Gajanan S. Joshi

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 Å 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.

Pyridyl Derivatives of Benzaldehyde as Potential Antisickling Agents

Compounds that bind to sickle hemoglobin (Hb S) producing an allosteric shift to the high‐affinity Hb S that does not polymerize are being developed to treat sickle cell anemia (SCA). In this study, three series of pyridyl derivatives of substituted benzaldehydes (Classes I–III) that combine structural features of two previously determined potent antisickling agents, vanillin and pyridoxal, were synthesized. When analyzed with normal human whole blood, the compounds form Schiff‐base adducts with Hb and left shift the oxygen equilibrium curve (OEC) to the more soluble high‐affinity Hb, more than vanillin or pyridoxal. Generally, Class‐I compounds with an aromatic aldehyde located ortho to the pyridyl substituent are the most potent, followed by the Class‐II compounds with the aldehyde at the meta‐position. Class‐III compounds with the aldehyde at the para position show the weakest activity. The structure–activity studies of these pyridyl derivatives of substituted benzaldehydes demonstrate significant allosteric potency that may be useful for treating SCA.

X-ray crystallographic analyses of symmetrical allosteric effectors of hemoglobin: compounds designed to link primary and secondary binding sites

The rational design and X-ray crystallographic analyses of two symmetrical allosteric effectors of hemoglobin (Hb) are reported. Compound design was directed by the previously solved co-crystal structure of one of the most potent allosteric effectors of Hb, 2-[4-[(3,5-dichlorophenylcarbamoyl)-methyl]-phenoxy]-2-methylpropionic acid (RSR4), which revealed two distinct binding sites for this compound in the Hb central water cavity. The primary binding site has been observed for all compounds of this structural class, which stabilize deoxy Hb by engaging in inter-dimer contacts with three of the four protein subunits. Interactions at the secondary binding site of RSR4 occur primarily between the beta(1) and beta(2) subunits and serve to further constrain the deoxy state. Based on these observations, it was hypothesized that compounds with the ability to simultaneously span and link both of these sites would possess increased potency, but at a lower molar concentration than RSR4. Two symmetrical compounds were designed and synthesized based on this hypothesis. The symmetrical effector approach was taken to minimize the number of compound orientations needed to successfully bind at either of the distinct allosteric sites. X-ray crystallographic analyses of these two effectors in complex with Hb revealed that they successfully spanned the RSR4 primary and secondary binding sites. However, the designed compounds interacted with the secondary binding site in such a way that intra-dimer, as opposed to inter-dimer, interactions were generated. In agreement with these observations, in vitro evaluation of the symmetrical effectors in Hb solution indicated that neither compound possessed the potency of RSR4. A detailed analysis of symmetrical effector-Hb contacts and comparisons with the binding contacts of RSR4 are discussed.