Rohinton Edalji

Hemoglobin covalently bridged across the polyphosphate binding site

Abstract Hemoglobin has been cross-linked covalently by reaction with 2-nor-2-formylpyridoxal 5′-phosphate followed by reduction with sodium borohydride. It is shown that a bridge is formed across the polyphosphate binding site between the two β chains. The modified hemoglobin binds oxygen cooperatively with a greatly decreased affinity demonstrating that the cross-link stabilizes the deoxy conformation but does not prevent the conformational change associated with oxygenation.

Oxygen affinity as an index of hemoglobin S polymerization: A new micromethod☆

Abstract The decrease in oxygen affinity with increasing hemoglobin concentration, which occurs in solutions of pure hemoglobin S, can be used to determine the minimum concentration at which polymerization of the deoxy form takes place. On this basis a very sensitive method for measuring the minimum gelling concentration has been developed. The influence of temperature, pH, and other hemoglobins on the end points obtained by this method is described. In all cases excellent agreement with the minimum gelling concentration determined directly on larger samples was observed. The results of this investigation demonstrate that the decrease in oxygen affinity of red cells containing hemoglobin S with increasing intracorpuseular hemoglobin concentration [May, A., and Huehns, E. R. (1975) Brit. J. Haematol.30, 317] is a direct consequence of the gelling properties of hemoglobin S alone.

Structure of MurF from Streptococcus pneumoniae co‐crystallized with a small molecule inhibitor exhibits interdomain closure

In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small‐molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X‐ray crystallography, which reveal the multidomain structure of a MurF–inhibitor complex in a compact conformation that differs dramatically from related structures. The lead molecule is bound in the substrate‐binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure‐based design to explore the suitability of MurF as a pharmaceutical target.

Structure-directed discovery of potent non-peptidic inhibitors of human urokinase that access a novel binding subsite

BACKGROUND Human urokinase-type plasminogen activator has been implicated in the regulation and control of basement membrane and interstitial protein degradation. Because of its role in tissue remodeling, urokinase is a central player in the disease progression of cancer, making it an attractive target for design of an anticancer clinical agent: Few urokinase inhibitors have been described, which suggests that discovery of such a compound is in the early stages. Towards integrating structural data into this process, a new human urokinase crystal form amenable to structure-based drug design has been used to discover potent urokinase inhibitors. RESULTS On the basis of crystallographic data, 2-naphthamidine was chosen as the lead scaffold for structure-directed optimization. This co-crystal structure shows the compound binding at the primary specificity pocket of the trypsin-like protease and at a novel binding subsite that is accessible from the 8-position of 2-napthamidine. This novel subsite was characterized and used to design two compounds with very different 8-substituents that inhibit urokinase with K(i) values of 30-40 nM. CONCLUSIONS Utilization of a novel subsite yielded two potent urokinase inhibitors even though this site has not been widely used in inhibitor optimization with other trypsin-like proteases, such as those reported for thrombin or factor Xa. The extensive binding pockets present at the substrate-binding groove of these other proteins are blocked by unique insertion loops in urokinase, thus necessitating the utilization of additional binding subsites. Successful implementation of this strategy and characterization of the novel site provides a significant step towards the discovery of an anticancer clinical agent.

Ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design

The DVD-IgTM protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain. To explore this area, we prepared a series of DVD-Ig proteins with binding specificities toward TNFα and an alternate therapeutic target. Kinetic measurements of TNFα binding to this series of DVD-Ig proteins were used to probe the effects of variable domain position and linker design on ligand on- and off-rates. We found that affinities for TNFα are generally lower when binding to the inner domain than to the outer domain and that this loss of affinity is primarily due to reduced association rate. This effect could be mitigated, to some degree, by linker design. We show several linker sequences that mitigate inner domain affinity losses in this series of DVD-Ig proteins. Moreover, we show that single chain proteolytic cleavage between the inner and outer domains, or complete outer domain removal, can largely restore inner domain TNFα affinity to that approaching the reference antibody. Taken together, these results suggest that a loss of affinity for inner variable domains in this set of DVD-Ig proteins may be largely driven by simple steric hindrance effects and can be reduced by careful linker design.

Intermolecular effects in the polymerization of hemoglobin S.

Myoglobin and isolated α and β subunits of hemoglobin A were found to have the same effect on the polymerization of hemoglobin S as intact hemoglobin A. This demonstrates that copolymerization of hemoglobins A and S need not be invoked to explain the gelling behavior of such mixtures, but that it can be adequately accounted for by the effect of the added hemoglobin on the activity coefficient of hemoglobin S. In addition, a hemoglobin tetramer in which an αAβA dimer is covalently linked to an αAβS dimer between the β chains was found to gel on deoxygenation. Therefore, only one β6 valine per tetramer is necessary for polymerization.

Recombinant human prorenin from CHO cells: expression and purification.

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from ∼1–5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr ∼40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P6-P′3 sequence of human angiotensinogen.

High-level expression of recombinant human FK-binding protein from a fusion precursor

The human peptidyl-prolyl isomerase FK-binding protein (FKBP) was cloned as a fusion partner with CMP-KDO synthetase (CKS), and the resultant construct was characterized as an improved high-expression source for FKBP. The CKS-FKBP fusion was expressed as a soluble protein at levels approaching 1 gm/L inEscherichia coli fermentations. The fusion protein was purified to near homogeneity by a one-step ammonium sulfate fractionation of whole cell lysate. After selective cleavage, the fusion precursor produced yields approaching 300 mg of purified FKBP per liter of harvested culture, a ∼30 to 60-fold increase over that observed for a nonfusion construct. Selective cleavage of the fusion partners was accomplished using either hydroxylamine or specific, limited proteolysis. Once separated from the CKS fusion partner, the FKBP was isolated in a single step by either reversed-phase HPLC or chromatography on Q-Sepharose. For comparison of physical and chemical properties, a nonfusion construct of recombinant human FKBP was expressed inE. coli and isolated. The purified FKBPs exhibited expected SDS-PAGE molecular weights and N-terminal sequences. The proteins had similar proton NMR spectra and binding to [3H]FK-506. The fusion construct, CKS-FKBP, was also found to bind [3H]FK-506. These data indicate that FKBP fused to the C-terminus of CKS folds independently of the fusion partner and suggests the fused FKBP adopts a conformation resembling that of the native protein.

From bacterial genomes to novel antibacterial agents: discovery, characterization, and antibacterial activity of compounds that bind to HI0065 (YjeE) from Haemophilus influenzae.

As part of a fully integrated and comprehensive strategy to discover novel antibacterial agents, NMR‐ and mass spectrometry‐based affinity selection screens were performed to identify compounds that bind to protein targets uniquely found in bacteria and encoded by genes essential for microbial viability. A biphenyl acid lead series emerged from an NMR‐based screen with the Haemophilus influenzae protein HI0065, a member of a family of probable ATP‐binding proteins found exclusively in eubacteria. The structure–activity relationships developed around the NMR‐derived biphenyl acid lead were consistent with on‐target antibacterial activity as the Staphylococcus aureus antibacterial activity of the series correlated extremely well with binding affinity to HI0065, while the correlation of binding affinity with B‐cell cytotoxicity was relatively poor. Although further studies are needed to conclusively establish the mode of action of the biphenyl series, these compounds represent novel leads that can serve as the basis for the development of novel antibacterial agents that appear to work via an unprecedented mechanism of action. Overall, these results support the genomics‐driven hypothesis that targeting bacterial essential gene products that are not present in eukaryotic cells can identify novel antibacterial agents.

Oxygenation properties of hemoglobin variants with substitutions near the polyphosphate binding site

Two hemoglobin variants with substitutions at beta 79 and beta 80 were found to have an increased oxygen affinity, but a normal response to organic phosphates. These observations are interpreted in terms of salt bridges which are affected by the substitutions.