Biomedical subjects

D J Abraham

Publications and source records attributed to D J Abraham.

At least 19 recordsLinked to original sources

PubMed1995

How allosteric effectors can bind to the same protein residue and produce opposite shifts in the allosteric equilibrium.

Monoaldehyde allosteric effectors of hemoglobin were designed, using molecular modeling software (GRID), to form a Schiff base adduct with the Val 1 alpha N-terminal nitrogens and interact via a salt bridge with Arg 141 alpha of the opposite subunit. The designed molecules were synthesized if not available. It was envisioned that the molecules, which are aldehyde acids, would produce a high-affinity hemoglobin with potential interest as antisickling agents similar to other aldehyde acids reported earlier. X-ray crystallographic analysis indicated that the aldehyde acids did bind as modeled de novo in symmetry-related pairs to the alpha subunit N-terminal nitrogens. However, oxygen equilibrium curves run on solutions obtained from T- (tense) state hemoglobin crystals of reacted effector molecules produced low-affinity hemoglobins. The shift in the allosteric equilibrium was opposite to that expected. We conclude that the observed shift in allosteric equilibrium was due to the acid group on the monoaldehyde aromatic ring that forms a salt bridge with the guanidinium ion of Arg 141 alpha on the opposite subunit. This added constraint to the T-state structure that ties two subunits across the molecular symmetry axis shifts the equilibrium further toward the T-state. We tested this idea by comparing aldehydes that form Schiff base interactions with the same Val 1 alpha residues but do not interact across the dimer subunit symmetry axis (a new one in this study with no acid group and others that have had determined crystal structures). The latter aldehydes shift the allosteric equilibrium toward the R-state. A hypothesis to predict the direction in shift of the allosteric equilibrium is made and indicates that it is not exclusively where the molecule binds but how it interacts with the protein to stabilize or destabilize the T- (tense) allosteric state.

Aldehydes↗

PubMed1995

Bisaldehyde allosteric effectors as molecular ratchets and probes.

Four new series of monoaldehyde bisacids and bisaldehyde bisacids with varying chain lengths have been synthesized and evaluated as allosteric effectors of hemoglobin. Molecular modeling, oxygen equilibrium, and crystallographic studies were combined for structure/function studies. Crystallographic analyses of the bisaldehydes reveal that Schiff base interaction occurred exclusively between Val 1 alpha and Lys 99 alpha of the opposite alpha chain even though the two terminal Val 1 alpha nitrogens are ideally spaced to also form cross-links. The reason for the observed mode of binding appears to be the influence of chain direction set by key substitutions on the bisaldehyde molecule. Even longer chain derivatives that could overcome the direction set by the key functional groups bind in the same manner. These studies support the general conclusion that long flexible molecules prefer to bind along cavity walls, like double-sided molecular sticky tape, rather than span large open spaces with few chances for interaction. The cross-linked bisaldehydes bind at the same site when incubated under both allosteric states and exhibit reduced cooperativity with a significant decrease in oxygen affinity. The chain length acts as a molecular ratchet and dictates the degree of allosteric effect observed. The tighter the cross-link, the greater the constraint on the tense- (T-) state and the stronger the allosteric effect that is produced. The monoaldehyde bisacids bind in the same fashion with Schiff base formation at Val 1 alpha while the acid that replaces the second aldehyde moiety forms a salt bridge with Lys 99 alpha of the opposite subunit. This class of molecules has weaker allosteric effector activity as would be expected with replacement of one covalent bond by a salt bridge. The importance of Lys 99 alpha on the allosteric equilibrium is confirmed.

Aldehydes↗

PubMed1995

Intrinsic activity at the molecular level: E. J. Ariëns' concept visualized.

The concept of using affinity and intrinsic activity to analyze drug interactions with receptors has had a long history in pharmacological studies. In the simplest case, the biological response will be proportional to the amount of drug bound, i.e. its affinity. However, the biological response is also mediated by the ability of a drug when bound to exert its maximum effectiveness. This effectiveness is termed the intrinsic activity. Physicochemical processes have been thought to be at the basis of intrinsic activity. Detailed oxygen and solution binding experiments combined with X-ray crystallographic studies on allosteric effectors to hemoglobin demonstrate that these potential drug agents bind at the same site in hemoglobin with similar binding constants yet shift the allosteric equilibrium and the oxygen affinity of the T-structure by different degrees. Therefore some of the effectors with similar binding affinities for the same site exhibit varying degrees of affectiveness, i.e. they possess different intrinsic activities. The intrinsic activity of the effector is defined as the ratio of the oxygen affinity constant to the T-state with drug/oxygen affinity constant to the T-state without drug (KT+drug)/(KT control). The source of the intrinsic activity appears to be the ability of the effectors to interact with key residues such as Lys99 alpha at the binding site. These results suggest a general molecular mechanism for allosteric effector modulation of hemoglobin function that might be of use in other allosteric enzyme systems.

Allosteric Regulation↗

PubMed1995

Altered cytokine expression in T lymphocytes from human immunodeficiency virus Tat transgenic mice.

Examination of the interaction between human immunodeficiency virus (HIV) regulatory gene products and the host immune system is fundamental to understanding the pathogenesis of HIV and could reveal possible targets for therapeutic intervention in the treatment of AIDS. The HIV Tat gene is a potential candidate for this type of strategy. Transgenic mice can be used to investigate the in vivo effects of Tat on the developing and dynamic immune system and on cellular gene expression. Thus, we have generated transgenic mice that harbor the HIV type 1 Tat gene under the transcriptional control of the human CD2 gene regulatory elements. This expression cassette results in high-level, tissue-specific transcription of the transgene within the T-cell compartment. In this report, we demonstrate the effects of Tat on the in vivo immune system. CD2-Tat transgenic mice show no signs of aberrant thymic development and have normal levels of T-cell subsets in the thymus and peripheral lymphoid organs. However, activated T cells from transgenic mice contain increased levels of tumor necrosis factor beta mRNA as well as biologically active tumor necrosis factor protein and express elevated levels of transforming growth factor beta and interleukin-4 receptor mRNA. These increased cytokine levels do not appear to alter mitogen- or antigen-stimulated responses or induce the formation of dermal lesions in ageing mice. Such investigations should provide insight into the combination of host immune factors mediating pathogenesis in HIV infection.

Aging↗

PubMed1994

The effect of physical organic properties on hydrophobic fields.

Physical organic structural properties of small molecules and macromolecules such as bond count, branching and proximity between multiple polar fragments contribute significantly to measured hydrophobicity (log P). These structural properties are encoded in the Rekker and Leo methods of calculating log P as structural-dependent factors. Regardless of the size of the atom primitive set, methods predicting log P with only atom primitives can miss subtle structural detail within series of related compounds. The HINT (Hydropathic INTeractions) model for inter- and intramolecular noncovalent interactions calculates atom-based hydrophobic constants, but uses all Leo-type factors in the calculation rather than a large set of atom primitives. Two types of applications of HINT are discussed: evaluation of the binding of an inhibitor (A74704) to HIV-1 protease, where it is shown that modeling of the protonation state (i.e., Asp25, Asp125) in the protein can strongly influence perceived substrate binding; and the use of HINT to calculate a third (hydropathic) field for CoMFA can yield a statistically enhanced and predictive model for molecular design.

Chemical Phenomena↗

PubMed1994

Evaluating docked complexes with the HINT exponential function and empirical atomic hydrophobicities.

Methods that predict geometries of ligands binding to receptor molecules can facilitate ligand discovery and yield information on the factors governing complementarity. Here, the use of atomic hydrophobicities in evaluating binding modes has been examined with four ligand-receptor complexes of known structure. In each system, hundreds of hypothetical binding orientations were generated with DOCK and evaluated using the HINT (Hydropathic INTeractions) exponential function and atomic hydrophobic constants. In three of the four systems, the experimental binding mode received the best HINT score; in the fourth system, the experimental binding mode scored only slightly lower than a similar, apparently reasonable orientation. The HINT function may be generally useful as a scoring method in molecular docking.

Binding Sites↗

PubMed1994

Effect of distamycin on chlorambucil-induced mutagenesis in pZ189: evidence of a role for minor groove alkylation at adenine N-3.

Previous work has shown that the bifunctional alkylating agent chlorambucil induces thermolabile adenine adducts and that the predominant chlorambucil-induced mutations in shuttle vector pZ189 are transversions at AT base pairs. In order to assess the role of thermolabile adducts in generating these transversions, pZ189 was treated with chlorambucil in the presence of distamycin, which specifically blocks formation of thermolabile adenine adducts. Analysis of the mutations resulting from replication of the damaged vector in human 293 cells showed that base substitutions at AT base pairs were specifically suppressed in concert with suppression of thermolabile adducts at specific sites in the supF target gene, strongly supporting a role for these adducts in mutagenesis. Since there is considerable evidence that these adducts are N-3 alkylations, a computer graphics model of such an adduct was constructed. Modeling studies indicated that the adduct could be formed with little distortion of the DNA helix. Analysis of the adduct using the HINT (Hydropathic INTeractions) program was consistent with the proposal that favorable hydrophobic interactions of the phenyl ring of chlorambucil with the wall of the minor groove may promote adenine N-3 alkylation by this drug.

Adenine↗

PubMed1993

Effect of local change in O2 saturation of hemoglobin on cerebral vasodilation from hypoxia and hypotension.

We tested the effect of certain newly synthesized allosteric modifiers of hemoglobin on the dilation induced by arterial hypoxia, arterial hypotension, and arterial hypercapnia in cerebral arterioles of anesthetized cats equipped with cranial windows for the observation of the cerebral microcirculation. The allosteric modifiers of hemoglobin are isomers of 2-(aryloxy)-2-methylpropionic acid. They shift the oxygen dissociation of hemoglobin to the right, thereby facilitating the local release of oxygen. When these compounds were applied topically by superfusion at a rate of 1 ml/min in a concentration of 0.1 mM, they had no significant effect on baseline arteriolar diameter but reduced significantly the vasodilation from arterial hypoxia and arterial hypotension. They did not influence the vasodilation from arterial hypercapnia. Spectrophotometric measurements of optical densities from pial veins 50-80 microns in diameter indicated that the superfusion with the allosteric compounds reduced hemoglobin oxygen saturation both during room air breathing and during hypoxia. We conclude that the vasodilations from arterial hypoxia and arterial hypotension are mediated by local oxygen-dependent mechanisms. The allosteric modifiers of hemoglobin may be useful as tools in investigating oxygen-dependent mechanisms.

Animals↗

PubMed1993

Enhanced oxygenation in vivo by allosteric inhibitors of hemoglobin saturation.

The in vivo effects on hemoglobin (Hb)-O2 affinity and tissue PO2 were investigated after intraperitoneal administration of 2-[4-(((dichloroanilino)-carbonyl)methyl)phenoxyl]-2-methyl propionic acid (RSR4; 150 mg/kg) or its 3,5-dimethyl derivative (RSR13; 300 mg/kg) in C3Hf/Sed mice. The Hb-O2 dissociation curve was plotted from tail vein blood samples using an O2 dissociation analyzer before and up to 160 min after compound administration. Twenty to 40 min after injection, the PO2 at 50% saturation of hemoglobin (Hb P50) increased by a mean of 25% (range 18-31%) after RSR4 and 53% (range 36-76%) after RSR13. Tissue PO2 was continuously measured using an O2 microelectrode in thigh muscle before and up to 40 min after RSR4 or RSR13 injection. Twenty to 40 min after administration, tissue PO2 increased by a mean of 78% (range 30-127%) after RSR4 and 66% (range 39-97%) after RSR13 administration in anesthetized mice. No change in tissue PO2 was seen in anesthetized controls.

Aniline Compounds↗

PubMed1992

Allosteric modifiers of hemoglobin: 2-[4-[[(3,5-disubstituted anilino)carbonyl]methyl]phenoxy]-2-methylpropionic acid derivatives that lower the oxygen affinity of hemoglobin in red cell suspensions, in whole blood, and in vivo in rats.

Two new potent allosteric effectors of hemoglobin, RSR-4 [2-[4-[[(3,5-dichloroanilino)carbonyl]-methyl]phenoxy]-2- methylpropionic acid] and RSR-13 [2-[4-[[(3,5-dimethlanilino)carbonyl]methyl]-phenoxy]-2-methylp rop ionic, are compared to the previously reported compounds L3,5 and L3,4,5 [Lalezari, I., Lalezari, P., Poyart, C., Marden, M., Kister, J., Bohn, B., Fermi, G., & Perutz, M. F. (1990) Biochemistry 29, 1515]. Unlike L3,5 and L3,4,5, RSR-4 and RSR-13 are less impeded by physiological concentrations of serum albumin. RSR-4 has also been shown to be more effective than L3,5 in shifting the allosteric equilibrium of bovine Hb toward the low-affinity T-state. X-ray crystal studies show that both RSR-4 and RSR-13 bind to only one pair of symmetry-related sites in the Hb central water cavity whereas previous studies on L3,5 and L3,4,5 demonstrated a second pair of symmetry-related binding sites near Arg 104 beta. Three major interactions between these allosteric effectors and Hb include the acid group with the guanidinium group of C-terminal Arg 141 alpha, the effector's amide oxygen with the ammonium ion of Lys 99 alpha, and the phi electrons of the halogenated or methylated aromatic ring and Asn 108 beta. No explanation has been found for the difference in number of binding sites observed for RSR-4 and RSR-13 (two sites) compared to L3,5 and L3,4,5 (four sites); also no correlation has been made between the number of binding sites and degree of allosteric shift in the oxygen equilibrium curve.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1992

X-ray diffraction study of di and tetra-ligated T-state hemoglobin from high salt crystals.

X-ray diffraction difference electron density maps at 3 A resolution obtained from di and tetra-ligated T-state hemoglobin (Hb) crystals are reported. Crystals isomorphous with native deoxyhemoglobin were obtained from ammonium sulfate solutions incubated with the synthetic allosteric effector RSR-56. RSR-56 binds at two symmetry-related Hb central water cavity sites and each molecule has major interactions with three different subunit side-chains; one effector with Arg141 alpha 2 HC3, Lys99 alpha 1 G6 and Asn108 beta 1 and the other with the symmetry related residues, Arg141 alpha 1 Lys99 alpha 2 and Asn108 beta 2. Crystals mounted in a nitrogen filled glove box were di-ligated as previously found with polyethyleneglycol Hb crystals. Crystals mounted in air under a layer of mother liquor were bright red and showed all four heme groups ligated. The difference electron density from the di-ligated crystals showed atomic movements to be restricted to the immediate neighborhood of the heme groups and the allosteric effector. By contrast, the tetra-ligated structure showed extended difference electron density near amino acid residues around both alpha and beta heme groups and along the alpha 1/beta 2 interface. Ligation of the beta heme group appears to magnify the difference density around the alpha heme groups. There is no evidence of breakage of the Bohr salt bridge, His146 beta HC3----Asp94 beta FG1, in the crystal. The observed difference electron density maps may help to clarify the way the allosteric mechanism is triggered.

Adult↗

PubMed1992

Physiologic and rheologic effects of the antisickling agent ethacrynic acid and its N-butylated derivative on normal and sickle erythrocytes.

Ethacrynic acid, a loop diuretic, has been shown to inhibit hemoglobin S polymerization. Until now, however, most studies were performed using purified solutions of hemoglobin S. The experiments reported here were designed to examine the effects of ethacrynic acid and its n-butryic acid derivative on the rheological and physiological properties of intact red blood cells. Using net and unidirectional flux measurements, both agents were shown to cause ion and water loss from normal and sickle erythrocytes. Since cell shrinkage adversely influences red cell rheology, it is unlikely that this class of compounds, despite its ability to inhibit hemoglobin S polymerization, will prove useful in the treatment of sickle cell disease.

Anemia, Sickle Cell↗

PubMed1992

KEY, LOCK, and LOCKSMITH: complementary hydropathic map predictions of drug structure from a known receptor-receptor structure from known drugs.

Three new routines (LOCK, KEY and LOCKSMITH) for the program HINT (hydrophobic interactions) are described and demonstrated. The KEY routine uses receptor structure to model the hydropathic profile of the ideal substrate for the receptor. The LOCK routine uses substrate or drug structure to model the hydropathic character of the receptor. LOCKSMITH is an algorithm designed to highlight the significant hydropathic features from a collection of agents. Ten allosteric modifiers of hemoglobin that have been characterized biologically and with X-ray diffraction to determine their protein binding sites/conformations illustrate the KEY and LOCKSMITH routines: The LOCKSMITH composite map correctly identifies the structural features and conformation of the more active modifiers. In addition, many hydropathic features of the "ideal" drug predicted by the KEY map overlap with actual structural features of the most active hemoglobin allosteric modifiers.

Algorithms↗

PubMed1992

The crystal state binding of dithionite to deoxy-hemoglobin.

The crystal state binding of sodium dithionite to deoxyhemoglobin is reported. Dithionite has been used extensively to deoxygenate hemoglobin and myoglobin and there has been considerable interest among users of dithionite about its effect on protein structure and binding site(s). We have determined that dithionite binds to deoxygenated hemoglobin crystals at the interface of two molecules in the crystal lattice. Specific residues involved in hydrogen bonds or salt interactions with dithionite include His116 and His117 of the beta 2 subunit and Lys16 of the alpha 1 subunit of the adjacent hemoglobin molecule. No binding was observed at the symmetry related His116 and 117 beta 1 residues. We have shown that dithionite does not affect the native hemoglobin structure or the binding of several allosteric inhibitors to hemoglobin and can be used to mount T state crystals in the air.

Binding Sites↗

PubMed1992

Expression and function of heterotypic adhesion molecules during differentiation of human skeletal muscle in culture.

The infiltration of skeletal muscle by leukocytes occurs in a variety of myopathies and frequently accompanies muscle degeneration and regeneration. The latter involves development of new myofibers from precursor myoblasts, and so infiltrating cells may interact with muscle at all stages of differentiation. The authors have investigated the surface expression of ligands for T-cell adhesion during the differentiation of human skeletal muscle in vitro. Myoblasts expressed low levels of ICAM-1 (CD54), which remained constant during muscle cell differentiation and could be induced by cytokines such as gamma-interferon. It is therefore likely that ICAM-1 is involved in the invasive accumulation of lymphocytes during skeletal muscle inflammation. In contrast, LFA-3 (CD58) was expressed at higher levels than ICAM-1 on myoblasts, decreased significantly during myogenesis, and was unaffected by immune mediators. Both ICAM-1 and LFA-3 were able to mediate T cell binding to myoblasts, whereas adhesion to myotubes was independent of the LFA-3 ligand. Although expressed throughout myogenesis, human leukocyte antigen class I and CD44 did not appear to mediate T cell binding. The expression of ligands that facilitate interaction of myogenic cells with lymphocytes may have important implications for myoblast transplantation.

Antibodies, Monoclonal↗

PubMed1991

4-[2-(o-carboxybenzamido)ethyl]phenoxyacetic acid dihydrate.

C18H17NO6.2H2O, Mr = 379.386, monoclinic, P21/c, a = 7.161 (5), b = 12.997 (2), c = 20.290 (3) A, beta = 99.22 (2) degrees, V = 1861.4 A3, Z = 4, Dx = 1.354 g cm-3, Cu K alpha radiation, lambda = 1.5418 A, mu = 9.2 cm-1, F(000) = 800, T = 293 K, final R(F) = 0.051 for 2262 observed reflections. The structure was solved by direct methods and compared to the previously determined structure of the analogous drug molecule, bezafibrate. The structural characteristics of the two compounds are then correlated to their relative activities as allosteric effectors of hemoglobin. The molecule adopts a synplanar-antiplanar conformation unlike other phenoxyacetic acids.

Bezafibrate↗

PubMed1991

X-ray diffraction study of the binding of the antisickling agent 12C79 to human hemoglobin.

The hemoglobin binding site of the antisickling agent 12C79 has been determined by x-ray crystallography. 12C79 is recognized as one of the first molecules to reach clinical trials that was designed, de novo, from x-ray-determined atomic coordinates of a protein. Several previous attempts to verify the proposed Hb binding sites via crystallographic studies have failed. Using revised experimental procedures, we obtained 12C79-deoxyhemoglobin crystals grown after reaction with oxyhemoglobin and cyanoborohydride reduction to stabilize the Schiff base linkage. The difference electron-density Fourier maps show that two 12C79 molecules bind covalently to both symmetry-related N-terminal amino groups of the hemoglobin alpha chains. This is in contrast to the original design that proposed the binding of one drug molecule that spans the molecular dyad to interact with both N-terminal alpha-amino groups.

Adult↗

PubMed1991

Vanillin, a potential agent for the treatment of sickle cell anemia.

Vanillin, a food additive, has been evaluated as a potential agent to treat sickle cell anemia. Earlier studies indicated that vanillin had moderate antisickling activity when compared with other aldehydes. We have determined by high performance liquid chromatography that vanillin reacts covalently with sickle hemoglobin (HbS) both in solution and in intact red blood cells. Hemoscan oxygen equilibrium curves show a dose-dependent left shift, particularly at low oxygen tensions. Rheologic evaluation (pO2 scan Ektacytometry) of vanillin-reacted HbS erythrocytes shows a dose-dependent inhibition of deoxygenation-induced cell sickling. Ektacytometry also suggests that vanillin may have a direct inhibitory effect on HbS polymer formation. Vanillin has no adverse effects on cell ion or water content. X-ray crystallographic studies with deoxyhemoglobin (HbA)-vanillin demonstrate that vanillin binds near His 103 alpha, Cys 104 alpha, and Gln 131 beta in the central water cavity. A secondary binding site is located between His 116 beta and His 117 beta. His 116 beta has been implicated as a polymer contact residue. Oxygen equilibrium, ektacytometry, and x-ray studies indicate that vanillin may be acting to decrease HbS polymerization by a dual mechanism of action; allosteric modulation to a high-affinity HbS molecule and by stereospecific inhibition of T state HbS polymerization. Because vanillin is a food additive on the GRAS (generally regarded as safe) list, and because it has little or no adverse effects at high dosages in animals, vanillin is a candidate for further evaluation as an agent for the treatment of sickle cell disease.

Anemia, Sickle Cell↗