Science
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Imidoesters might react in any of four ways Science ο New agents inhibit Wood cell sickling Oxygenated hemoglobin S Imidoesters might cross-link — groups within hemoglobin S and increase attraction for oxygen Imidoesters might cross-link • amino acids at interaction points between hemoglobin S molecules Low oxygen " ^ Deoxygenated hemoglobin S --. results in rearrangement of groups • within hemoglobin molecule • Parallel alignment of --i hemoglobin S molecules Imidoesters might inhibit these structural changes and gelation Imidoesters might cross-link membrane proteins, making membranes less permeable to ion leakage "Λ --Λ • " > | I Hemoglobin S molecules gel Cell membrane alteration Loss of potassium ions Sickling of cell Irreversible hemoglobin S loss Scientists at Lawrence Berkeley Laboratory find that three imidoesters retard sickling in vitro without side effects After recently announcing the effec- tiveness of dimethyladipimidate (DMA) for inhibiting sickling of red blood cells, in vitro, Lawrence Berkeley Laboratory scientists have discovered that three more compounds exhibit the same activity. All three compounds are imidoesters, one of them bifunctional and two monofunctional. The new compounds are being inves- tigated in vitro by the laboratory's membrane bioenergetics group, headed by Dr. Lester Packer. He is assisted by graduate student Edwin N. Bymun. The molecular defect in sickle cell anemia involves the single substitution of a molecule of valine for glutamic acid in the sixth position of the β chain of hemoglobin. Deoxygenated sickle erythrocytes (red blood cells) appear with the characteristic abnormal shape. In addition to their abnormal shape, sickled erythrocytes also exhibit altered properties such as decreased filterability and decreased viscosity. Some of the altered properties are traceable to the abnormal shape, whereas others are traceable to changes in the cell membranes. In the latter category is included increased potassi- um ion leakage from deoxygenated sickled cells. In oxygenated erythrocytes, hemo- globin molecules are dispersed ran- domly. When deoxygenated, the mole- cules align themselves in a more or less parallel array, eventually forming fila- ments and microtubules. In the sickled erythrocytes, gelation occurs during contact between the molecules, causing cell distortion, potassium ion leakage, and the characteristic sickle shape. The general object of chemical modifi- cation strategies to prevent sickling is the prevention of hemoglobin gelation, which appears to be the root of the trouble. Several attempts to inhibit sickling by modifying the hemoglobin molecule in vitro have been successful. Nitrogen mustard appears to inhibit hemoglobin gelation directly. Cyanate also appears to be effective in altering hemoglobin oxygen affinity. Although cyanate is a powerful antisickling agent, it has some undesirable side effects that have prompted the search for equally power- ful agents without the side effects. The first of these, discovered at Lawrence Berkeley Laboratory, was DMA. New compounds added to the list are the bifunctional imidoesters dimethylsub- erimidate (DMS), and dithio-bis-di- methylpropioimidate (DMSSP), which is effective by itself and breaks down into monofunctional fragments that are also effective. The monofunc- tional imidoester, ethyl acetimidate (EA), completes the list of new agents. In completely deoxygenated sickle erythrocytes, 50% inhibition of sickling occurs with less than 500 micromolar DMA or DMS. EA is almost as effec- tive. DMSSP has been found to be ef- fective in about the same concentra- tion range as DMA, DMS, and EA. However, DMSSP (which is a cleava- ble bifunctional reagent) inhibits sick- ling even after the dithiol bridge is broken by adding reduced thiols. Packer says that the effects of all these reagents in treating sickle eryth- rocytes are being investigated in terms of a number of structural and metabol- ic parameters. For example, studies with DMA show that the net loss of potassium from sickled cells, incubated under hypoxic conditions, is virtually eliminated. Potassium loss is regarded as a quantitative measure of the amount of sickling. The LBL workers have found that viscosities of sickle erythrocytes treated with such agents as DMA are in the range of normal cells. Scanning electron micrographs of normal and both treated and untreated sickle cells show that treatment with the imidoesters dramatically reduces the degree of sickling. Taken together with the viscosity and potassium leak- age data, the micrographs leave little doubt of the effectiveness of the imi- Continued on page 21 16 C&EN March 17, 1975
Transcript of Science
Imidoesters might react in any of four ways
Science ο
New agents inhibit Wood cell sickling
Oxygenated hemoglobin S
Imidoesters might cross-link — groups within hemoglobin S and increase attraction for oxygen
Imidoesters might cross-link • amino acids at interaction points between hemoglobin S molecules
Low oxygen
" ^ Deoxygenated hemoglobin S --. results in rearrangement of groups
• within hemoglobin molecule
• Parallel alignment of - - i hemoglobin S molecules
Imidoesters might inhibit these structural changes and gelation
Imidoesters might cross-link membrane proteins, making membranes less permeable to ion leakage
"Λ
--Λ
• " > |
I
Hemoglobin S molecules gel
Cell membrane alteration
Loss of potassium ions
Sickling of cell
Irreversible hemoglobin S loss
Scientists at Lawrence Berkeley Laboratory find that three imidoesters retard sickling in vitro without side effects
After recently announcing the effectiveness of dimethyladipimidate (DMA) for inhibiting sickling of red blood cells, in vitro, Lawrence Berkeley Laboratory scientists have discovered that three more compounds exhibit the same activity. All three compounds are imidoesters, one of them bifunctional and two monofunctional.
The new compounds are being investigated in vitro by the laboratory's membrane bioenergetics group, headed by Dr. Lester Packer. He is assisted by graduate student Edwin N. Bymun.
The molecular defect in sickle cell anemia involves the single substitution of a molecule of valine for glutamic acid in the sixth position of the β chain of hemoglobin. Deoxygenated sickle erythrocytes (red blood cells) appear with the characteristic abnormal shape. In addition to their abnormal shape, sickled erythrocytes also exhibit altered properties such as decreased filterability and decreased viscosity. Some of the altered properties are traceable to the abnormal shape,
whereas others are traceable to changes in the cell membranes. In the latter category is included increased potassium ion leakage from deoxygenated sickled cells.
In oxygenated erythrocytes, hemoglobin molecules are dispersed randomly. When deoxygenated, the molecules align themselves in a more or less parallel array, eventually forming filaments and microtubules. In the sickled erythrocytes, gelation occurs during contact between the molecules, causing cell distortion, potassium ion leakage, and the characteristic sickle shape. The general object of chemical modification strategies to prevent sickling is the prevention of hemoglobin gelation, which appears to be the root of the trouble.
Several attempts to inhibit sickling by modifying the hemoglobin molecule in vitro have been successful. Nitrogen mustard appears to inhibit hemoglobin gelation directly. Cyanate also appears to be effective in altering hemoglobin oxygen affinity. Although cyanate is a powerful antisickling agent, it has some undesirable side effects that have prompted the search for equally powerful agents without the side effects. The first of these, discovered at Lawrence Berkeley Laboratory, was DMA. New compounds added to the list are the bifunctional imidoesters dimethylsub-erimidate (DMS), and dithio-bis-di-methylpropioimidate (DMSSP), which is effective by itself and breaks
down into monofunctional fragments that are also effective. The monofunctional imidoester, ethyl acetimidate (EA), completes the list of new agents.
In completely deoxygenated sickle erythrocytes, 50% inhibition of sickling occurs with less than 500 micromolar DMA or DMS. EA is almost as effective. DMSSP has been found to be effective in about the same concentration range as DMA, DMS, and EA. However, DMSSP (which is a cleava-ble bifunctional reagent) inhibits sickling even after the dithiol bridge is broken by adding reduced thiols.
Packer says that the effects of all these reagents in treating sickle erythrocytes are being investigated in terms of a number of structural and metabolic parameters. For example, studies with DMA show that the net loss of potassium from sickled cells, incubated under hypoxic conditions, is virtually eliminated. Potassium loss is regarded as a quantitative measure of the amount of sickling. The LBL workers have found that viscosities of sickle erythrocytes treated with such agents as DMA are in the range of normal cells.
Scanning electron micrographs of normal and both treated and untreated sickle cells show that treatment with the imidoesters dramatically reduces the degree of sickling. Taken together with the viscosity and potassium leakage data, the micrographs leave little doubt of the effectiveness of the imi-
Continued on page 21
16 C&EN March 17, 1975
Scanning electron micrographs of sickle erythrocytes show oxygenated cells (top), deoxygenated cells (middle), and deoxygenated cells treated previously with dimethyladipimidate (DMA) under oxygenated conditions (bottom)
doesters in treating sickling in vitro, without noticeable side effects.
The mechanism of sickling inhibition by the imidates is unknown. However, Packer suggests some possibilities. He notes that prevention of sickling by the monofunctional EA reagent indicates that modification of hemoglobin oxygen affinity probably accounts for most of the sickling prevention. Chemical modification of cell membranes by both types of reagents, however, also may produce an effect on hemoglobin structure. Membrane modification is probably the principal reason for the reduction of potassium leakage in treated cells.
Packer believes that it is still too early to speculate about the possible clinical role for imidoesters in the treatment of sickle cell disease. But he does observe that the imidoesters act in concentrations that are several orders of magnitude lower than those employed with cyanate. The marked reduction in sickling and the apparent lack of major effects on cell metabolism after treatment suggest that the imidoesters may be used without seriously disturbing red cell physiology. Because sickling erythrocytes have lifetimes about half those of normal erythrocytes, it is possible that treatment with the agents may be useful in stabilizing the functioning life spans of such cells. Stabilization of normal cells also may be prolonged with the agents for in vitro storage. α
Technology
ASTM aims at commercial catalyst standards Some ordering of the sometimes chaotic affairs in the world of commercial catalyst production is expected from the deliberations of a new committee established by the American Society for Testing & Materials. The first working session of ASTM Technical Committee D-32 will convene on May 20 to develop standards for commercial catalyst materials and their use. The committee was organized in January
and is chaired by Dr. Arthur H. Neal of Esso Research Laboratories of Exxon Corp., Baton Rouge, La. Among the most urgent problems to be considered by the committee will be standards for auto emission catalysts.
The scope of Committee D-32 encompasses development of test methods and related reference materials, classifications, recommended practices, and nomenclature and definitions per-
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I I March 17, 1975 C&EN 21
