Cyanate and sickle-cell anemia

73

T I B S - April 1976

REVIE',VS Cyanate and sickle-cell anemia D.R. Harkness

Cyanate and protein reactions

The treatment o f sickle-cell patients with cyanate represents the first attempt to overcome the effects o f a deleterious mutation by direct chemical modification o f the mutant gene product. Human trials have revealed that oral administration o f cyanate is unsafe; complications arise that were not uncovered in animal studies.

(

~

ormalhemoglobinmolecule Hb(2echains,2~chains)

VAL HIS LEU THR PRO GLU N-termi e n~chai d onalfn

V:L

H~S L~U T:R

P~O V:L

7'

m

Sickle-celanemi l ahemoglobin Hbs(2achains,2abnormal~chains) Normal and sickle.cell mzemia hemoglobins showing (--~ the valine substitution at position, 6 on the ~chairL

Sickle-cell anemia occurs in persons who have inherited a mutant gene, from both parents, which directs the insertion of valine instead ofglutamic acid in the sixth position (from the valine terminus) of the fl chain of hemoglobin. When deoxygenated this mutant hemoglobin, designated Hb S, forms intermolecular aggregates which deform the erythrocytes and make them rigid. The shortened survival and abnormal flow properties of these cells result in the anemia and the vaso-occlusive phenomena which account for the majorD.R.H. is Professor of Medicine at the University of Miami School of Medicine, Miami, Fla. 33152, U.S.A.

The studies with urea prompted Cerami and Manning to examine the effects of cyanate on sickling and hemoglobin S gelation, since in solution cyanate is in equilibrium with urea. They found that cyanate was a much more effective inhibitor ofsickling than urea and that its effects were irreversible, whereas those of urea were reversible [6]. In an early experiment it was demonstrated that erythrocytes removed from patients with sickle-cell anemia, treated with cyanate in vitro and then returned to the patient after tagging with radioactive chromate, had a much longer survival than untreated cells [7].

ity of the clinical manifestations of this disease, including the episodic occurrence of generalized or localized severe skeletal pain, the so-called 'painful crisis'. In 1966 Murayama postulated that hydrophobic interaction between the N-terminal valine and the f16 valine played a role in the aggregation of deoxyhemoglobin S [1]. Later it was demonstrated that urea inhibits gelation of solutions of Hb S, which gave rise to the suggestion that intravenous urea might be a useful form of therapy for termination of painful crises [2]. Initial trials appeared encouraging, but later co-operative double-blind studies failed to confirm any clinical benefit from urea [3-5].

These observations stimulated countless studies on the pharmacology and toxicology of cyanate and its specific reaction with normal hemoglobin and Hb S and with proteins in general. This literature is too extensive to be reviewed in detail here and is adequately covered in recent reviews [8,9]. Cyanate reacts with functional groups of proteins: reversibly with the sulfhydryl group of cysteine, the carboxyl groups of glutamic and aspartic acids, the imidazole nitrogen of histidine and the phenolic oxygen of tyrosine, and irreversibly with unprotonated amino groups [1014]. At the normal intracellular pH of 7.2, reaction with ~ amino groups is markedly favored over that with e amino groups since for most proteins the pK3 values for these groups are approximately 7.5 and 9.5, respectively. For hemoglobin this difference in reactivity is nearly 100-fold. Furthermore, reaction with hemoglobin is favored over reaction with other proteins since the pKa of the ~ amino group of the ~ chain of hemoglobin is lower than that of most other proteins [15]. Cyanate has been used to specifically modify the ~ amino groups of hemoglobins in elegant studies which demonstrated the site of carbamino formation [16,17]. The pK~ of amino groups has been determined by the kinetics of reactivity with cyanate [18]. The reaction product of an amino group with cyanate is stable. Upon acid hydrolysis the carbamylated N-terminal amino acid cyclizes, cleaving the terminal peptide bond and releasing the N-terminal amino acid as its hydantoin derivative. This reaction has been utilized for the identification of N-terminal amino acids in proteins and polypeptides [19]. Manning et al. [20] developed a sensitive gas chromatographic assay for quantitation of valine hydantoin which has been widely employed in clinical and laboratory studies to measure the extent of hemoglobin carbamylation. The N-terminal amino acid of both the • and fl chains of human hemoglobin is valine. The carbamylation

TIBS - April 1976

74

H 0

I

C-H

II I

'l

R - N - C - - C - - N H 2 + HN=C=O H

pH 7-8

H 0

CH

H

~IH

I

I

,

H

R-N~C-C--N-C--NH

2

O , H+

HaC\ H /CH-C

H3C R-NH~z+H- N

C=O

I

\¢/ N-H II O

Fig. 1. Reaction of cyanaCe with Ihe ~ amino group of the N-terminal amino acid (in this case valine) of a poO'peptide followed by elimination of valine hydantoh~ by acid catalysis.

reaction and acid catalyzed formation o f valine hydantoin are shown in Fig. I. Although initially it was thought that carbamylated S hemoglobin would not gel, this has been disproven. It is now generally agreed that the anti-sickling action o f cyanate results from its effects upon the functional properties of hemoglobin [21,22]. Carbamylation increases the oxygen affinity of hemoglobin, making it less likely that erythrocytes containing Hb S will deliver enough oxygen in vivo to form sufficient deoxyhemoglobin S to promote intracellular aggregation and sickling. Cyanate reacts equally with the ct amino groups o f both the ct and fl chains of oxyhemoglobin; in deoxyhemoglobin, reaction with the ct chain is favored [23]. Carbamylation o f the cc chain contributes more to the increase of affinity for oxygen than reaction with the fl chain [16,24,25]. Clinical applications

Although many proteins are carbamylated by cyanate in vitro, in the lower doses thought appropriate for clinical application, there appeared to be no adverse effects o f cyanate in animals [26-28]. The single major effect o f cyanate observed in animals is the left shift in the oxygen dissociation curve, which causes an increase in red cell mass analogous to that in persons with mutant hemoglobins which possess high oxygen affinity. The initial clinical trials with sodium cyanate were started by workers at the

Rockefeller University in 1971. Studies were conducted on oral and intravenous administration of the drug as well as in vitro 'extracorporeal' carbamylation of blood, then returned to the patient. Early studies designed to study toxicity o f the drug administered orally to persons with sickle cell anemia revealed that there were significant dose-related levels o f hemoglobin carbamylation, increases in red cell survival and red cell mass, and decreases in reticulocyte counts and serum bilirubin [29,30]. In patients receiving doses above 30 mg per kg per day, nausea, epigastric pain, retrosternal distress, tiredness and drowsiness were observed but these symptoms tended to diminish with time. Although these initial studies had not been designed to determine the effects of cyanate upon the incidence o f painful crises, a retrospective analysis indicated that there were fewer crises in the patients who had higher hemoglobin carbamylation [29]. In 16 patients observed for an average of 11.1 months and having an average carbamylation o f 0.16 moles N C O - / m o l e hemoglobin tetramer, the incidence of crises was 3.6 per year whereas 15 patients treated for the same period of time who had an average carbamylation o f 0.39 experienced 2.1 crises per year. In a group of nine untreated patients observed for an average o f 9.6 months, there was an average of six crises per year. In April 1973 we started a prospective double-blind cross-over study to determine the efficacy o f sodium cyanate for the prevention of painful crises in patients with sickle cell anemia. Nineteen symptomatic patients were randomized into two groups and begun on either placebo or NaNCO, 30 mg per kg per day, taken in a single dose at bedtime. At the end of six months the medications were switched. Both patients and physicians were blinded. Patients were seen every two weeks, assessed clinically and various laboratory tests performed including complete blood counts, determination o f whole blood Pso (mm Hg at which hemoglobin is 50% saturated with 02) and hemoglobin carbamylation. Patients were instructed to come to the emergency room for any episode of pain lasting over four hours where they were evaluated by a physician participating in the study. If-pain persisted for more than eight hours in the energency the patient was admitted to the hospital. There were marked variations in the mean levels o f carbamylation achieved by these 19 patients while taking cyanate. Although some patients undoubtedly did not adhere strictly to the dosage schedule, other factors have been shown to affect the amount o f drug absorbed [31]. The effects upon hemotologic parameters were

similar to those reported by the Rdckefeller group (Table I). In general, the magnitude of change for each patient was related to the degree o f hemoglobin carbamylation. Despite the hemotologic improvement in these patients while taking cyanate, there was no decrease in the number o f episodes of painful crises (Table II), their duration or severity. The reasons for failure o f cyanate to decrease the symptoms o f sickle-cell anemia in spite o f the rise in red cell mass are not clear. A clinical trial with androgens, which expand the red cell mass but have no effect on sickling, had to be discontinued because several patients had an increase in frequency and severity o f crises thought to result from the greater blood viscosity as the packed cell volume increased [32]. With cyanate it is possible that there is a direct counterbalancing of the increased oxygen affinity by the increase in viscosity resulting in lack o f benefit to the patient in terms o f incidence o f painful crises. Whether long-term cyanate therapy might decrease the cumulative organ damage resulting from repeated occurrences o f small areas o f infarction is not known. T o x i c i t y problems

Significant toxicities have occurred in patients taking cyanate, including a reversible peripheral neuropathy involving both sensory and motor nerves [33], cataracts TABLE I Mean percentage change in hematologic data in 19 patients who received oral sodium cyanate for 6 months Mean

Range

Cgrbamylation* Ps0** Hemoglobin

0.48 - 9.3%

0.15 to 0.84 -18to0

concentration

+ 10 % - 19 %

- 2 to +24 -54 to +21

-12 % + 7.9°0 +47 %

-50 to +50 -20 to +31 -20 to + 150

Reticulocytecount Total bilirubin concentration Red-cell volume Red-cell half-life

*Molesof valine hydantoin per mole of hemoglobin tetramer. **Seetext for definition.

TABLE lI Incidence of painful crises in 19 patients who completed one year on double-blindcyanate study

Class Ill Class IV Total

Placebo

Cyanate

22 (2) 11 (2) 33 (4)

23 (2) 17 (6) 40 (8)

Class III = emergencyroom visit; class IV = hospital-

ization. Numbers in parentheses represent crises with concomitant ba~cterialinfection.

75

T I B S - A p r i l 1976

[34] and weight loss. Clinical toxicity observed in the Rockefeller University studies was more severe in the patients taking higher doses of cyanate. However, even at the lower dosage employed in our study, four developed sub-clinical n e u r o p a t h y as evidenced by delayed nerve conduction velocity, seven had weight loss greater than 50~, of body weight and one patient developed large bilateral cataracts. These complications of cyanate therapy have lead to the termination of all clinical trials employing oral sodium cyanate. Clinical trials with cyanate using batch in vitro treatment o f the patient's blood followed by its return to the patient's circulation are currently in progress. This is a very tedious form o f therapy, both for the patient and the physician, but it does have the advantage that very little free cyanate is given to the patient, thereby minimizing the likelihood of toxicity. In ten patients in w h o m 201'o of the circulating red cell mass was removed and treated weekly or biweekly for 12 m o n t h s with sufficient N a N C O to yield a carbamylation o f the treated cells o f 1.5_+_0.4 moles N C O / m o l e hemoglobin tetramer, there was a decrease in crises from 0.25 per m o n t h before treatment to 0.08 per m o n t h during treatment [35]. This study d e m o n strates the feasibility of this approach to therapy. However, one cannot conclude that this form of therapy actually decreases the incidence ofcrises since the study lacks blinded controls. It has been established that sodium cyanate is unsafe for oral use. Even if cyanate eventually proves to be o f little or no value as a form o f extracorporeal treatment for sickle cell anemia, the enthusiasm generated by its potential has stimulated significant research. It represents the first time that attempts have been made to overcome the effects of a deleterious m u t a t i o n by direct chemical modification o f the m u t a n t gene product. It has created a great stimulus to studies on chemical modification o f the hemoglobin molecule and the search for other anti-sickling compounds. In addition, valuable experience has been gained in m e t h o d s o f assessing therapy in this complex disease, and the impetus was provided for the development of suitable m e a n s for extracorporeal treatment o f blood. Finally, although the potential toxicities from administration of a chemical which reacts with proteins in general (though favoring modification o f hemoglobin) were anticipated, they were not uncovered in animal studies. Only after their observation in m a n has it been possible to find suitable animal models for cyanate-induced n e u r o p a t h y and cataracts. The cyanate experience not only inspired m u c h research, but also emphasized

Fig. 2. Electron micrograph ~/" a normal human ervthrocyte magn(fication I 1,000 x ( Electron micrograph supplied b)" M. ,4. Gokbnan and R. C. Le(l~ Papanieolaon Cancer Research Institute. Florida).

Fig. 3. Electron micrograph of a sickled eo'throcrte from a patient with siekle-cell anaemia - magnification I0,000 x ( Electron micrograph supplied by J. T. Thornthwaite oral R.C. Le(f. Papanicolann Cancer Research Institute. Florida).

the need to proceed with caution when evaluating such highly reactive and nonspecific chemicals in man. References

1 Murayama, M. (1966) Science 153, 145-149 2 Nalbandian, R. M., Henry, R. L., Barnhart, M. 1., Nichols, B. M. and Camp, F. R. (1970) U.S. Army Medical Research Laboratory Report No 896 3 Kraus, A. P., Robinson, H., Cooper, M. R., Felts, J.H., Rhyne, A.L., Porter, F.S., Rosse, W.F. and Grush, O.C. (1974) J. Amer. Med. Ass. 228, 1120-1124

4 McCurdy, P.R., Binder, R.A., Mahmood, L., Bullock, W. H., Hudson, R. L., Jilly, P.N., Scott, R.B., Garren, T.A., Schmitz, T.H. and Westerman, M. P. (1974) J. Amer. Med. Ass. 228, 11251128

5 Rhodes. R.S., Revo, L., Hara, S., Hartmann, R.C. and Van Eys, J. (1974) J. Amer. Med. Ass. 228, 1129-1131 6 Cerami, A. and Manning, J. M. (1971) Proc. Nat. Acad. Sci. USA 68, 1180-1183 7 Gillette, P.N., Manning, J.M. and Cerami, A. (1971) Proc. Nat. Acad. Sci. USA 68, 2791-2793 8 Manning, J.M., Cerami, A., Gillette, P.N., deFuria, F.G. and Miller, D.R. (1974) Advan. En.-ymol. 40, 1-27 9 Harkness, D.R. and Roth, S. (1975) in Progress in Hematology(Brown, E. B., ed.) Vol. 9, pp. 157184, Grune and Stratton, New York 10 Smyth, D.G. (1967) J. Biol. Chem. 242, 15791591 I 1 Stark, G. R. (1964)J. Biol. Chem. 239, 1411-1414 12 Stark, G. R. (1965) Biochemistry 4, 588-595 13 Stark, G. R. (1965) Biochemistry 4, 2363-2367 14 Stark, G.R., Stein, W.H. and Moore, S. (1960)

T I B S - A p r i l 1976

76 J. Biol. Chem. 235, 3177-3181 15 Hill, R.J. and Davis, R.U. (1967) J. BioL Chem. 242, 2005-2012 16 Kilmartin, J.V. and Rossi-Bernardi, L. (1971) Bioehem. J. 124, 31~5 17 Kilmartin, J.V., Fogg, J., Luzzana, M.and RossiBernardi, L. (1973) J. Biol. Chem. 248, 7039-7043 18 Gardner, M.H., Garner, W.H. and Gurd, F. R. N. (1973) J. Biol. Chem. 248, 5451-5455 19 Stark, G.R. and Smyth, D.G. (1963) J. Biol. Chem. 238, 214-226 20 Manning, J. M., Lee, C.K., Cerami, A. and Gillette, P.N. (1973) J. Lab. Clin. Med. 81,941-945 21 Diederich, D. (1972) Biochem. Biophys. Res. Commun. 46, 1255-1261 22 May, A., Bellingham, A.J.. Huehns, E.R. and Beaven, G. H. (1972) I.zmcet I, 658-661 23 Njikam, N., Jones, W. M., Nigen, A. M., Gillette, P.N., Williams, R.C. and Manning, J.M. (1973) J. Biol. Chem. 248, 8052-8056 24 Nigen, A.M., Njikam, N., Lee, C.K. and Manning, J. M. (1974) J. Biol. Chem. 249, 6611-6616 25 Rossi, F., Perrella, M., Bresciani, D., Guglielmo, G. and Rossi-Bernardi, L. (1975) FEBS la,tt. 55, 99-101 26 Cerami, A., Allen, T.A., Graziano, J.H., deFuria, F.G., Manning, J. M. and Gillette, P. N. (1973) J. PharmacoL Exp. Ther. 185, 653-666 27 Graziano, J. H., Thornton, Y. S., Leong, J. K. and Cerami, A. (1973) J. Pharmacol. Exp. Ther. 185, 667-675 28 Leong, J.K., Grady, R.W., Herbert, J., Graziano, J. H., Cerami, A., Judge, M. and Quater-

main, D. (1974) J. PlaarmacoL Exp. Ther. 191, 60-67 29 Gillette, P.N., Peterson, C.M., Lee, Y.S. and Cerami, A. (1974) New Eng. J. Med. 290,654-659 30 Gillette, P.N., Lee, Y.S. and Peterson, C.M. (1973) in Progress ira Hematology (Brown, E.B., ed.) Vol. 8, pp. 181-190, Grune and Stratton, New York 31 Nigen, A.M., Manning, J.M., Peterson, C.M. and White, J. M. (1975) J. Pharmocol. Exp. Ther. 195, 333-339 32 Zanger. B., Alfrey, C.P., Mclntire, L.V. and Leverett, L. B. (1974)J. Lab. Cl#a. Med. 84, 88990 I 33 Peterson, C. M., Tsairis, P., Ohnishi, A., Lu, Y. S., Grady, A., Cerami, A. and Dyck, P.J. (1974) Ann. Intern. Med. 81, 152-158 34 Nicholson, D. H., Harkness, D, R., Bensen, W.E. and Peterson, C. M. (1976) Arch. Ophthahnol. (in the press) 35 Diederich, D., Gill, P., Trueworthy, R. and Larsen, W. (1975) in Erythroeyte Structure and Function (Brewer, G.J., ed.) Progr. Clin. Biol. Res., Vol. I, pp. 379-398, Allen R. Liss, Inc., New York.

Intranuclear messenger-ribosome complexes and protein synthesis Jo Alene Goidl

Recent reports are reviewed, two o f which provMe evidence o f the association o f ribosornal R N A or ribosornes with messenger-like R N A within the e u k a r y o t i c cell nucleus. The significance o f these findings is related to the theory o f nucleolus-dependent transport o f genetic htformation. The possibility o f intranuclear prote#7 synthesis is also considered.

The concept of controlled co-ordinated transfer of messenger R N A and ribosomes from the nucleus to the cytoplasm o f e u k a ryotic cells has been considered for m a n y years. However, the generally accepted view contends that message and ribosomes arrive in the cytoplasm independently of one another. Two recent reports now suggest that at least some messenger R N A ( m R N A ) m a y be associated with ribosomes, or their precursors, within the cell nucleus and may therefore arrive in the cytoplasm as a m R N A - r i b o s o m e complex. J.A.G. is at the Fels Research Institute, Temple Universio'. Philudelphia. Pa.. U.S.A.

Heterokaryons made by the fusion of two different cell types have been used by n u m e r o u s investigators to examine the processes by which genetic information is expressed. Harris and his co-workers [1] used a m o u s e cell-hen erythrocyte hybrid to follow the appearance of hen-specific antigens on the surface of the heterokaryon. Birds are a m o n g the few species that have nucleated erythrocytes but in mature erythrocytes the nucleus is generally inactive. In the heterokaryon the erythrocyte is reactivated gradually after fusion. During the period of reactivation R N A synthesis resumed in the erythrocyte nuclei but new hen-specific antigens did not appear on the cell surface. However, when

nucleoli began to develop in the erythrocyte nuclei, hen-specific antigens were detected on the cell surface. Similar results were obtained when chick embryos were used as the source oferythrocytes although the sequence o f events 6ccurr~d more rapidly. Other chick-specific proteins including several soluble enzymes - were examined with comparable results. The chick protein was demonstratable in the heterokaryon cytoplasm only when nucleoli appeared in the erythrocyte nuclei. Thus, it seemed likely that the ability o f the erythrocyte nucleus to determine the synthesis o f a protein was conditional on the development o f the nucleolus. The major nuclear species o f R N A which will be mentioned in this review are heterogeneous nuclear R N A ( h n R N A ) , ribosomal precursor R N A and ribosomal R N A (rRNA). H n R N A is characterized by its rapid labeling and polydispersity of size which is generally o f high molecular weight. It is thought to be the primary precursor to partially processed m R N A (prem R N A ) as well as to cytoplasmic messenger R N A ( m R N A ) . Analysis of the R N A species synthesized in the two types o f nuclei o f the heterokaryon revealed rapidly labeled, polydisperse R N A and r R N A in the m o u s e nuclei but only polydisperse R N A in the erythrocyte nuclei before nucleolar development. As expected, r R N A was synthesized in the erythrocyte nuclei when they developed nucleoli. However, gene transcripts were apparently synthesized prior to nucleolar development but these 'messages' were translated into specific proteins only after the appearance o f nucleoli. To determine whether there was a functional relationship between the simultaneous appearance o f nucleoli and chickspecific proteins, the effect o f inactivation o f the nucleolus after the appearance o f chick proteins was examined. Nucleoli were irradiated with a m i c r o b e a m o f ultraviolet light. W h e n an extranucleolar region o f the erythrocyte nucleus or one o f the two nucleoli in the erythrocyte nucleus was irradiated, the enzymatic activity of the chick protein was indistinguishable from the activity in unirradiated cells. However, when a solitary nucleus was irradiated enzymatic activity progressively decreased to the level observed in non-hybridized m o u s e cells. It seemed the nucleolus was indeed necessary for the continued, as well as initial, expression of structural genes. The possibility that the nucleolar involvement in structural gene expression might be related to the flow o f genetic information from the nucleus to the cytoplasm was examined by irradiating the nucleolus of m o n o n u c l e a t e d cells and subsequently exposing the cells to a radioactive R N A