In recent work, we have found that, under solution conditions, several nitroxide-labeled analogs of phenylalanine and tryptophan all exhibit specific binding to hemoglobin (Hb) at a region close to the beta-chain N-termini -- a location that was not observed in previous x-ray co-crystallization studies by others. Quantitative structure-activity relationship (QSAR) analysis of a series of ring-modified phenylalanine and tryptophan analogs that inhibit sickle hemoglobin (HbS) aggregation also indicates that the indole exhibits significant stereospecificity in binding to Hb; bromination at the 5-position substantially increases activity of the amino acid alone or incorporated at specific positions in dipeptides. In this project, we propose to build on this initial foundation, and to utilize a systematic structurally based approach for the design of antisickling agents with high inhibitory activity, and high specific affinity for hemoglobin. The basic goals of this project are to experimentally determine the detailed binding site location and binding stereochemistry for aromatic inhibitors of HbS gelation through high field nuclear magnetic resonance (NMR) measurements, to utilize theoretical predictions based on distance geometry analysis of known inhibitors and molecular dynamics sampling of binding regions to refine the binding site location, to design improved inhibitor structures with enhanced Hb binding affinity and specificity over serum albumin using computer-aided molecular modeling, to develop practical routes for their synthesis, and, if needed, to design modifications to permit ready erythrocyte membrane traversal. We anticipate an iterative process in which the data from experimental studies will be used as a base for the design of new inhibitors; the best predictions will be synthesized and tested, and succeeding cycles of refinement will proceed until an inhibitor with appropriate antigelation activity, specific hemoglobin affinity and membrane permeability is developed.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL045977-04
Application #
2222599
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1991-02-01
Project End
1996-01-31
Budget Start
1994-02-01
Budget End
1995-01-31
Support Year
4
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612
Li, M; Lin, Z; Johnson, M E (1995) Photoaffinity labelling of cyanomethaemoglobin with derivatives of tryptophan and 5-bromotryptophan. Biochem J 308 ( Pt 1):251-60
Prabhakaran, M; Johnson, M E (1993) Molecular dynamics of sickle and normal hemoglobins. Biopolymers 33:735-42
Kar, L; Lai, C S; Wolff, C E et al. (1993) 1H NMR-based determination of the three-dimensional structure of the human plasma fibronectin fragment containing inter-chain disulfide bonds. J Biol Chem 268:8580-9
Sherman, S A; Johnson, M E (1993) Derivation of locally accurate spatial protein structure from NMR data. Prog Biophys Mol Biol 59:285-339
Eason, M G; Liggett, S B (1992) Subtype-selective desensitization of alpha 2-adrenergic receptors. Different mechanisms control short and long term agonist-promoted desensitization of alpha 2C10, alpha 2C4, and alpha 2C2. J Biol Chem 267:25473-9
Manavalan, P; Prabhakaran, M; Johnson, M E (1992) Location of potential binding sites on deoxy hemoglobin for the design of antigelling agents. J Mol Biol 223:791-800
Kar, L; Matsumura, P; Johnson, M E (1992) Bivalent-metal binding to CheY protein. Effect on protein conformation. Biochem J 287 ( Pt 2):521-31
Kar, L; De Croos, P Z; Roman, S J et al. (1992) Specificity and affinity of binding of phosphate-containing compounds to CheY protein. Biochem J 287 ( Pt 2):533-43