Incidence of Infection in Young Children with Sickle Cell Disease in Africa

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The overall goals of this project are to further elucidate the mechanism of polymerization of deoxy Hb S by exploring the role of amino acids at protein-protein interaction sites of Hb S polymers and to develop structurebased anti-sickling agents, including peptides, that will inhibit Hb S polymerization and ameliorate sickle cell disease (SCD). We recently found that the homogeneous nucleation process of Hb C-Harlem (c_:13,.6GI,-_-_w7,.3 A,_A,o) is about 10:?-fold slower than that of Hb S. In addition, our differential interference contrast (DIC and electron microscopic studies of Hb C-Harlem showed crystal formation but no formation of fiber or othe polymerization products characteristic of Hb S, such as macrofibers or bundles. These findings indicate itae influence of 1373on properties of Hb S polymers and led to our hypotheses regarding the role of amino acids at the axial and lateral contacts of hemoglobin polymers. We hypothesize that (1) movement of Hb S molecules within nuclei prior to polymer formation is controlled by synergistically distributed interaction energies of lateral and axial contacts, e.g., amino acids at 136,t322 and 1373by hydrophobic, electrostatic and hydrogen bond interactions, respectively, and (2) enhancement of the B73 Asp hydrogen bond interaction with 134Thr in I-t5 S polymers promotes 14-stranded fiber formation, while weakening this interaction leads to two-stranded crystal formation. To test these hypo;heses, we will use mutagenesis strategies to engineer Hb S variants with mutations at critical interaction sites. We will evaluate kinetic and thermodynamic properties as well as polymer structure of the resultant hemoglobin variants. In specific aim 2, based on the basic information of the proteinprotein interactions of Hb S polymers we will identify and develop structure-based agents, including peptides, which interact with axial and lateral contact sites of Hb S polymers. We will first perform an antipolymerization test of peptide mapping in the A-helix domains containing 134Thr, which is expected to strongly bind the E-helix domain containing 1373Asp or 1373His. Using computer-based structural analysis, we will seek structure-based chemicals or peptides that interact with 1373Asp on the E-helix and/or 134Thr on the Ahelix to inhibit Hb S nucleation and polymerization. We will also use phage display technologies to produce peptides that bind to the E-helix domain and screen these peptides to find those with better binding properties to the 1373Asp in the E-helix than the native A-helix domains of Hb S. The identified peptides then will be tested for anti-polymerization properties using in vitro assays. We believe that simultaneous introduction by viral vectors of these peptides (blocking critical protein-protein interactions) and of y-globin chains (to form Hb F) in gene therapy of SCD wilt result in the generation of a novel therapeutic way for treating this disorder.