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2,3-Diarylfuro[2,3-b]pyridine-4-amines are a novel class of potent and selective inhibitors of Lck. The discovery, synthesis, and structure activity relationships of this series of inhibitors are reported. The most promising compounds were also profiled to deduce their pharmacokinetic properties.

Hemoglobin Wayne (Hb Wayne) is a frame-shift, elongated alpha-chain variant that exists in two forms, with either asparagine or aspartic acid as residue 139. Oxygen equilibrium studies showed that stripped Hb Wayne Asn and Hb Wayne Asp possessed high oxygen affinity (P 1/2 = 0.60 and 0.23 mmHg at pH 7, respectively), were non-co-operative and have a markedly reduced Bohr effect (-delta log P 1/2/pH (7 to 8) = 0.34 and 0.10, respectively). Adding organic phosphate results in a decreased oxygen affinity and increased Bohr effect for both Hbs Wayne. The overall rate of carbon monoxide binding at pH 7 (l' = 5.6 X 10(6) M-1 S-1) was similar for both stripped Hbs Wayne and was 25-fold more rapid than that of stripped Hb A. When organic phosphate was added, Hb Wayne Asn exhibited a homogeneous slower rate of carbon monoxide binding (l' = 2.6 X 10(6) M-1 S-1), whereas Hb Wayne Asp showed heterogeneous binding (l' = 6.1 X 10(6) and 2.6 X 10(6) M-1 S-1 for fast and slow phases, respectively). The rates of overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement for both Hbs Wayne were found to be slow compared to Hb A and uniquely different from each other. Similarly, sedimentation velocity experiments indicated that, although Hb Wayne Asn and Hb Wayne Asp were both less tetrameric than Hb A, each hemoglobin exhibited a distinct degree of oxygen-linked subunit dissociation. These observed differences in the allosteric properties of Hb Wayne Asn and Hb Wayne Asp appeared to be directly attributable to residue 139. The equilibrium and kinetic data are consistent with the X-ray diffraction analysis of Hb Wayne Asp, which shows that the C terminus of the deoxytetramers are severely disordered, a condition that results in major destabilization of the T conformation and disruption of normal hemoglobin function.

Hemoglobin Alberta has an amino acid substitution at position 101 (Glu----Gly), a residue involved in the alpha 1 beta 2 contact region of both the deoxy and oxy conformers of normal adult hemoglobin. Oxygen equilibrium measurements of stripped hemoglobin Alberta at 20 degrees C in the absence of phosphate revealed a high affinity (P50 = 0.75 mm Hg at pH 7), co-operative hemoglobin variant (n = 2.3 at pH 7) with a normal Bohr effect (- delta log P50/delta pH(7-8) = 0.65). The addition of inositol hexaphosphate resulted in a decrease in oxygen affinity (P50 = 8.2 mm Hg at pH 7), a slight increase in the value of n and an enhanced Bohr effect. Rapid mixing experiments reflected the equilibrium results. A rapid rate of carbon monoxide binding (l' = 7.0 X 10(5) M-1 S-1) and a slow rate of overall oxygen dissociation (k = 15 s-1) was seen at pH7 and 20 degrees C in the absence of phosphate. Under these experimental conditions the tetramer stability of liganded and unliganded hemoglobin Alberta was investigated by spectrophotometric kinetic techniques. The 4K4 value (the liganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta was found to be 0.83 X 10(-6) M compared to a 4K4 value for hemoglobin A of 2.3 X 10(-6) M, indicating that the Alberta tetramer was less dissociated into dimers than the tetramer of hemoglobin A. The values of 0K4 (the unliganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta and hemoglobin A were also measured and found to be 2.5 X 10(-8) M and 1.5 X 10(-10) M, respectively, demonstrating a greatly destabilized deoxyhemoglobin tetramer for hemoglobin Alberta compared to deoxyhemoglobin A. The functional and subunit dissociation properties of hemoglobin Alberta appear to be directly related to the dual role of the beta 101 residue in stabilizing the tetrameric form of the liganded structure, while concurrently destabilizing the unliganded tetramer molecule.

The kinetics of assembly have been monitored spectrophotometrically for normal and variant human oxyhemoglobins in 0.1 M Tris, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4, at 21.5 degrees C. Oxyhemoglobin versus oxy chain static difference spectra were performed and revealed subtle but significant absorption changes in both the visible and Soret regions. Kinetic experiments were performed by rapidly mixing equivalent (in heme) concentrations of alpha and beta A chains and following the change in absorbance at 583 nm with time. Over a protein concentration range of 10-100 microM in heme prior to mixing, these time courses were homogeneous and followed first-order kinetics, yielding a value of 0.069 s-1 for the apparent rate constant of dissociation of oxygenated beta A chain tetramers. Under these conditions, the overall assembly of oxyhemoglobins S (beta 6Glu----Val) and N-Baltimore (beta 95Lys----Glu) were also governed by the rates of dissociation of their respective oxygenated beta S and beta N-Baltimore chain tetramers with the apparent first-order rate constants of 0.044 and 0.15 s-1, respectively. In the Soret region, the alpha, beta monomer combination reaction could be observed if the protein concentration (heme basis) was lowered and if protein nonequivalency (beta chain exceeded alpha chain concentration) mixing experiments were performed. A kinetic oxyhemoglobin A, oxy-alpha, oxy-beta A monomer difference spectrum could be generated, and simple second-order kinetics were observed (415 nm) yielding rate constants of 2.3, 3.3, and 4.8 X 10(5) M-1 s-1 for the assembly of oxyhemoglobins S, A, and N-Baltimore, respectively. To our knowledge, this is the first kinetic study to reveal significant differences between the rate of association of alpha and beta monomers of hemoglobin A and those of two distinctly charged hemoglobin variants.