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

Children'S Hospital Of Philadelphia, Philadelphia PA

Investigators

Linked publications & trials

Abstract

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