Multiplex immune serum biomarker profiling in sarcoidosis and systemic sclerosis.

Multiplex protein technology has the potential to identify biomarkers for the differentiation, classification and improved understanding of the pathogenesis of interstitial lung disease. The aim of this study was to determine whether a 30-inflammatory biomarker panel could discriminate between healthy controls, sarcoidosis and systemic sclerosis (SSc) patients independently of other clinical indicators. We also evaluated whether a panel of biomarkers could differentiate between the presence or absence of lung fibrosis in SSc patients. We measured 30 circulating biomarkers in 20 SSc patients, 21 sarcoidosis patients and 20 healthy controls using Luminex bead technology and used Fisher's discriminant function analysis to establish the groups of classification mediators. There were significant differences in median concentration measurements between study groups for 20 of the mediators but with considerable range overlap between the groups, limiting group differentiation by single analyte measurements. However, a 17-analyte biomarker model correctly classified 90% of study individuals to their respective group and another 14-biomarker panel correctly identified the presence of lung fibrosis in SSc patients. These findings, if they are corroborated by independent studies in other centres, have potential for clinical application and may generate novel insights into the modulation of immune profiles during disease evolution.

Overview of pathogenesis of systemic sclerosis.

The aetiology of SSc is subject to ongoing research, as the precise events that underlie the development of this disease remain unclear. The pathogenesis is known to involve endothelium, epithelium, fibroblasts, innate and adaptive immune systems and their component immunological mediators. Endothelial cell damage may be the initiating factor, but the precise triggering event(s) remain elusive. Angiogenesis also appears to be dysregulated. Vasculopathy shows similarities in different organs (e.g. pulmonary arterial hypertension, renal disease, digital tip ulcers). Endothelin-1 is a potent mediator of vasculopathy, and hence represents a highly relevant target for intervention of vascular features in SSc.

Endothelin receptor selectivity: evidence from in vitro and pre-clinical models of scleroderma.

Scleroderma [systemic sclerosis (SSc)] is a spectrum of connective tissue diseases characterized by micro- and macro-vasculopathy, inflammation and autoimmunity and tissue remodelling that often leads to excessive scarring and fibrosis in both interstitial and vascular compartments. Pre-clinical investigations and gene association studies have led to improved understanding of the cell and molecular mechanisms underlying disease pathogenesis and to the identification of key molecular candidates that may represent potentially useful disease biomarkers and effective therapeutic targets. Studies on the endothelin (ET) system, pre-dominantly ET-1 and the cell surface receptors [type A (ET(A))] and type B (ET(B))], have provided evidence for an important role of this system in the vascular and fibrotic pathologies in SSc. To date, promising clinical results, utilizing dual/mixed ET receptor antagonism have been obtained in two of the vascular complications associated with SSc, ischaemic digital ulceration and pulmonary arterial hypertension. Evidence suggests that ET-1 is able to activate and re-program the functional phenotypes of vascular smooth muscle cells, microvascular pericytes and tissue fibroblasts into pro-fibrogenic cell populations with myofibroblasts-like properties. The impact of receptor-selective, over mixed-receptor, antagonism has also been studied in vitro with respect to cell differentiation and proliferation, extracellular matrix synthesis, production and deposition and in pathological cellular contraction. However, the complexity of the ET system, potential for receptor cross-talk, interactions with down-stream signal transduction cascades, as well as the potent inter-relationships with other important ligand-receptor pathways have made in vivo studies difficult to unravel. Moreover, little information is available on the role of the ET system and receptor selectivity in the recruitment and activation of mesenchymal progenitor cells in tissue remodelling and fibrosis or on the early inflammatory response. Here, we discuss the available pre-clinical evidence for the role of the ET system in tissue repair, scarring and fibrosis, using the connective tissue diseases SSc and model systems of fibrogenesis.

Nitric oxide synthase expression and activity in the tight-skin mouse model of fibrosis.

OBJECTIVES: Nitric oxide (*NO) is an important physiological signalling molecule and a potent vasodilator. We have previously demonstrated abnormal *NO metabolism in the plasma of patients with systemic sclerosis (SSc; scleroderma), a disease that features vascular dysfunction as well as collagen overproduction and fibrosis. The aim of the present study was to examine nitric oxide synthase (NOS) expression and activity and assess the potential role of antioxidants in the scleroderma-like syndrome of the tight-skin 1 (TSK-1/+) mouse, an experimental animal model for fibrosis. METHODS: Skin, lung or plasma was taken from TSK-1/+ (n = 15) and wild-type (WT; n = 12) littermate mice. Type 1 collagen, endothelial NOS (eNOS), haemoxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) protein and gene expression were determined by western blot and reverse transcriptase-polymerase chain reaction. eNOS expression was further determined by immunohistochemistry. NOS activity was evaluated by conversion of [14C] L-arginine to [14C] L-citrulline. Levels of circulating plasma nitrite/nitrate (NO(x)) were also measured. Total antioxidant activity was evaluated by ABTS+ production (ABTS = 2,2'-azino-bis-[3-ethylbenz-thiazoline-6-sulphonic acid). RESULTS: In the skin, eNOS was present in the epidermal layer, hair follicles and also in the endothelial cells lining the blood vessels. Expression of both the eNOS protein and gene was significantly reduced in TSK-1/+ skin tissue, while type 1 collagen protein was elevated compared with WT. Furthermore, there was decreased NOS activity in TSK-1/+ skin tissue; however, there was no measurable difference in plasma NO(x). Correspondingly, the protective antioxidant enzyme HO-1 and the associated transcription factor Nrf2 showed reduced protein and gene expression levels in TSK-1/+ skin, while there was also less total antioxidant activity. In TSK-1/+ lung tissue, however, we observed no difference in collagen protein expression, *NO metabolism or HO-1 expression and total antioxidant activity compared with WT. CONCLUSIONS: The findings suggest that there is also abnormal *NO metabolism in the TSK-1/+ mouse model of fibrosis, particularly in the skin, while expression and activity of protective antioxidants are reduced. The TSK-1/+ mouse may also be useful for testing treatments that target vascular endothelial cell function in patients with SSc.

X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with FMN at 1.8 A resolution.

BACKGROUND: Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate (PLP), a cofactor used by many enzymes involved in amino acid metabolism. The enzyme oxidizes either the 4'-hydroxyl group of pyridoxine 5'-phosphate (PNP) or the 4'-primary amine of pyridoxamine 5'-phosphate (PMP) to an aldehyde. PNPOx is a homodimeric enzyme with one flavin mononucleotide (FMN) molecule non-covalently bound to each subunit. A high degree of sequence homology among the 15 known members of the PNPOx family suggests that all members of this group have similar three-dimensional folds. RESULTS: The crystal structure of PNPOx from E. coli has been determined to 1.8 A resolution. The monomeric subunit folds into an eight-stranded beta sheet surrounded by five alpha-helical structures. Two monomers related by a twofold axis interact extensively along one-half of each monomer to form the dimer. There are two clefts at the dimer interface that are symmetry-related and extend from the top to the bottom of the dimer. An FMN cofactor that makes interactions with both subunits is located in each of these two clefts. CONCLUSIONS: The structure is quite similar to the recently deposited 2.7 A structure of Saccharomyces cerevisiae PNPOx and also, remarkably, shares a common structural fold with the FMN-binding protein from Desulfovibrio vulgaris and a domain of chymotrypsin. This high-resolution E. coli PNPOx structure permits predictions to be made about residues involved in substrate binding and catalysis. These predictions provide testable hypotheses, which can be answered by making site-directed mutants.

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.

Methylphenylmercury: a novel heavy atom reagent for protein crystallography.

Methylphenylmercury reacts with two normally inaccessible cysteine residues in crystals of carbonmonoxyhaemoglobin, but not with the third, normally reactive one. It may, therefore, be useful in the preparation of new heavy atom derivatives for protein crystallography.

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.

Hydropathic analysis of the non-covalent interactions between molecular subunits of structurally characterized hemoglobins.

The software program, HINT (Hydropathic INTeractions), which characterizes non-polar-non-polar, polar-polar, and non-polar-polar interactions, has been used to examine subunit interface associations involved in the hemoglobin allosteric transition at a residue and atomic level. HINT differs from many other computational programs in that it is based not on a statistical method or a force-field but employs parameters experimentally determined from solvent transfer experiments. The main focus of this study is to compare HINT scores that are based upon experimentally and thermodynamically derived measurements with experimentally determined thermodynamic results. The HINT analysis yields a good first-order approximation of experimentally measured energies for these interactions as determined by free energies of dimer-tetramer assembly for mutant hemoglobins. The results provide a framework for understanding subunit stabilities based upon individual atom interactions and repulsions. HINT, in agreement with previous analyses, indicates that: (1) the alpha1beta1 and alpha2beta2 subunit contacts are stabilized via several polar and many hydrophobic interactions with few repulsive contact areas in both the T (deoxyhemoglobin) and R (oxyhemoglobin) structures; (2) the alpha1alpha2 subunit contacts are primarily stabilized by polar salt bridge linkages in both T and R states; and (3) the alpha1beta2 and alpha2beta1 contacts have both strong positive and negative interactions in both T and R states with few hydrophobic interactions. The HINT scoring methodology provides a quantitative characterization of the major role of the alpha1beta2 and alpha2beta1 interfaces in the T-->R quaternary transition. HINT also confirms the stronger hydrogen bond formation in mutant Hb Rothschild (Trp 37beta-->Arg) with Asp94alpha1 that gives rise to a low-affinity (deoxy) hemoglobin. HINT shows that the stabilization of the alpha1beta2 interface with mutant Hb Ypsilanti (Asp99alpha-->Tyr) produces a high-affinity (oxy) hemoglobin by reducing hydrophobic-polar contacts in the R state. HINT interaction maps also identified specific sites for mutagenesis at the alpha1beta2 interface that can be explored to shift the allosteric equilibrium in either direction. In addition, the HINT program provides useful diagnostic data for checking the quality of refined crystallographic structures.

Function and regulation of the murine lymphocyte CD2 receptor.

The CD2 receptor on T-lymphocytes plays a major part in mediating adhesive interactions via the LFA-3 ligand and in transducing signals for lymphocyte activation. In this study the expression, function, and internalization of the CD2 receptor was investigated in resting and activated murine T-cells. Surface iodination of intact lymphocytes showed that both types of cell expressed this antigen as a single polypeptide of 63 KDa, and flow cytometry analysis demonstrated that there was four times as much CD2 on lymphoblasts as on resting cells. Moreover, the CD2 receptor had a more prominent role in the adhesion of the activated lymphocytes to extravascular cells than in the binding of resting cells. Only activated lymphocytes internalized CD2, in the presence or absence of the anti-CD2 monoclonal antibody (mAb) 12-15, more than 80% of the 12-15/CD2 complex being removed from the cell surface within 24 hr. Application of 125I-labelled mAb 12-15 followed by subcellular fractionation on Percoll gradients showed that the complex was internalized initially into a low-density compartment and subsequently transported to heavy-density organelles, in which it was degraded. Immunogold electron microscopy revealed that immediately after the initial binding of mAb 12-15 to the lymphoblasts, the gold particles were localized in clusters exclusively at the plasma membrane. After a short period of culture, the mAb 12-15/CD2 complex was detected in small vesicles near the cell surface. Immunogold staining for a lysosomal enzyme beta-glucuronidase (Gus), for the lysosomal membrane protein LAMP-1, and for the mannose 6-phosphate targetting receptor (MPR) showed that the complex was transported from the endosomal compartment to lysosomal organelles in the activated T-cell. Although mAb 12-15 bound to CD2 in resting T-lymphocytes, in these cells the complex remained associated with the plasma membrane compartment only, even after prolonged culture. These data show that activated but not resting lymphocytes endocytosed the receptor, thereby regulating the expression of this antigen at the plasma membrane. This suggests that the endocytic and lysosomal compartments of lymphocytes have major roles in immune functions, by controlling the level of receptors at the lymphocytes cell surface and thus their response to cytokines and inflammatory mediators as well as their direct interaction with other cells.

Synthesized allosteric effectors of the hemoglobin molecule: a possible mechanism for improved erythrocyte oxygen release capability in hemoglobinopathy H disease.

Patients with the nondeletion genotype of hemoglobinopathy H (HbH or beta4) disease have higher proportions of HbH and more severe tissue hypoxia than patients with the deletion genotype. Because these patients' red blood cells (RBCs) contain mainly two Hb species, HbH and HbA, the high proportion of HbA can be exploited by lowering its oxygen affinity; this would probably increase oxygen delivery to the RBCs and improve the patients' clinical phenotype. Allosteric effectors that induce a low-affinity Hb may be useful in this regard. We investigated the effect of a bezafibrate derivative, RSR-4, on the oxygen affinity of RBCs and purified hemolysates containing HbA and HbH. This allosteric effector crosses RBC membranes and binds reversibly to the alpha-chains of deoxy-Hb, decreasing hemoglobin oxygen affinity. The blood used was obtained from a patient with HbH disease (alphaTSaudi homozygote) whose HbH level was 33.5% as measured by high-performance liquid chromatography. Oxygen binding studies were performed in RBCs and purified hemolysates. RBCs incubated in the presence of 500 microM RSR-4 (2-[[[(3,5-dichloroanilino)-carbonyl]methyl]phenoxy]-2-methylpropi onic acid) in standard conditions (pH 7.4, 0.14 M NaCl, 37 degrees C) displayed an increase in their P50 value from 14.5 to 35.2 mm Hg. Oxygen binding studies in purified stripped hemolysates (pH 7.2, 0.1 M NaCl, 25 degrees C) showed that addition of both 500 microM RSR-4 and 1 mM of 2,3 diphosphoglycerate (DPG) led to an 11-fold decrease in oxygen affinity, whereas the addition of the natural effector DPG or RSR-4 alone produced a 2.7- and 5.7-fold decrease, respectively. In both cases, the oxygen equilibrium curves (OECs) were biphasic due to the presence of the noncooperative, high-oxygen-affinity HbH (beta4) component. After addition of RSR-4, the lower part of the OEC (corresponding to HbH) was not shifted compared with the upper part (corresponding to HbA). These results were confirmed by kinetic studies of CO recombination. Both experiments demonstrated that RSR-4 does not affect beta4 Hb. Our findings provide an experimental model for lowering the oxygen affinity of HbA in HbH-containing cells and suggest that the oxygen delivery capability of the latter would be thereby improved.

The X-ray structure determination of bovine carbonmonoxy hemoglobin at 2.1 A resoultion and its relationship to the quaternary structures of other hemoglobin crystal froms.

Crystallographic studies of the intermediate states between unliganded and fully liganded hemoglobin (Hb) have revealed a large range of subtle but functionally important structural differences. Only one T state has been reported, whereas three other quaternary states (the R state, B state, and R2 or Y state) for liganded Hb have been characterized; other studies have defined liganded Hbs that are intermediate between the T and R states. The high-salt crystal structure of bovine carbonmonoxy (CO bovine) Hb has been determined at a resolution of 2.1 A and is described here. A detailed comparison with other crystallographically solved Hb forms (T, R, R2 or Y) shows that the quaternary structure of CO bovine Hb closely resembles R state Hb. However, our analysis of these structures has identified several important differences between CO bovine Hb and R state Hb. Compared with the R state structures, the beta-subunit N-terminal region has shifted closer to the central water cavity in CO bovine Hb. In addition, both the alpha- and beta-subunits in CO bovine Hb have more constrained heme environments that appear to be intermediate between the T and R states. Moreover, the distal pocket of the beta-subunit heme in CO bovine Hb shows significantly closer interaction between the bound CO ligand and the Hb distal residues Val 63(E11) and His 63(E7). The constrained heme groups and the increased steric contact involving the CO ligand and the distal heme residues relative to human Hb may explain in part the low intrinsic oxygen affinity of bovine Hb.

High-resolution crystal structure of deoxy hemoglobin complexed with a potent allosteric effector.

The crystal structure of human deoxy hemoglobin (Hb) complexed with a potent allosteric effector (2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-2-methylpropionic acid) = RSR-13) is reported at 1.85 A resolution. Analysis of the hemoglobin:effector complex indicates that two of these molecules bind to the central water cavity of deoxy Hb in a symmetrical fashion, and that each constrains the protein by engaging in hydrogen bonding and hydrophobic interactions with three of its four subunits. Interestingly, we also find that water-mediated interactions between the bound effectors and the protein make significant contributions to the overall binding. Physiologically, the interaction of RSR-13 with Hb results in increased oxygen delivery to peripheral tissues. Thus, this compound has potential therapeutic application in the treatment of hypoxia, ischemia, and trauma-related blood loss. Currently, RSR-13 is in phase III clinical trials as a radiosensitizing agent in the treatment of brain tumors. A detailed structural analysis of this compound complexed with deoxy Hb has important implications for the rational design of future analogs.

Fibroblast matrix gene expression and connective tissue remodeling: role of endothelin-1.

This study examines endothelin-induced modulation of extracellular matrix synthesis and remodeling by fibroblasts, and its potential role in the pathogenesis of systemic sclerosis (scleroderma). Endothelin-1 promoted fibroblast synthesis of collagen types I and III, but not fibronectin, by a mechanism dependent upon both ETA and ETB receptors. Conversely, endothelin-1 inhibited both protein expression of matrix metalloproteinase 1 and zymographic activity exclusively via ETA receptors. A dual regulatory role for endothelin-1 in transcriptional regulation was suggested by the ability of endothelin-1 to enhance steady-state levels of collagen mRNA and activate the proalpha2(I) collagen (Col1a2) promoter, but in contrast to reduce matrix metalloproteinase 1 transcript expression and suppress transcription of a human matrix metalloproteinase 1 promoter reporter construct in transient transfection assays. Although endothelin-1 significantly enhanced remodeling of three-dimensional collagen lattices populated by normal fibroblasts, this was not observed for lattices populated by systemic sclerosis fibroblasts. Promotion of matrix remodeling was dependent upon ETA receptor expression and was blocked by specific inhibitors of tyrosine kinases or protein kinase C. Reverse transcriptase polymerase chain reaction, S1 nuclease, and functional cell surface binding studies showed that normal and systemic sclerosis fibroblasts express both ETA and ETB receptors (predominantly ETA), but that ETA receptor mRNA levels and ETA binding sites on fibroblasts cultured from systemic sclerosis skin biopsies are reduced by almost 50%. Endothelin-1 is thus able to induce a fibrogenic phenotype in normal fibroblasts that is similar to that of lesional systemic sclerosis fibroblasts. Moreover, reduced responsiveness to exogenous endothelin-1 in systemic sclerosis suggests that downstream pathways may have already been activated in vivo. These data further implicate dysregulated endothelin-receptor pathways in fibroblasts in the pathogenesis of connective tissue fibrosis.

Swainsonine affects the processing of glycoproteins in vivo.

Rats, sheep and guinea pigs treated with swainsonine excrete 'high mannose' oligosaccharides in urine. The major rat and guinea pig oligosaccharide is (Man)5GlcNAc, whereas sheep excrete a mixture of oligosaccharides of composition (Man)2-5GlcNAc2 and (Man)3-5GlcNAc. The presence of these oligosaccharides suggests that Golgi alpha-D-mannosidase II as well as lysosomal alpha-D-mannosidase is inhibited by swainsonine resulting in storage of abnormally processed asparagine-linked glycans from glycoproteins. Altered glycoprotein processing appears to have little effect on the health of the intoxicated animal, but the accompanying lysosomal storage produces a disease state.

A phase I study of RSR13, a radiation-enhancing hemoglobin modifier: tolerance of repeated intravenous doses and correlation of pharmacokinetics with pharmacodynamics.

PURPOSE: Preclinical studies indicate that RSR13 oxygenates and radiosensitizes hypoxic solid tumors by decreasing the oxygen (O(2))-binding affinity of hemoglobin (Hb). A Phase I open-label, multicenter dose and frequency escalation study was conducted to assess the safety, tolerance, pharmacokinetics, and pharmacodynamic effect of daily RSR13 administration to cancer patients receiving concurrent palliative radiotherapy (RT). METHODS AND MATERIALS: Eligibility criteria included the following: ECOG performance status < or =2; resting and exercise arterial oxygen saturation (SaO(2)) > or =90%; an indication for palliative RT, 20-40 Gy in 10-15 fractions. RSR13 was administered i.v. via central vein over 60 min immediately before RT. Patients received supplemental O(2) via nasal cannula at 4 L/min during RSR13 infusion and RT. Plasma, red blood cell (RBC), and urine RSR13 concentrations were assayed. The pharmacodynamic effect of RSR13 on Hb-O(2) binding affinity was quantified by multipoint tonometry and expressed as an increase in p50, defined as the partial pressure of O(2) that results in 50% SaO(2). The RSR13 dose in the first cohort was 75 mg/kg once a week for two doses; successive cohorts received higher, more frequent doses up to 100 mg/kg/day for 10 days during RT. RESULTS: Twenty patients were enrolled in the study. Repeated daily doses of RSR13 were generally well tolerated. Two adverse events of note occurred: (1) A patient with pre-existing restrictive lung disease had transient persistent hypoxemia after the sixth RSR13 dose; (2) a patient with a recurrent glioma receiving high-dose corticosteroids had edema after the seventh RSR13 dose, likely due to the daily high-volume fluid infusions. Both patients recovered to baseline status with conservative management. Maximum pharmacodynamic effect occurred at the end of RSR13 infusion and was proportional to the RBC RSR13 concentration. After an RSR13 dose of 100 mg/kg, the peak increase in p50 averaged 8.1 mm Hg, consistent with the targeted physiologic effect, and then diminished with a half-life of approximately 5 h. CONCLUSIONS: RSR13 was well tolerated in daily doses up to 100 mg/kg administered for 10 days during RT. The combined administration of RSR13 with 4 L/min supplemental O(2) yielded pharmacodynamic conditions in which hypoxic tumor radiosensitization can occur. Ongoing Phase II and Phase III studies are evaluating the combination of RT and RSR13 for selected indications, including primary brain tumors, brain metastases, and non-small-cell lung cancer.

Design, synthesis, and testing of potential antisickling agents. 10. (2,2-Dimethylchroman-6-yl)alkanoic acids.

Five (2,2-dimethylchroman-6-yl)alkanoic acids were synthesized and tested for antigelling activities. It was envisioned that these agents might bind via hydrophobic bonding to nonpolar sites of the "donor-acceptor" regions of hemoglobin S. Several (2,2-dimethylchroman-6-yl)alkanoic acids containing 1-4 carbon atoms on the side-chain residue were designed to interact at the acceptor site, were synthesized, and were found to be moderately potent antigelling agents. The weak activity observed for two of the acids at low concentrations is rationalized in terms of weak binding affinities or multiple binding to active and nonactive sites. The effect of these compounds on shifting the allosteric equilibrium was small or negligible. The low toxicity of one of the (2,2-dimethylchroman-6-yl)alkanoic acids demonstrates the potential use of yet another class of compounds that can be modified in the development of antisickling agents.

Neurochemistry of aging. 1. Toxins for an animal model of Alzheimer's disease.

A chronic deficiency in central cholinergic function has been implicated in a number of neuropsychiatric diseases including Alzheimer's disease. Until recently, animal models that simulate the neurochemical conditions that appear to cause these diseases in humans, as a result of a direct manipulation of the central cholinergic system, were not available. Over the past few years, however, we have been successful in developing a cholinotoxin, 1-ethyl-1-(2-hydroxyethyl)aziridinium chloride (AF64A), which has the potential to serve as a novel compound in developing animal models of human brain disorders in which a cholinergic hypofunction has been implicated. In this paper are described the design, synthesis, and testing of several structural analogues of AF64A as potential cholinotoxins, by evaluating them for their ability to inhibit high-affinity choline transport and their affinity toward brain muscarinic receptors. One of the compounds, 1-cyclopropyl-1-(2-hydroxyethyl)aziridinium chloride (i.e. aziridine analogue of 13) was found to have a remarkably high affinity (about 40 times higher than AF64A) toward brain muscarinic receptors.

Allosteric modifiers of hemoglobin. 2. Crystallographically determined binding sites and hydrophobic binding/interaction analysis of novel hemoglobin oxygen effectors.

The protein-bound conformations of six new allosteric effectors similar to bezafibrate that markedly decrease the oxygen affinity of hemoglobin have been determined by X-ray crystallography. Comparisons are made with the bound conformations of three urea analogues reported by Lalezari, Perutz, and co-workers. All six new molecules bind at the same site previously observed for bezafibrate and exhibit a wide range of allosteric activity. Unlike the urea derivatives, which show two binding sites for the most potent derivatives, only one of the six new molecules (one with moderate allosteric activity) exhibits a second binding site. A new computer program, HINT (hydrophobic interactions), has been created and utilized to identify the major interactions between small molecules and the protein. The three strongest interactions identified by HINT involve Arg 141 alpha with the acid of the analogues, Lys 99 alpha with the bridging amide carbonyl, and the amide NH of the side chain of Asn 108 beta with the halogenated aromatic ring.

Neurochemistry of aging. 2. Design, synthesis, and biological evaluation of halomethyl analogues of choline with high affinity choline transport inhibitory activity.

The design, synthesis, and testing of several halomethyl analogues of choline and acetylcholine as potential cholinotoxins is described. The compounds were evaluated for their ability to inhibit high-affinity choline transport and their affinity toward postsynaptic muscarinic receptors. Among the analogues tested, bromomethyl and iodomethyl analogues of choline were found to be the most potent inhibitors of the high affinity choline transport system. Introduction of a beta-methyl group in the halomethyl analogues drastically reduced their potencies. The bromomethyl and iodomethyl analogues were further investigated for their effects on choline acetyltransferase activity, acetylcholinesterase activity and QNB binding. Neither compound possesses significant ability to alter any of the above cholinergic markers, except at very high concentrations. These results suggest that the bromomethyl and iodomethyl choline analogues may be used as specific inhibitors of the presynaptic high-affinity choline transport system.