid nwt.al~olism might prevent 1:irus multiplic,at,ion, MT have searched for substalwts capnblc of inhibiting enzymes inwl\-ed in biological tr:tnsfornlat,iotls of polynucleotides. Similar views have bee11 advanced by Kradolfcr (5) in an attempt to find a possible correlation between antivirus and antienzyme activity. In the course of this investigation WC have directed our attJent~ion to the degradative phase of nucleic acid metabolism. Although a number of enzymes are involved in the breakdown of nucleic acids, the primary objective of the present study was the inhibition of ribonwlease. Investigations on the inhibition of ribonuclease have been reported by ZGllner and Fellig (6), and by Roth (7). Recently Vandendricswhc (8) described the diff’erential inhibitory activities exerted by several 366
polywids 011 t,he depolymerizing ability to split cayclic nurleotidce.
:rt%ivit.y of rihonuclense,
and on it,s
.411 wlutiotw ittf.etded for t hc enz~.me assxy were prepared with g$l:rss-distillctl ‘I’0 :I mixture of 1 ml. of ritwnuc~lc;ttc? holution ant1 4 ml. of 0.1 III wetat c ikrtffrr. IJH 5.0, were ddecl 1 ml. of lruffer icontrol) or of I,uffered irihihitor snlut,iotr, :rttd f,he tubes were plied in 3 30” bath. One-milliliter poltions of the enL\.ttte .solutiotl were then d&t1 to each t.ut)c. ‘Ten minutes after each :ddilion, 2:nll. diquots were removed ant1 mixed with 2-ml. portions of 2.570t,richloro:weti~~ :tcitl wtlt,:lining 0.2570 of utxiyl acetate :lftrr 30 min. the suspensions were cetttrifugctl ant1 filt.crect, and 2-d. :rliquots of t.lre ~uperri:~tnnts were digested wit IL wlfuric :ici(l :~nd hyclroyeu I)eroxi(le for dctrrmimttion accortlitig to Fi,kr antI \v:I~c:I~.
~UiJtJ:L~~O\\-
(10,.
( ‘f’fd:~in ittltiltitors,
wlictl
l)t’rsrtlt
iti altlttwi:rlrlc
cottc:etit,r.:lt.iorts,
tctlcl 1.0 c:iUsc:
368
HEYMANN
ET AL.
precipitation by MacFadyen’s solution of the normally soluble products of I1N:~se action. This “coprecipitation” simulates inhibitor activity. To avoid possible errors, separate controls were included in which the inhibitor solution was added to IO-min. digests of ribonucleate and RNase, and the “inhibition” value obtained was subtracted from the experimental value. Frequently, the coprecipitation noted in such controls exceeded that occurring in the actual inhibition experiment in that the corrected inhibition was seen to pass through a maximum with increasing inhibitor concentration. Therefore, correction of the copr&pit,nt,iorl error occasionally led to low estimates of inhibitory power, but in most instances coprecipitation was negligible at effective concentrations of inhibitor. Inhibitors were examined at concentrations of 1.2, 12, and 121 pg./ml. to 01). tain an approximate est,imate of potency. Since more detailed st,ud,v of selected samples showed that an approximately linear relationship exists between per cent inhibition and the logarithm of the concentration, the concentration yielding 50% inhibition (ZC’,o) was determined for each compound by graphical interpolation.
Precision of Results Upon repeated determinations of an ZC~O value, the results lay wit,hin one-half to twice the previously found figure. This variability results primarily from the photometer readings which vary by f0.02 density units = f2 pg. of phosphorus in serial determinations of identical standards. The total soluhlc phosphorus in n complete digest ranged from 20 to 30 fig&ml. aliquot. l’reincubation of enzyme and inhibitor for various periods of time w:ts Gt.hout influence on the ZCjo . The effects of changes in pH, electrolyte concentration, and presence of proteins on inhibitory action were studied in separat,e experiments (see below). RESULTS
According to Ziillner and Fellig (6), and Roth (7), heparin is a powerful inhibitor of ribonuclease. In our experiments ICsO-values for this substance varied between 12 and 60 pg./ml. for samples of different provenience. In view of this high activity, we tested other anticoagulants, such as crude hirudine, dicoumarol, bis(4-hydroxycoumarinyl)acetic acid, phenylindane-1 ,3-dione, potassium oxalate, and sodium humate (11). With the exception of sodium humate, none of the afore-mentioned anticoagulants demonstrated any activity as an inhibitor of RNase. A. Acidic Polymers Related to Humic Acids Noting recurring prepared quinone exhibited
that heparin as well as humic acid is a polymer with frequently groups of pronounced and moderate acidity, respectively, we polymers by the interaction of formaldehyde w&h hydrosulfonic acid (12) or gentisic acid (13, 14). These compounds ICho-values of 55 pg./ml. and 5 pg./ml., respectively. On the
INHIBITION
OF
RNASE
BY
ACIDIC
POLYMERS
XI)
hasis of these findings 64 additional phenolic and related polymers were investigated. From a chemical point of view the substances may be divided into six groups according to the starting materials used, and, in each group, whcrcrer applicable, three modes of polymerization will be considered. The subst,ances are arranged accordingly in Table I, which records the IC”5,,-values found. E\-idewe for t,hc polymcria nature of the substances prepared consists of t,he following general points: (a) production of highly colored, wateriIwoluble but base-soluble substances from soluble starting materials 01 101~ molecular weight by procedures known to yield polymers in :wdogous cases; (0) failure of the products to diffuse through cclloph:u~e membranes, a criterion applied to a considerable number of the products; (c) sedimentation experiments in an ultracentrifuge as applied t,o sclectcd substances, particularly polysulfonic acids for which the soluljility criterion mcnt~ioncd in (a) fails. Thus, orienting sedimentationwtc mwsuremcnts for compound R 37 with the aid of an improvised light-:rbsorpt,ion m&hod revealed a sedimenting boundary with 820 = 1.(i Svcdberg ant1 obvious signs of high polydispersity. Compound f 1%in :qucous solution at pH 7 exhibited a slowly moving boundary, I)ut paper electrophoresis at pII 7-7.5, although twhnically unsatist’acatory, rcvwled the presence of a multiplicitIy of substances. Compound B 12 uxs srdimentable, but its higher homolog, the product obtained f’mn reaction of formaldehyde w&h 2, K-dihydroxy4methylbenzenesulfonic: acid \vas not’, nor was the product resulting from formaldehyde and 4,5-dihydroxybenzene-I ,2-disulfonic acid. The reaction of formaldchyde and sulfosalicylic acid gave a crystalline substawe of low molecular \vcight. Undoubtedly, the substawes obtained from formaldehyde and ?,-hytlrosybeI~zeIlesulfoni~ acid, or hydroquinone-2,5-disulfonic acid are 1l0t polymeric. Thr nat,ure of the monomers is the primary basis for the chemical c*lassification and characterization of the polymers. Yrom analogy to i,hc st,ructures of the phenol-aldehyde resins, of emeraldine and its relat,ives, and of the “synthetic humic acids” (15), plausible structures may bc deduced, but no experimental work was done to confirm these inferences or to find possible differences in fine structure that might, pertain to the biological activity of the substances. Sinw t’hc acidic polymers were intended for tests in biological systems, it \V:IS desirable to study the influence of environmental factors on th(i 11I\‘nsc inhibition by these substances. Enzyme-polyanion interaction
Anti-RNase
and Antivirus
Activities of Acidic _____-
Pol?ynlers Effect
Serial number
Polymerizing polymerizing
aldehyde RCHO, othr agent or nature of monomer
‘ink%&, / IGO, pg./1711.
on viruses
’
-’ OH
Group
I. Polymers
of ,/,+COOlI
I II \/ OH (a) Aldcl~ydc RCRO,
1 la 2 3 4 5 6 7 8 9 10 11 12
polymers
R=
H (basic crtt:tlysis) H (acidic catcdysis) 4-Nitro-l-fury1 1-Thienyl CHO4-Kitrophenyl l-Fury1 3,4-Dichlorophenyl Phenyl Methyl 3.Methoxy-4-sulfol)llc~~~l p-Dimet~hyltllninol,hcn3-1 4-Pyridyl
-1 2 3 3 4 9 10 24 25 30 34 111:1ct. Inaci,.
(b) Osidntive
16 17
# la + gcntisic :tcid (!hlorohydroquirlorlc (‘Hz0
Group II. Polymers
+
acid
1
1
$- 1
,I
Imp of 3,5-, 3,4- :md 2,3-dih~droxyl)ellzoic ltnd related compounds HO / COOH of 0\ OH ((0
polymers
Alticliy~e
370
( I
+ + + + +-
i I I 1 1
l~olymcrs
gcnt’isic
1. Polymers
+ T -
+ ~~~ acids,
L-~ +
Serial number
= I
= R&S? Iir lhibition,
Polymerizing aldehyde RCHO, other polymerizing agent or nature of monomer
Effect on viruses
IGO, pg./ml.
RCHO, R=
1s 19 20 a1 228 23
-
1 2 3 7 12 Illact.
HLThienyl (~‘HOl’henyl 4.Nitro~l~furyl 4.l’yridyl
+ -
(c) ‘~ripherlylmeth~lle 24
#20 + gentisic
polymer
acid \ 2. l’olymers
I 2 __~ COOH
1
+/
-+--
of HO 0 / OH
(u) Aldchyde
polymers
RCHO, R=
25 26 27 -~~~ --~-
1.l”Ury1 I’henyl H-.-__
3 1 5 ~~ (h) 0sitl:ttive
I 7
~ +
polymc~
-i + + OH (u) Aldehyde RCHO, R=
X0 31 32 33
polymers
+-
H4-Nitro-l-fury1 CHOLThienyl 371
372
HEYMANN TABLE
Serial number
Polymerizing polymerizing
ET AL.
I-Continued
aldebyde RCHO, other agent or nature of monomer
(b) Oxidative 34
Ii&O~
Ill+/+ 4. Related
35 36
polymer
polymers
1 CH?O + hydroquinone-2,5-diacetic CH& + homogcntisic acid Group
III.
Polymers
acid
+
1 3
of dihydroxybenzenesulfonic
acids
OH (CL) Aldehyde
RCHO,
37 37a 38 30 40 41 42 (u’)
polymers
R=
13 55 18 32 300 Inact. Inact.
H-(Na salt) H-(free acid) 4-Nitro-l-fury1 l-Fury1 Methyl 4.Pyridyl CHOPolymers
of
of formaldehyde
and various
dihydroxybenzenesulfonic
acids
SOiH
__.___ 43 44 45 37 37a 46 47
.--~------
RI=Rz=OH R,=R,=OH R,=RI=OH,
Ita=Cl
vs. vs. Itl=Ra=OH, R,=R,=OH,
R.,=(:H:~ R:,=SO,H
3 4 6
+ -
+
13 55 19 72
V.S.
V.S.
-
TABLE Serial number
I-Continwd Effect on viruses
Polymerizing aldehyde RCHO, other polymerizing agent or nature of monomer
Influenza
(b) Oxidative 48
polymer
acid + KkWs
Hydroquinonesulfonic
(c) Triphcnglmethnne 49 Group
Hydroquinonesulfonic IV.
Polymers
acid +
;6,In
~>600
1
-
/
-
+
~
-t
polymer I
2
of aminocarhoxylic and aminosulfonic with FeS04 and Hz02
acids,
hlonomer
50 51 52 .A ‘8 Group
Anthranilic acid 2,4-Diaminobenzoic acid 3,5-Diaminobenzoic acid 2,4-Diaminobenzenesulfonic V. I’olvmers
5 7 9 15
acid
of substituted hydroquinones preparctl T
OH Rl=Rs=(:l R,=U R,=R,=(:l R,= (:Hc RI=Cl, +CHIO Sodium humate Group
from VI.
lignite l’yrrole-quinone 111
---
Vaccinia
polymers
-
+
prepared
I / i
-
/
+
I)v oxidation
with
374
HEYMANN
TABLE Serial number
T
ET
AL.
I-Continued Effect
Polymerizing polymerizing
on viruses
aldehyde RCHO, other agent or nature of monomer Vaccinia
R,=O, R,=N-ChH, R,=ClN<, Rz=R,=U Rl=O, Rz=Rn=(:Hs R,=O, li~=R.i=(:l R,=O, 1tc= -ioH,)2(:ooH II, = ( )
61 62 63 (i-1 65 66
U If no designation
is indicated,
3 3
x
Concentration
-
-
II
of RNaee Inhibition
on pI1 Per cent
(‘om~Kxmd
-
R=H. TABIX
Dependence
-
*5 4 27 ’ 160 1
inhibition
ICao at pH 5
5
0
I I 1 IO 2
x0 54 82 95 x5
66 34 81 x7 X6
at pH 7
8
pg./ml. 60
60 I 6 56
12.4 6.2 62 12.4 3
43 15 54 32 61
28 9 357 22 45
has been shown to depend strongly on pII and ionic strength. Ziillner and Vellig (G) observed the pH dependence of RNase inhibition by heparin, while other variables were studied by AZyrb&ck and Persson (16) in the system, heparin-fi-amylase. 1. Eflect of pH Changes. The effect of pH changes upon the antiRKase activity of a number of polymers was studied by measuring the degree of inhibition obtained for a given concentration of inhibitor at pH 5.0, 6.0, 7.0, and 8.0 (each value f0.05 pFI unit). ‘I’he results appear ilk Table II. With rising pTI, the concentrations of sodium humate and compound # 1 required to give a finite and measurable response were substantially higher than the ICsO , whereas compounds $46 and F$56 appeared to be less affected. If the p1-I of an alrnost completely inhibited reaction mixture is raised, at least part of the reactivity is restored. A digestion mixture containing the standard concentration of RKnse, and 12.4 pg./ml. of sodium humate (%60) in Verona1 buffer pH 5 (8 ml.) an inhibition of 88% by comparison with a suitable control. III a second ml. of buffer pH 5 was added at time 0, followed by 4 ml. of buffer $1 8 min. of incuhntion, giving rise t,o a final pH of 7. The digestion was :tllowed
RNA, showed series 1 after IO to pro-
TABLE Effect of 0.9% XaCl Compound
ry
III
on the Inhibitory
Concentration
Action cent inhibition \Vith SaCl Without NaCl Per
pg./d.
60 1 Sod. hepnrin 13:~13707~’ I&L 13707 u I’olyure:~
I’mn
72.4 6“ 72.4 12.-I 1.2
3,J’-di:tn~it~otlil,t:rleyl~
teed for m addit,iond 10.min. period. inhibition of 579& wits otmervetl.
86, 88, 8!J so, 92 SO !J-I 60 ‘2,2’--I 1‘~sullonic .
By coniprrison
6-l) 73, 51) ii, 92 2-I 7s ‘24 acid
\Vilh :I suitahlc
~ont.lol,
:w
2. Changes in Eleckrolyte Concentration. The results obtained for four polymers in experiments in which the electrolyte concentration of the medium was altered are recorded in Table 111. Sodium humate and compound B 1 are less influenced by salt than is sodium heparin, although it should be not.ed that compound B 1 \vas present in significantly higher concentration. The results obtained with the polyurea from 4,4’-diaminodibenzyl-2,2’-disulfonic acid (Ra 13707) indicate that the sulfat,e groups of heparin are not neccssnrily associated wit.h the anti-inhibitory effect of salt; the results indicate that the salt effect becomes more significant at lower inhibitor concentrations. The effect is self-limiting as seen from the behavior of compound B 1 (82 pg./ml.) in the presence of increasing concentrations of salt (0.9, 1.8, 3.6 %), which do not, greatly affect the inhibition (93, 86, 78 %, respectively). 3. Influence of Serum Albumin. Since acidic polymers possess considerable affinity for the basic sites of any protein, the effect of bovine serum albumin upon their antienzyme activities was examined. The albumin used (Armour, Lot ~Pl3706) possessed a weak ribonuclease activity, which was measurable when t’he protein was incorporated in the standard digestion mixture in concentrations of 10 mg./ml. and 1 mg./ml., but which became negligible at greater dilution. Albumin abolished the a,ction of normally effective inhibitor levels when present in the standard digestion mixture in concentrations higher than 100 pg./ml. Moreover, at this dilution the potency of the inhibitors was markedly reduced, as shown in Table LV. Evidently there exist no striking differences in the eff’ect of albumin upon the anti-1tNase activit,y of various classes of compounds.
376
HEYMANN
ET
TABJ,E Inhibitory
ICsa In presence of 1y&;l.
In absence of albumin pg./??tl.
Fluid
Dilution
Ratio: ICao inayb;t3~1Cao in
az.lml.
1 4 13 9 25
55 1 37 5 8
of Yolk-Sac
IV
Concentration in Presence OT Absence of 100 pg./ml. Bovine Serum Albuinin
Compound x
Efect
.%I,.
20 52 110 90 250
0.05 0.08 0.12 0.10 0.10
TABLE V and Amniotic Fluid on Inhibitory (4 b&ml.) Per cent inhibition Yolk-sac fluid
of egg fluid
Action
of Compd.
#l
observed in presence of Amniotic fluid
0 29 0 56 17 58 In absence of egg fluid 61
I:10 1:lOO 1 : 1000
4. InJluence of Yolk-Sac Fluid and Amniotic Fluid. Since the action of certain acidic polymers against influenza virus when introduced into the chorioallantoic sac of infected embryonated eggs was abolished when injected into the yolk sac, the effect of yolk-sac and amniotic fluids on RNase inhibition was examined. As is shown in Table V, both substances diminish the RNase inhibitory action of polymers, however, to different degrees. The anti-inhibitory effect of amniotic fluid begins to disappear when the material is present in a dilution of 1: 100, whereas that of yolk-sac liquid still persists in a dilution of 1: 1000. B. Anti-RNase
Activity of Other Acidic Polymers
In addition to the acidic polymers prepared for the present study we have examined a number of other acidic polymers with frequently recurring acidic groups. Suramin (Bayer 205) exists in solution in micellar aggregates; the substance has been shown to inhibit enzymes, e.g., urease (17). Suramin inhibited RNase at 140 pg./ml. Chitin sulfate’ 1 We thank chitin sulfate.
Dr.
I. B. Cushing,
Abbott
Laboratories,
for a sample
of sodium
INHIBITION
OF
ItN.4SE
BY
ACIDIC
POLYMEltS
3’7
7
(Is), known to possess a heparin activity of 32 units/mg., was similar to heparin in anti-RNase activity. Chondroitin sulfate proved to be inactive, in agreement with the results of Zijllner and I’ellig (6). iYo noteworthy activity was observed with carboxymethylcellulose, p&in, or rennin; tannic and alginic acids were of questionable activity. Neher and Kradolfer (19) have reported that polymers arising from the action of phosgene on aromatic diaminocarhoxylic and sulfonic acids inhibit influenza virus multiplication in eggs as indicated by hemagglutination titration, and have referred to the anti-Rn’ane activity of some of these polymers. We hare examined most of the polymers mentioned by Neher and Kradolfer as well as a number of related compounds. While none of the monomers, from which t’hese sulfonic polymers were derived, possessed activity, very potent TZNase inhibitors were found among the polyureas. For example, 4,4’-diaminostilbene2,2’-disulfonic acid was inact,ive, but the polyurea prodwed from it, inhibited RSase at 40 pg./ml. The polyurea from 4 ,I’-diaminodibenzyl%,2’-disulfonic acid (Ba 13707), differing from the foregoing by the absence of t,he &ilbene unsaturabion, inhibits ilt 1 pg./ml., :11d the same potency was observed with the polymer from benzidinc-3,s ,3’, 3’t,ctrnsulfonic acid. \veI’c hs expected for neutral polysacrharides, sar11ples of tirxt~r:tlls in:wtive regardless of t,hc molecular weight of t,he sample investigated. C. Anti-RNasc! Activity of Nonpolymeric S’ubstanccs A number of nonpolymeric substances have been reported as RNase inhibitors in the literature. Cupric sulfate (20) was effectfive in our experiments at a level of 200 pg./ml. An anti-RNasc effect>of sulfhydryl reagents has been reported by Ledoux (21); however, the results have recently been questioned by several nut,hors (22). We found N-ethyl muleimide t’o be devoid of inhibitory power. Converxcly, we could confirm the activity of lilac-leaf extract reported by Bernheimer and Steele (23). Sodium dodecyl sulfate has been used t)o inhibit RNuse during isolation of RXJA from natural material (21, 25); in our assay m&hod strong inhibition was observable at about 300 pg./ml. Roth (7) found I hat 2-aminobenzimidazole produced 50 % inhibition at the relatively high concentration of 1.7 X IOF M (2.26 mg./ml.) in media containing 0.04 Pg./ml. of RNasc. h number of substances report,ed to be effective against wrtain viruses \vcre tested. Among the antibiotics, penicillin has been shown by histo-
378
HEYMANN
ET AL.
chemical means to inhibit RNase (26), but we did not observe any inhibitory power in vitro. None of the following substances inhibits the action of RNase: representatives of the tetracycline group, magnamycin, puromycin, streptomycin, chloramphenicol, patulin, aaaserine, cycloserine, 5,6-dichloro-1-/3-n-ribofuranosylbenzimidaaole, and the 4,5, 6-trichloroanalog2 (27)) 6-mercaptopurine, “Benuron” (a polysaccharide of bacterial origin), butanedione monoxime monothiosemicarbazone (28)) and isatine-3-thiosemicarbazone (28, 29). The only apparent exception was 5-(2’ ,4’-dichlorophenoxy)thio-(2)uraciF (30, 31) which gave 50% inhibition of RNase at about 120 pg./ml. Ot’her thiouracils examined were inactive. The singular nature of the observation is receiving further study. As detailed in the section Results, a number of hydroxybenzenesulfonic acids failed to yield polymers, and these substances are, not unexpectedly, inactive as inhibitors of ribonuclease. Moreover, about 200 diversified organic substances of low molecular weight and comprising many classes including acids and bases of the aliphatic, aromatic, and heterocyclic series, quinones, purines, pyrimidines, dyes, and detergents gave uniformly negative results. Specific inhibitors of RNase of low molecular weight are evidently rare, the only possible exception found in the present experiments being the afore-mernioned t,hiouracil derivative. DISCUSSION
Among the 66 acidic polymers tested, a considerable number displayed marked anti-RNase activity, exceeding, in many instances, that of heparin. In Groups I and II, condensat’ion with formaldehyde yields the most active polymers with the except’ion of the derivatives of 3,4-dihydroxybenzoic acid (compounds A 25, 26 27), although even in this case the formaldehyde derivative is of high, but not of maximal potency. Aromatic and heterocyclic aldehydes yield compounds of intermediate activities, while introduction of a nuclear nitro group leads to slight potentint,ion. Basic aldehydes give rise to extremely weak or inactive compounds as shown by the results obtained with compounds B 11, 12, 23, 41. 2 We thank Dr. I(. Folkers, Pvlerck 8: Co., for kindly benzimidazoles mentioned. 3 We are obliged to Dr. G. H. Hitchings, Burroughs kind donation of a sample of this thiouracil.
supplying
samples of the
Wellcome
& Co., for his
Such polymers may be internally neutralized, lacking t’he anionic character required for RNase inhibition. Oxidative polymerization in general yields highly active polymers of the “humic acid type ” in (;roups I, II, and, of course, V, which is composed entirely of this type of substance. Only the aulfonic acid derivative, compound %48, differs in this respect. The triphenylmethane polymers represent an increase in complexity over their diphenylmethane properties acprecursors, but no noteworthy alteration of inhibitory companies this change (compounds # 1 and 16, 27 and 29, 1 and 49). The sulfonic acid compounds (Group III) exhibit a greater variation of :&ivity wit)h structure than do the ot’her classes. The six aldehydc polymers examined conform t,o the generalization expressed above, with the exception of glyoxnl, which surprisingly gave rise t,o a11 inactive product. The second section of Group III includes only formaldehyde polymers derived from mrious sulfonic acids. Cross-comparisons of compounds # 1 and 37, 6 and 39, 2 and 33, 30 and 43 indicate t,hnt, t,hr iulfonic acids t,end to be slighhly less potent t,han the carboxyl analogs. Group I\’ contains a number of soluble polymers presumably co11+t,itut,cd in :I manner analogous to emeraldinr; here again the slightly tlcprcssing effect of :I sulfonica acid group is evident. Group \’ comprises t)hc highly active synthetic hutnic acids as ~~11 as sodium humatc of natural wigill. Unfortun:~tely, the structural features of humic acids or of the compounds in Group VI have not been clcnrly cstnblishrd, and it is not, profitable to associate any one of t#he proposed structures with anl i-RIiasc activit,y. An indication of :I relation of structure to activity is given by ;~JI obscrvat~iotl dctailrtl ill Iht> sc(%iotl, K~s~l!s rcgardillg the polyurew: wtlwivably the mow flesi\)lc dilwiixyl polymer is c3p:tl)la of :t more illtjitnntc approat%h to the ellzymc thall the more rigid stilbcllc polymer. ,211illtcrcstitlg problem is that, of selcct,ivity or specificity of tht: alitiwzymc activity of the aridic* polymers under diwussioll. The differcllt rc~sponscsto sodium chloride shown in Table III indicate that, the afinities for the prot’ein, IQNasc, of closely rclatBed polymers \vit,h similar functional groups are not identical; this is also borne out l)y the small but definite gradation of ntlti-ItSwe act,ivities. On the other hand, the results in Table IV indicate t,tint now of the cotupoun& possesses :t prc’fr>relltinl affinity for l~X\‘;wc? iu the prwcncc of :L -&O-fold ex(‘(:s~ of SCi’um albumin. &Iow cTit~iw1 csperimeirts :~re iI1 I)rogress itr order to tlctwmine \\hctlicr wlwti\~c~ affinities call tw demonstrated irk &o. I~:th cl nl. (13), and ~~\billyi-Szcbchcly et nb. (14), h:rvtb &on-11 t,h:lt,
380
HEYMAh-N
El- .4L.
soluble phenolic polymers arc inhibitors of hynluronidase; furthermore, Rogers and Spensley (12)) working on similar substances, emphasized the macroanionic nature of t.hese enzyme inhibitors. Diczfaluzy et al. (32) examined polymeric acids as inhibitors of phosphatase, hexokinase, /3-amylase, and also hyaluronidase. The polymers resulting from oxidation of gentisic acid were shown to inhibit hyaluronidase by Roseman and Dorfman (33) as well as by Forrest rt al. (34), who also called attention to the similarity between these polymers and humic acid. In view of the marked anti-Wase activity encountered among the acidic polymers, we have investigated their action upon a number of other enzymes. At the present tirne, it is known that deoxyribonucleasc, phosphorylase,* the adenosinetripepsin, lysozyme, polynucleotide phosphatase of myeloblastic fowl leucosis virus, and that in the venom of Crotalus adamanteus are capable of inhibition. Moreover, several of these polymers are anticoagulants in oitro and in v&o, presumably because of the inhibition of one or more cnxymes involved in the clotting mechanism. Antivirus
Activity of Polyncric
ItNase Irlhibitors
The purpose of t.hese experiments \vas to determine \+*hether compounds of high anti-ItKasc activity were capable of interfering with virus multiplication. In this connection it is of interest to note that Takemoto et al. described an antiviral act,ivity of a “polymerized benzenesulfonic acid sodiurn salt” (35). Consequently, many of the polymers described in this paper, as well as a number of other substances known to ha,ve anti-RNase activity, were tested against various viruses and bacteriophages. The details of these investigations are being submitted elsewhere for publication and iwlude a critical examination regarding the mechanism by which the substances act against viruses. It seemed appropriate to indicate in Table I those compounds that had some effect in the hostvirus systems most extensively employed in the study: embryonatetl eggs infected with 103 median egg infective doses per egg of influenza virus strain RR, , or with 50 LDsO per egg of vaccinia virus. The compounds were given in approximately the maximum t’olerated doses, from 0.75 to 10 mg./egg. Activity denotes absence or marked reduction of chicken red cell a.gglut~inins after 24 hr. of incubation iu the case of 4 ]‘riv;rtc cornrr~utlicwt,ion lcge of Medicine.
~IYJIU Dr.
hl. Shchelin,
Xew
York
Urliversity, (‘ul-
INHIBITION
OF
RNASE
BY
ACIDIC
POLYMF:RS
381
influenza virus, and an increase in the average survival t’ime over the controls of 3 days or more. Of the substances examined, 17 showed activity in the influenza test, and 27 in the vaccinia test. All of the active compounds are potent inhibitors of RNase with the possible exception of compound B 53, a moderately active inhibitor. On the ot’her hand, all of t,hc noninhibitory polymers tested in the virus assays were without effect; however, a number of highly active RNnse inhibitors (lCSO < 5 pg./ml.) were likewise devoid of antiviral potency. Within the present series of acidic polymers, ability to inhibit RNase thus appears to be a necessary but not a sufficient prerequisite for activity in the virus tests. It is of int,erest t,hnt four compounds, L 14, 49, 56, and 16, were able to prevent the appearance of hemagglutinins when 10” median egg infective doses per egg were given. The far feebler aldehyde polymers ,$ 1 and 13 are derived from t’he same monomers as arc x114 and 49, respectively, but the latter are product’s of oxidative or “triphenylmethane” polymerization. Since these modes of preparation also pertain to # 16 and 56, the suggestion is made that the increased activity of the four compounds i: probably connected with 6he more complex, possibly cross-linked nLolerul:lr makeup one may expect from the type of polymrrization wnction employed.
As macroanions, the polymers discussed possess a broad affinity for the basic sites of all proteins (36). Therefore their inhibitory action on R.Kase cannot well be highly specific, but a variety of biological systems are likely to be affected by them. Moreover, the RiYases in the hostvnrus system may differ in their properties and action from the pancreatic l
1. The anti-RNase activity of GGacidic polymers of largely synthetic origin has been investigated. In the test system used, activities of halfinhibition at concentrations of I pg./ml. in protein-free media were frequent. The degree of inhibition is decreased markedly by proteins, vld to a varying but lesser extent by salts, or increase of pII.
382
HEYMANR- ET AI,.
2. Numerous other polymers, as well as nonpolymeric subst,ances, have been similarly tested, with negative results in almost all instances. 3. Among the acidic polymers, ;L number of t,he highly active ItNasc inhibitors have antiinfluenza and antivaccinia activities in eggs. The possible relationship between anti-R.Knse activity and virus inhibition in embryonated eggs is discussed. I~F,P~CTUCN(~S 1
rwiew wilh httliny rcfercncw , HPC:IIowrmIss, :I rcccnt Suclcic Acitls” (( ‘h:~r&f, 15.:IIICII):~vitlsorl ) .I. K., fVlS.),
Academic 2. 3. 4. 5. 6.
1~. T)., i/r “‘l’lw II, 1,. 461 li’.
For
KUSKEE,
H~nfis,
l’ress Inc., Sew 11. I%:., ANI) I'ARUEE,
H., Cold Spring
yo&, N. le., 1!).55. 11. B., HiOchirU. f?l ~iO&S.
Vol. n&r
19, 236 (1!).5(;).
Harbor
Symposia Qucwl. Viol. 12, 104 (194i). CASPERSSON, 1‘~., AM) THORWON, K. G., Min. Wochwhr. 31, 205 (1953). KRADOLFER, F., Schweiz, Z. Al/gem. J’dhol. 7~. Rakteriol. 17, 520 C19.51). %oLLNER, N., AXI) I"ELLIG, J., Awl. J. Physiol. 173, 223 (1953). i. Row, J. S., .4 wh. Riochem. Riophys. 44, 265 (1953). 8. V.~NI~~~NI~RIESS~~I~E, I,., ilrch. Biochem. Bioph,+s. 65, 34i (1!)66). 9. KFNITZ.
M.,J. Gen. I'hysiol. 24, 15 (1940,. (:. H., AXI) SI'BRAI~OIV, JV,. d. Rio/. ('hoa. 66,376 (1!)25J. 11. ADLER, 1, ., .4x11 \VIE:~IIO\~SKY. W., A rd. rrptl. I'athol. I'hurr,~rckol.92, 22 (lW2). H. J., ,\SU ~PESSJXY, I'. ('.. i?iochir/~. rt Uioph!/s. .;1clu 13, 2% (l!)r,-I); 12. IioGERS.
10. FISKE,
Sutrtr.e 173, 1190 (1954).
15. ~~;RDTMAN,
Il.,
ASU (:RANATIJ,
?rl., AC/U C'hetu. #%and.
8, 811 (3!)54),
arid rarlirt
paper cited thrc. 16. MYRB;~CK, K., .4sD I'ERSSON, B., AIkiC Kemi 5, 477 (1%2). li. Wi~~.s,~.n.,Uiochetrl.J. 57, 109 (1954). 18. (:~XIING. I. B., I>AvJ~, 11. I'., li~a~ovir,, 1:. .J., AND RI.~c('oRuI-oI),\I,~,
1). \v.,
J. ilm. Chew. Sot. 76, 1590 (1954). 19. NEIIER, It., .~ND KRADOJSER, E'., %. SuturJorsch. lob, 191 11!).55). 20. %JTTJ,E, K., J. Hiol. Chem. 163, 111 (1946). 21. l,E~orx. I,., Biochim. ct f~iophqs. .4rfrr 11, 517 (1!)63); 13, 121, 537 (1!)51); 14, 265 (l!G4).
A. W., .4x1) STEELE, 89, 123 (1955).
23. BERNIIEIXER,
.J. hr., JR.,
t'mc.
sot.
Ezpll.
Biol.
.lIed.
24. KAY, I<. It. hr., ASI) ~h)UNCE, A. I,., J. Am. Chew. SW. 76, 4011 (1953). 25. (:RESTFIRLI), A. M., SMITH, K. (:., ASI) ALLEN, F. w., ,I. Bid. Chrm. 216, lS.5 (10.55).
OF
BIOCtI~MIS’TRY
ASI)
HIOI’IIYSICH
73, :%jti--:%j:j (1068)
The Inhibition of Ribonuclease by Acidic Polymers and Their Use as Possible Antiviral Agents Hans Heymann, Z. R. Gulick, C. J. De Boer, G. de Stevens and R. L. Mayer
Invasion of I~schcrichia coli ~11s by the T-coliphagcs in\‘olv(:s, ill general, a cytological disorganization of the host c*ell, particularly of its deoxyribonucleic acid (DNA) (1). A n increase in intracellular deoxyribonuclease (DNase) activity upon ‘I’z-bacteriophage invasion has been demonstrated, although it has been st,ressed that a necessary connection between activated DiYnse and phage multiplicat,ion htls not been established (2). T,ess-detailed biochemical illformation is available regarding viral invasion of animal cells, but an at,t,ack on, and a reorganization of the protein-synthesizing system, gencrnlly believed to be ribonwleoprotein, has heel1 recognized (3, 4) for, c.g., vwcinia virus grown in cmhryonated (‘ggs. On the hypothesis that interference with trwleic: acid nwt.al~olism might prevent 1:irus multiplic,at,ion, MT have searched for substalwts capnblc of inhibiting enzymes inwl\-ed in biological tr:tnsfornlat,iotls of polynucleotides. Similar views have bee11 advanced by Kradolfcr (5) in an attempt to find a possible correlation between antivirus and antienzyme activity. In the course of this investigation WC have directed our attJent~ion to the degradative phase of nucleic acid metabolism. Although a number of enzymes are involved in the breakdown of nucleic acids, the primary objective of the present study was the inhibition of ribonwlease. Investigations on the inhibition of ribonuclease have been reported by ZGllner and Fellig (6), and by Roth (7). Recently Vandendricswhc (8) described the diff’erential inhibitory activities exerted by several 366
polywids 011 t,he depolymerizing ability to split cayclic nurleotidce.
:rt%ivit.y of rihonuclense,
and on it,s
.411 wlutiotw ittf.etded for t hc enz~.me assxy were prepared with g$l:rss-distillctl ‘I’0 :I mixture of 1 ml. of ritwnuc~lc;ttc? holution ant1 4 ml. of 0.1 III wetat c ikrtffrr. IJH 5.0, were ddecl 1 ml. of lruffer icontrol) or of I,uffered irihihitor snlut,iotr, :rttd f,he tubes were plied in 3 30” bath. One-milliliter poltions of the enL\.ttte .solutiotl were then d&t1 to each t.ut)c. ‘Ten minutes after each :ddilion, 2:nll. diquots were removed ant1 mixed with 2-ml. portions of 2.570t,richloro:weti~~ :tcitl wtlt,:lining 0.2570 of utxiyl acetate :lftrr 30 min. the suspensions were cetttrifugctl ant1 filt.crect, and 2-d. :rliquots of t.lre ~uperri:~tnnts were digested wit IL wlfuric :ici(l :~nd hyclroyeu I)eroxi(le for dctrrmimttion accortlitig to Fi,kr antI \v:I~c:I~.
~UiJtJ:L~~O\\-
(10,.
( ‘f’fd:~in ittltiltitors,
wlictl
l)t’rsrtlt
iti altlttwi:rlrlc
cottc:etit,r.:lt.iorts,
tctlcl 1.0 c:iUsc:
368
HEYMANN
ET AL.
precipitation by MacFadyen’s solution of the normally soluble products of I1N:~se action. This “coprecipitation” simulates inhibitor activity. To avoid possible errors, separate controls were included in which the inhibitor solution was added to IO-min. digests of ribonucleate and RNase, and the “inhibition” value obtained was subtracted from the experimental value. Frequently, the coprecipitation noted in such controls exceeded that occurring in the actual inhibition experiment in that the corrected inhibition was seen to pass through a maximum with increasing inhibitor concentration. Therefore, correction of the copr&pit,nt,iorl error occasionally led to low estimates of inhibitory power, but in most instances coprecipitation was negligible at effective concentrations of inhibitor. Inhibitors were examined at concentrations of 1.2, 12, and 121 pg./ml. to 01). tain an approximate est,imate of potency. Since more detailed st,ud,v of selected samples showed that an approximately linear relationship exists between per cent inhibition and the logarithm of the concentration, the concentration yielding 50% inhibition (ZC’,o) was determined for each compound by graphical interpolation.
Precision of Results Upon repeated determinations of an ZC~O value, the results lay wit,hin one-half to twice the previously found figure. This variability results primarily from the photometer readings which vary by f0.02 density units = f2 pg. of phosphorus in serial determinations of identical standards. The total soluhlc phosphorus in n complete digest ranged from 20 to 30 fig&ml. aliquot. l’reincubation of enzyme and inhibitor for various periods of time w:ts Gt.hout influence on the ZCjo . The effects of changes in pH, electrolyte concentration, and presence of proteins on inhibitory action were studied in separat,e experiments (see below). RESULTS
According to Ziillner and Fellig (6), and Roth (7), heparin is a powerful inhibitor of ribonuclease. In our experiments ICsO-values for this substance varied between 12 and 60 pg./ml. for samples of different provenience. In view of this high activity, we tested other anticoagulants, such as crude hirudine, dicoumarol, bis(4-hydroxycoumarinyl)acetic acid, phenylindane-1 ,3-dione, potassium oxalate, and sodium humate (11). With the exception of sodium humate, none of the afore-mentioned anticoagulants demonstrated any activity as an inhibitor of RNase. A. Acidic Polymers Related to Humic Acids Noting recurring prepared quinone exhibited
that heparin as well as humic acid is a polymer with frequently groups of pronounced and moderate acidity, respectively, we polymers by the interaction of formaldehyde w&h hydrosulfonic acid (12) or gentisic acid (13, 14). These compounds ICho-values of 55 pg./ml. and 5 pg./ml., respectively. On the
INHIBITION
OF
RNASE
BY
ACIDIC
POLYMERS
XI)
hasis of these findings 64 additional phenolic and related polymers were investigated. From a chemical point of view the substances may be divided into six groups according to the starting materials used, and, in each group, whcrcrer applicable, three modes of polymerization will be considered. The subst,ances are arranged accordingly in Table I, which records the IC”5,,-values found. E\-idewe for t,hc polymcria nature of the substances prepared consists of t,he following general points: (a) production of highly colored, wateriIwoluble but base-soluble substances from soluble starting materials 01 101~ molecular weight by procedures known to yield polymers in :wdogous cases; (0) failure of the products to diffuse through cclloph:u~e membranes, a criterion applied to a considerable number of the products; (c) sedimentation experiments in an ultracentrifuge as applied t,o sclectcd substances, particularly polysulfonic acids for which the soluljility criterion mcnt~ioncd in (a) fails. Thus, orienting sedimentationwtc mwsuremcnts for compound R 37 with the aid of an improvised light-:rbsorpt,ion m&hod revealed a sedimenting boundary with 820 = 1.(i Svcdberg ant1 obvious signs of high polydispersity. Compound f 1%in :qucous solution at pH 7 exhibited a slowly moving boundary, I)ut paper electrophoresis at pII 7-7.5, although twhnically unsatist’acatory, rcvwled the presence of a multiplicitIy of substances. Compound B 12 uxs srdimentable, but its higher homolog, the product obtained f’mn reaction of formaldehyde w&h 2, K-dihydroxy4methylbenzenesulfonic: acid \vas not’, nor was the product resulting from formaldehyde and 4,5-dihydroxybenzene-I ,2-disulfonic acid. The reaction of formaldchyde and sulfosalicylic acid gave a crystalline substawe of low molecular \vcight. Undoubtedly, the substawes obtained from formaldehyde and ?,-hytlrosybeI~zeIlesulfoni~ acid, or hydroquinone-2,5-disulfonic acid are 1l0t polymeric. Thr nat,ure of the monomers is the primary basis for the chemical c*lassification and characterization of the polymers. Yrom analogy to i,hc st,ructures of the phenol-aldehyde resins, of emeraldine and its relat,ives, and of the “synthetic humic acids” (15), plausible structures may bc deduced, but no experimental work was done to confirm these inferences or to find possible differences in fine structure that might, pertain to the biological activity of the substances. Sinw t’hc acidic polymers were intended for tests in biological systems, it \V:IS desirable to study the influence of environmental factors on th(i 11I\‘nsc inhibition by these substances. Enzyme-polyanion interaction
Anti-RNase
and Antivirus
Activities of Acidic _____-
Pol?ynlers Effect
Serial number
Polymerizing polymerizing
aldehyde RCHO, othr agent or nature of monomer
‘ink%&, / IGO, pg./1711.
on viruses
’
-’ OH
Group
I. Polymers
of ,/,+COOlI
I II \/ OH (a) Aldcl~ydc RCRO,
1 la 2 3 4 5 6 7 8 9 10 11 12
polymers
R=
H (basic crtt:tlysis) H (acidic catcdysis) 4-Nitro-l-fury1 1-Thienyl CHO4-Kitrophenyl l-Fury1 3,4-Dichlorophenyl Phenyl Methyl 3.Methoxy-4-sulfol)llc~~~l p-Dimet~hyltllninol,hcn3-1 4-Pyridyl
-1 2 3 3 4 9 10 24 25 30 34 111:1ct. Inaci,.
(b) Osidntive
16 17
# la + gcntisic :tcid (!hlorohydroquirlorlc (‘Hz0
Group II. Polymers
+
acid
1
1
$- 1
,I
Imp of 3,5-, 3,4- :md 2,3-dih~droxyl)ellzoic ltnd related compounds HO / COOH of 0\ OH ((0
polymers
Alticliy~e
370
( I
+ + + + +-
i I I 1 1
l~olymcrs
gcnt’isic
1. Polymers
+ T -
+ ~~~ acids,
L-~ +
Serial number
= I
= R&S? Iir lhibition,
Polymerizing aldehyde RCHO, other polymerizing agent or nature of monomer
Effect on viruses
IGO, pg./ml.
RCHO, R=
1s 19 20 a1 228 23
-
1 2 3 7 12 Illact.
HLThienyl (~‘HOl’henyl 4.Nitro~l~furyl 4.l’yridyl
+ -
(c) ‘~ripherlylmeth~lle 24
#20 + gentisic
polymer
acid \ 2. l’olymers
I 2 __~ COOH
1
+/
-+--
of HO 0 / OH
(u) Aldchyde
polymers
RCHO, R=
25 26 27 -~~~ --~-
1.l”Ury1 I’henyl H-.-__
3 1 5 ~~ (h) 0sitl:ttive
I 7
~ +
polymc~
-i + + OH (u) Aldehyde RCHO, R=
X0 31 32 33
polymers
+-
H4-Nitro-l-fury1 CHOLThienyl 371
372
HEYMANN TABLE
Serial number
Polymerizing polymerizing
ET AL.
I-Continued
aldebyde RCHO, other agent or nature of monomer
(b) Oxidative 34
Ii&O~
Ill+/+ 4. Related
35 36
polymer
polymers
1 CH?O + hydroquinone-2,5-diacetic CH& + homogcntisic acid Group
III.
Polymers
acid
+
1 3
of dihydroxybenzenesulfonic
acids
OH (CL) Aldehyde
RCHO,
37 37a 38 30 40 41 42 (u’)
polymers
R=
13 55 18 32 300 Inact. Inact.
H-(Na salt) H-(free acid) 4-Nitro-l-fury1 l-Fury1 Methyl 4.Pyridyl CHOPolymers
of
of formaldehyde
and various
dihydroxybenzenesulfonic
acids
SOiH
__.___ 43 44 45 37 37a 46 47
.--~------
RI=Rz=OH R,=R,=OH R,=RI=OH,
Ita=Cl
vs. vs. Itl=Ra=OH, R,=R,=OH,
R.,=(:H:~ R:,=SO,H
3 4 6
+ -
+
13 55 19 72
V.S.
V.S.
-
TABLE Serial number
I-Continwd Effect on viruses
Polymerizing aldehyde RCHO, other polymerizing agent or nature of monomer
Influenza
(b) Oxidative 48
polymer
acid + KkWs
Hydroquinonesulfonic
(c) Triphcnglmethnne 49 Group
Hydroquinonesulfonic IV.
Polymers
acid +
;6,In
~>600
1
-
/
-
+
~
-t
polymer I
2
of aminocarhoxylic and aminosulfonic with FeS04 and Hz02
acids,
hlonomer
50 51 52 .A ‘8 Group
Anthranilic acid 2,4-Diaminobenzoic acid 3,5-Diaminobenzoic acid 2,4-Diaminobenzenesulfonic V. I’olvmers
5 7 9 15
acid
of substituted hydroquinones preparctl T
OH Rl=Rs=(:l R,=U R,=R,=(:l R,= (:Hc RI=Cl, +CHIO Sodium humate Group
from VI.
lignite l’yrrole-quinone 111
---
Vaccinia
polymers
-
+
prepared
I / i
-
/
+
I)v oxidation
with
374
HEYMANN
TABLE Serial number
T
ET
AL.
I-Continued Effect
Polymerizing polymerizing
on viruses
aldehyde RCHO, other agent or nature of monomer Vaccinia
R,=O, R,=N-ChH, R,=ClN<, Rz=R,=U Rl=O, Rz=Rn=(:Hs R,=O, li~=R.i=(:l R,=O, 1tc= -ioH,)2(:ooH II, = ( )
61 62 63 (i-1 65 66
U If no designation
is indicated,
3 3
x
Concentration
-
-
II
of RNaee Inhibition
on pI1 Per cent
(‘om~Kxmd
-
R=H. TABIX
Dependence
-
*5 4 27 ’ 160 1
inhibition
ICao at pH 5
5
0
I I 1 IO 2
x0 54 82 95 x5
66 34 81 x7 X6
at pH 7
8
pg./ml. 60
60 I 6 56
12.4 6.2 62 12.4 3
43 15 54 32 61
28 9 357 22 45
has been shown to depend strongly on pII and ionic strength. Ziillner and Vellig (G) observed the pH dependence of RNase inhibition by heparin, while other variables were studied by AZyrb&ck and Persson (16) in the system, heparin-fi-amylase. 1. Eflect of pH Changes. The effect of pH changes upon the antiRKase activity of a number of polymers was studied by measuring the degree of inhibition obtained for a given concentration of inhibitor at pH 5.0, 6.0, 7.0, and 8.0 (each value f0.05 pFI unit). ‘I’he results appear ilk Table II. With rising pTI, the concentrations of sodium humate and compound # 1 required to give a finite and measurable response were substantially higher than the ICsO , whereas compounds $46 and F$56 appeared to be less affected. If the p1-I of an alrnost completely inhibited reaction mixture is raised, at least part of the reactivity is restored. A digestion mixture containing the standard concentration of RKnse, and 12.4 pg./ml. of sodium humate (%60) in Verona1 buffer pH 5 (8 ml.) an inhibition of 88% by comparison with a suitable control. III a second ml. of buffer pH 5 was added at time 0, followed by 4 ml. of buffer $1 8 min. of incuhntion, giving rise t,o a final pH of 7. The digestion was :tllowed
RNA, showed series 1 after IO to pro-
TABLE Effect of 0.9% XaCl Compound
ry
III
on the Inhibitory
Concentration
Action cent inhibition \Vith SaCl Without NaCl Per
pg./d.
60 1 Sod. hepnrin 13:~13707~’ I&L 13707 u I’olyure:~
I’mn
72.4 6“ 72.4 12.-I 1.2
3,J’-di:tn~it~otlil,t:rleyl~
teed for m addit,iond 10.min. period. inhibition of 579& wits otmervetl.
86, 88, 8!J so, 92 SO !J-I 60 ‘2,2’--I 1‘~sullonic .
By coniprrison
6-l) 73, 51) ii, 92 2-I 7s ‘24 acid
\Vilh :I suitahlc
~ont.lol,
:w
2. Changes in Eleckrolyte Concentration. The results obtained for four polymers in experiments in which the electrolyte concentration of the medium was altered are recorded in Table 111. Sodium humate and compound B 1 are less influenced by salt than is sodium heparin, although it should be not.ed that compound B 1 \vas present in significantly higher concentration. The results obtained with the polyurea from 4,4’-diaminodibenzyl-2,2’-disulfonic acid (Ra 13707) indicate that the sulfat,e groups of heparin are not neccssnrily associated wit.h the anti-inhibitory effect of salt; the results indicate that the salt effect becomes more significant at lower inhibitor concentrations. The effect is self-limiting as seen from the behavior of compound B 1 (82 pg./ml.) in the presence of increasing concentrations of salt (0.9, 1.8, 3.6 %), which do not, greatly affect the inhibition (93, 86, 78 %, respectively). 3. Influence of Serum Albumin. Since acidic polymers possess considerable affinity for the basic sites of any protein, the effect of bovine serum albumin upon their antienzyme activities was examined. The albumin used (Armour, Lot ~Pl3706) possessed a weak ribonuclease activity, which was measurable when t’he protein was incorporated in the standard digestion mixture in concentrations of 10 mg./ml. and 1 mg./ml., but which became negligible at greater dilution. Albumin abolished the a,ction of normally effective inhibitor levels when present in the standard digestion mixture in concentrations higher than 100 pg./ml. Moreover, at this dilution the potency of the inhibitors was markedly reduced, as shown in Table LV. Evidently there exist no striking differences in the eff’ect of albumin upon the anti-1tNase activit,y of various classes of compounds.
376
HEYMANN
ET
TABJ,E Inhibitory
ICsa In presence of 1y&;l.
In absence of albumin pg./??tl.
Fluid
Dilution
Ratio: ICao inayb;t3~1Cao in
az.lml.
1 4 13 9 25
55 1 37 5 8
of Yolk-Sac
IV
Concentration in Presence OT Absence of 100 pg./ml. Bovine Serum Albuinin
Compound x
Efect
.%I,.
20 52 110 90 250
0.05 0.08 0.12 0.10 0.10
TABLE V and Amniotic Fluid on Inhibitory (4 b&ml.) Per cent inhibition Yolk-sac fluid
of egg fluid
Action
of Compd.
#l
observed in presence of Amniotic fluid
0 29 0 56 17 58 In absence of egg fluid 61
I:10 1:lOO 1 : 1000
4. InJluence of Yolk-Sac Fluid and Amniotic Fluid. Since the action of certain acidic polymers against influenza virus when introduced into the chorioallantoic sac of infected embryonated eggs was abolished when injected into the yolk sac, the effect of yolk-sac and amniotic fluids on RNase inhibition was examined. As is shown in Table V, both substances diminish the RNase inhibitory action of polymers, however, to different degrees. The anti-inhibitory effect of amniotic fluid begins to disappear when the material is present in a dilution of 1: 100, whereas that of yolk-sac liquid still persists in a dilution of 1: 1000. B. Anti-RNase
Activity of Other Acidic Polymers
In addition to the acidic polymers prepared for the present study we have examined a number of other acidic polymers with frequently recurring acidic groups. Suramin (Bayer 205) exists in solution in micellar aggregates; the substance has been shown to inhibit enzymes, e.g., urease (17). Suramin inhibited RNase at 140 pg./ml. Chitin sulfate’ 1 We thank chitin sulfate.
Dr.
I. B. Cushing,
Abbott
Laboratories,
for a sample
of sodium
INHIBITION
OF
ItN.4SE
BY
ACIDIC
POLYMEltS
3’7
7
(Is), known to possess a heparin activity of 32 units/mg., was similar to heparin in anti-RNase activity. Chondroitin sulfate proved to be inactive, in agreement with the results of Zijllner and I’ellig (6). iYo noteworthy activity was observed with carboxymethylcellulose, p&in, or rennin; tannic and alginic acids were of questionable activity. Neher and Kradolfer (19) have reported that polymers arising from the action of phosgene on aromatic diaminocarhoxylic and sulfonic acids inhibit influenza virus multiplication in eggs as indicated by hemagglutination titration, and have referred to the anti-Rn’ane activity of some of these polymers. We hare examined most of the polymers mentioned by Neher and Kradolfer as well as a number of related compounds. While none of the monomers, from which t’hese sulfonic polymers were derived, possessed activity, very potent TZNase inhibitors were found among the polyureas. For example, 4,4’-diaminostilbene2,2’-disulfonic acid was inact,ive, but the polyurea prodwed from it, inhibited RSase at 40 pg./ml. The polyurea from 4 ,I’-diaminodibenzyl%,2’-disulfonic acid (Ba 13707), differing from the foregoing by the absence of t,he &ilbene unsaturabion, inhibits ilt 1 pg./ml., :11d the same potency was observed with the polymer from benzidinc-3,s ,3’, 3’t,ctrnsulfonic acid. \veI’c hs expected for neutral polysacrharides, sar11ples of tirxt~r:tlls in:wtive regardless of t,hc molecular weight of t,he sample investigated. C. Anti-RNasc! Activity of Nonpolymeric S’ubstanccs A number of nonpolymeric substances have been reported as RNase inhibitors in the literature. Cupric sulfate (20) was effectfive in our experiments at a level of 200 pg./ml. An anti-RNasc effect>of sulfhydryl reagents has been reported by Ledoux (21); however, the results have recently been questioned by several nut,hors (22). We found N-ethyl muleimide t’o be devoid of inhibitory power. Converxcly, we could confirm the activity of lilac-leaf extract reported by Bernheimer and Steele (23). Sodium dodecyl sulfate has been used t)o inhibit RNuse during isolation of RXJA from natural material (21, 25); in our assay m&hod strong inhibition was observable at about 300 pg./ml. Roth (7) found I hat 2-aminobenzimidazole produced 50 % inhibition at the relatively high concentration of 1.7 X IOF M (2.26 mg./ml.) in media containing 0.04 Pg./ml. of RNasc. h number of substances report,ed to be effective against wrtain viruses \vcre tested. Among the antibiotics, penicillin has been shown by histo-
378
HEYMANN
ET AL.
chemical means to inhibit RNase (26), but we did not observe any inhibitory power in vitro. None of the following substances inhibits the action of RNase: representatives of the tetracycline group, magnamycin, puromycin, streptomycin, chloramphenicol, patulin, aaaserine, cycloserine, 5,6-dichloro-1-/3-n-ribofuranosylbenzimidaaole, and the 4,5, 6-trichloroanalog2 (27)) 6-mercaptopurine, “Benuron” (a polysaccharide of bacterial origin), butanedione monoxime monothiosemicarbazone (28)) and isatine-3-thiosemicarbazone (28, 29). The only apparent exception was 5-(2’ ,4’-dichlorophenoxy)thio-(2)uraciF (30, 31) which gave 50% inhibition of RNase at about 120 pg./ml. Ot’her thiouracils examined were inactive. The singular nature of the observation is receiving further study. As detailed in the section Results, a number of hydroxybenzenesulfonic acids failed to yield polymers, and these substances are, not unexpectedly, inactive as inhibitors of ribonuclease. Moreover, about 200 diversified organic substances of low molecular weight and comprising many classes including acids and bases of the aliphatic, aromatic, and heterocyclic series, quinones, purines, pyrimidines, dyes, and detergents gave uniformly negative results. Specific inhibitors of RNase of low molecular weight are evidently rare, the only possible exception found in the present experiments being the afore-mernioned t,hiouracil derivative. DISCUSSION
Among the 66 acidic polymers tested, a considerable number displayed marked anti-RNase activity, exceeding, in many instances, that of heparin. In Groups I and II, condensat’ion with formaldehyde yields the most active polymers with the except’ion of the derivatives of 3,4-dihydroxybenzoic acid (compounds A 25, 26 27), although even in this case the formaldehyde derivative is of high, but not of maximal potency. Aromatic and heterocyclic aldehydes yield compounds of intermediate activities, while introduction of a nuclear nitro group leads to slight potentint,ion. Basic aldehydes give rise to extremely weak or inactive compounds as shown by the results obtained with compounds B 11, 12, 23, 41. 2 We thank Dr. I(. Folkers, Pvlerck 8: Co., for kindly benzimidazoles mentioned. 3 We are obliged to Dr. G. H. Hitchings, Burroughs kind donation of a sample of this thiouracil.
supplying
samples of the
Wellcome
& Co., for his
Such polymers may be internally neutralized, lacking t’he anionic character required for RNase inhibition. Oxidative polymerization in general yields highly active polymers of the “humic acid type ” in (;roups I, II, and, of course, V, which is composed entirely of this type of substance. Only the aulfonic acid derivative, compound %48, differs in this respect. The triphenylmethane polymers represent an increase in complexity over their diphenylmethane properties acprecursors, but no noteworthy alteration of inhibitory companies this change (compounds # 1 and 16, 27 and 29, 1 and 49). The sulfonic acid compounds (Group III) exhibit a greater variation of :&ivity wit)h structure than do the ot’her classes. The six aldehydc polymers examined conform t,o the generalization expressed above, with the exception of glyoxnl, which surprisingly gave rise t,o a11 inactive product. The second section of Group III includes only formaldehyde polymers derived from mrious sulfonic acids. Cross-comparisons of compounds # 1 and 37, 6 and 39, 2 and 33, 30 and 43 indicate t,hnt, t,hr iulfonic acids t,end to be slighhly less potent t,han the carboxyl analogs. Group I\’ contains a number of soluble polymers presumably co11+t,itut,cd in :I manner analogous to emeraldinr; here again the slightly tlcprcssing effect of :I sulfonica acid group is evident. Group \’ comprises t)hc highly active synthetic hutnic acids as ~~11 as sodium humatc of natural wigill. Unfortun:~tely, the structural features of humic acids or of the compounds in Group VI have not been clcnrly cstnblishrd, and it is not, profitable to associate any one of t#he proposed structures with anl i-RIiasc activit,y. An indication of :I relation of structure to activity is given by ;~JI obscrvat~iotl dctailrtl ill Iht> sc(%iotl, K~s~l!s rcgardillg the polyurew: wtlwivably the mow flesi\)lc dilwiixyl polymer is c3p:tl)la of :t more illtjitnntc approat%h to the ellzymc thall the more rigid stilbcllc polymer. ,211illtcrcstitlg problem is that, of selcct,ivity or specificity of tht: alitiwzymc activity of the aridic* polymers under diwussioll. The differcllt rc~sponscsto sodium chloride shown in Table III indicate that, the afinities for the prot’ein, IQNasc, of closely rclatBed polymers \vit,h similar functional groups are not identical; this is also borne out l)y the small but definite gradation of ntlti-ItSwe act,ivities. On the other hand, the results in Table IV indicate t,tint now of the cotupoun& possesses :t prc’fr>relltinl affinity for l~X\‘;wc? iu the prwcncc of :L -&O-fold ex(‘(:s~ of SCi’um albumin. &Iow cTit~iw1 csperimeirts :~re iI1 I)rogress itr order to tlctwmine \\hctlicr wlwti\~c~ affinities call tw demonstrated irk &o. I~:th cl nl. (13), and ~~\billyi-Szcbchcly et nb. (14), h:rvtb &on-11 t,h:lt,
380
HEYMAh-N
El- .4L.
soluble phenolic polymers arc inhibitors of hynluronidase; furthermore, Rogers and Spensley (12)) working on similar substances, emphasized the macroanionic nature of t.hese enzyme inhibitors. Diczfaluzy et al. (32) examined polymeric acids as inhibitors of phosphatase, hexokinase, /3-amylase, and also hyaluronidase. The polymers resulting from oxidation of gentisic acid were shown to inhibit hyaluronidase by Roseman and Dorfman (33) as well as by Forrest rt al. (34), who also called attention to the similarity between these polymers and humic acid. In view of the marked anti-Wase activity encountered among the acidic polymers, we have investigated their action upon a number of other enzymes. At the present tirne, it is known that deoxyribonucleasc, phosphorylase,* the adenosinetripepsin, lysozyme, polynucleotide phosphatase of myeloblastic fowl leucosis virus, and that in the venom of Crotalus adamanteus are capable of inhibition. Moreover, several of these polymers are anticoagulants in oitro and in v&o, presumably because of the inhibition of one or more cnxymes involved in the clotting mechanism. Antivirus
Activity of Polyncric
ItNase Irlhibitors
The purpose of t.hese experiments \vas to determine \+*hether compounds of high anti-ItKasc activity were capable of interfering with virus multiplication. In this connection it is of interest to note that Takemoto et al. described an antiviral act,ivity of a “polymerized benzenesulfonic acid sodiurn salt” (35). Consequently, many of the polymers described in this paper, as well as a number of other substances known to ha,ve anti-RNase activity, were tested against various viruses and bacteriophages. The details of these investigations are being submitted elsewhere for publication and iwlude a critical examination regarding the mechanism by which the substances act against viruses. It seemed appropriate to indicate in Table I those compounds that had some effect in the hostvirus systems most extensively employed in the study: embryonatetl eggs infected with 103 median egg infective doses per egg of influenza virus strain RR, , or with 50 LDsO per egg of vaccinia virus. The compounds were given in approximately the maximum t’olerated doses, from 0.75 to 10 mg./egg. Activity denotes absence or marked reduction of chicken red cell a.gglut~inins after 24 hr. of incubation iu the case of 4 ]‘riv;rtc cornrr~utlicwt,ion lcge of Medicine.
~IYJIU Dr.
hl. Shchelin,
Xew
York
Urliversity, (‘ul-
INHIBITION
OF
RNASE
BY
ACIDIC
POLYMF:RS
381
influenza virus, and an increase in the average survival t’ime over the controls of 3 days or more. Of the substances examined, 17 showed activity in the influenza test, and 27 in the vaccinia test. All of the active compounds are potent inhibitors of RNase with the possible exception of compound B 53, a moderately active inhibitor. On the ot’her hand, all of t,hc noninhibitory polymers tested in the virus assays were without effect; however, a number of highly active RNnse inhibitors (lCSO < 5 pg./ml.) were likewise devoid of antiviral potency. Within the present series of acidic polymers, ability to inhibit RNase thus appears to be a necessary but not a sufficient prerequisite for activity in the virus tests. It is of int,erest t,hnt four compounds, L 14, 49, 56, and 16, were able to prevent the appearance of hemagglutinins when 10” median egg infective doses per egg were given. The far feebler aldehyde polymers ,$ 1 and 13 are derived from t’he same monomers as arc x114 and 49, respectively, but the latter are product’s of oxidative or “triphenylmethane” polymerization. Since these modes of preparation also pertain to # 16 and 56, the suggestion is made that the increased activity of the four compounds i: probably connected with 6he more complex, possibly cross-linked nLolerul:lr makeup one may expect from the type of polymrrization wnction employed.
As macroanions, the polymers discussed possess a broad affinity for the basic sites of all proteins (36). Therefore their inhibitory action on R.Kase cannot well be highly specific, but a variety of biological systems are likely to be affected by them. Moreover, the RiYases in the hostvnrus system may differ in their properties and action from the pancreatic l
1. The anti-RNase activity of GGacidic polymers of largely synthetic origin has been investigated. In the test system used, activities of halfinhibition at concentrations of I pg./ml. in protein-free media were frequent. The degree of inhibition is decreased markedly by proteins, vld to a varying but lesser extent by salts, or increase of pII.
382
HEYMANR- ET AI,.
2. Numerous other polymers, as well as nonpolymeric subst,ances, have been similarly tested, with negative results in almost all instances. 3. Among the acidic polymers, ;L number of t,he highly active ItNasc inhibitors have antiinfluenza and antivaccinia activities in eggs. The possible relationship between anti-R.Knse activity and virus inhibition in embryonated eggs is discussed. I~F,P~CTUCN(~S 1
rwiew wilh httliny rcfercncw , HPC:IIowrmIss, :I rcccnt Suclcic Acitls” (( ‘h:~r&f, 15.:IIICII):~vitlsorl ) .I. K., fVlS.),
Academic 2. 3. 4. 5. 6.
1~. T)., i/r “‘l’lw II, 1,. 461 li’.
For
KUSKEE,
H~nfis,
l’ress Inc., Sew 11. I%:., ANI) I'ARUEE,
H., Cold Spring
yo&, N. le., 1!).55. 11. B., HiOchirU. f?l ~iO&S.
Vol. n&r
19, 236 (1!).5(;).
Harbor
Symposia Qucwl. Viol. 12, 104 (194i). CASPERSSON, 1‘~., AM) THORWON, K. G., Min. Wochwhr. 31, 205 (1953). KRADOLFER, F., Schweiz, Z. Al/gem. J’dhol. 7~. Rakteriol. 17, 520 C19.51). %oLLNER, N., AXI) I"ELLIG, J., Awl. J. Physiol. 173, 223 (1953). i. Row, J. S., .4 wh. Riochem. Riophys. 44, 265 (1953). 8. V.~NI~~~NI~RIESS~~I~E, I,., ilrch. Biochem. Bioph,+s. 65, 34i (1!)66). 9. KFNITZ.
M.,J. Gen. I'hysiol. 24, 15 (1940,. (:. H., AXI) SI'BRAI~OIV, JV,. d. Rio/. ('hoa. 66,376 (1!)25J. 11. ADLER, 1, ., .4x11 \VIE:~IIO\~SKY. W., A rd. rrptl. I'athol. I'hurr,~rckol.92, 22 (lW2). H. J., ,\SU ~PESSJXY, I'. ('.. i?iochir/~. rt Uioph!/s. .;1clu 13, 2% (l!)r,-I); 12. IioGERS.
10. FISKE,
Sutrtr.e 173, 1190 (1954).
15. ~~;RDTMAN,
Il.,
ASU (:RANATIJ,
?rl., AC/U C'hetu. #%and.
8, 811 (3!)54),
arid rarlirt
paper cited thrc. 16. MYRB;~CK, K., .4sD I'ERSSON, B., AIkiC Kemi 5, 477 (1%2). li. Wi~~.s,~.n.,Uiochetrl.J. 57, 109 (1954). 18. (:~XIING. I. B., I>AvJ~, 11. I'., li~a~ovir,, 1:. .J., AND RI.~c('oRuI-oI),\I,~,
1). \v.,
J. ilm. Chew. Sot. 76, 1590 (1954). 19. NEIIER, It., .~ND KRADOJSER, E'., %. SuturJorsch. lob, 191 11!).55). 20. %JTTJ,E, K., J. Hiol. Chem. 163, 111 (1946). 21. l,E~orx. I,., Biochim. ct f~iophqs. .4rfrr 11, 517 (1!)63); 13, 121, 537 (1!)51); 14, 265 (l!G4).
A. W., .4x1) STEELE, 89, 123 (1955).
23. BERNIIEIXER,
.J. hr., JR.,
t'mc.
sot.
Ezpll.
Biol.
.lIed.
24. KAY, I<. It. hr., ASI) ~h)UNCE, A. I,., J. Am. Chew. SW. 76, 4011 (1953). 25. (:RESTFIRLI), A. M., SMITH, K. (:., ASI) ALLEN, F. w., ,I. Bid. Chrm. 216, lS.5 (10.55).