Structural modification of azolylacryloyl derivatives yields a novel class of covalent modifiers of hemoglobin as potential antisickling agents.

The intracellular polymerization and the concomitant sickling processes, central to the pathology of sickle cell disease, can be mitigated by increasing the oxygen affinity of sickle hemoglobin (HbS). Attempts to develop azolylacryloyl derivatives to covalently interact with ßCys93 and destabilize the low-O2-affinity T-state (deoxygenated) HbS to the polymer resistant high-O2-affinity R-state (liganded) HbS were only partially successful. This was likely due to the azolylacryloyls carboxylate moiety directing the compounds to also bind in the central water cavity of deoxygenated Hb and stabilizing the T-state. We now report a second generation of KAUS compounds (KAUS-28, KAUS-33, KAUS-38, and KAUS-39) without the carboxylate moiety designed to bind exclusively to ßCys93. As expected, the compounds showed reactivity with both free amino acid l-Cys and the Hb ßCys93. At 2 mM concentrations, the compounds demonstrated increased Hb affinity for oxygen (6% to 15%) in vitro, while the previously reported imidazolylacryloyl carboxylate derivative, KAUS-15 only showed 4.5% increase. The increased O2 affinity effects were sustained through the experimental period of 12 h for KAUS-28, KAUS-33, and KAUS-38, suggesting conserved pharmacokinetic profiles. When incubated at 2 mM with red blood cells from patients with homozygous SS, the compounds inhibited erythrocyte sickling by 5% to 9%, respectively in correlation with the increase Hb-O2 affinity. These values compare to 2% for KAUS-15. When tested with healthy mice, KAUS-38 showed very low toxicity.

Identification of a novel class of covalent modifiers of hemoglobin as potential antisickling agents.

Aromatic aldehydes and ethacrynic acid (ECA) exhibit antipolymerization properties that are beneficial for sickle cell disease therapy. Based on the ECA pharmacophore and its atomic interaction with hemoglobin, we designed and synthesized several compounds - designated as KAUS (imidazolylacryloyl derivatives) - that we hypothesized would bind covalently to ßCys93 of hemoglobin and inhibit sickling. The compounds surprisingly showed weak allosteric and antisickling properties. X-ray studies of hemoglobin in complex with representative KAUS compounds revealed an unanticipated mode of Michael addition between the ß-unsaturated carbon and the N-terminal αVal1 nitrogen at the α-cleft of hemoglobin, with no observable interaction with ßCys93. Interestingly, the compounds exhibited almost no reactivity with the free amino acids, L-Val, L-His and L-Lys, but showed some reactivity with both glutathione and L-Cys. Our findings provide a molecular level explanation for the compounds biological activities and an important framework for targeted modifications that would yield novel potent antisickling agents.

Novel bifunctional alkaline protease inhibitor: protease inhibitory activity as the biochemical basis of antifungal activity.

An alkaline protease inhibitor (API) from a Streptomyces sp. NCIM 5127 was shown to possess antifungal activity against several phytopathogenic fungi besides its antiproteolytic (anti-feedent) activity [J. V. Vernekar et al. (1999) Biochem. Biophys. Res. Commun. 262, 702-707]. Based on the correlation between antiproteolytic and antifungal activities in several tests such as copurification, heat inactivation, chemical modification, and its binding interaction with the fungal protease, we demonstrate, for the first time, that the dual function of API is a consequence of its ability to inhibit the essential alkaline protease. The parallel enrichment of both the functions during purification together with the heat inactivation of API leading to the concomitant loss of the two activities suggested their presence on a single molecule. Chemical modification of API with NBS resulted in the complete loss of antiproteolytic and antifungal activities, with no gross change in conformation implying the involvement of a Trp residue in the active site of the inhibitor and the presence of a single active site for the two activities. Treatment of API with DTT abolished both the activities although the native structure of API remained virtually unaffected, indicating the catalytic role of the disulfide bonds. Inactivation of API either by active site modification or by conformational changes leads to the concurrent loss of both the antiproteolytic and antifungal activities. Experimental evidences presented here serve to implicate that the antifungal activity of API is a consequence of its protease inhibitory activity.

Evidence for tryptophan in proximity to histidine and cysteine as essential to the active site of an alkaline protease.

The presence, microenvironment, and proximity of an essential Trp with the essential His and Cys residues in the active site of an alkaline protease have been demonstrated for the first time using chemical modification, chemo-affinity labeling, and fluorescence spectroscopy. Kinetic analysis of the N-bromosuccinimide- (NBS) or p-hydroxymercuribenzoate- (PHMB) modified enzyme from Conidiobolus sp. revealed that a single Trp and Cys are essential for activity in addition to the Asp, His, and Ser residues of the catalytic triad. Full protection by casein against inactivation of the enzyme by NBS and quenching of Trp fluorescence upon binding of the enzyme with NBS, substrate (sAAPF-pNA), or inhibitor (SSI) confirmed participation of the Trp residue at the substrate/inhibitor binding site of the alkaline protease. Comparison of the K(sv) values for the charged quenchers CsCI (1.66) and KI (7.0) suggested that the overall Trp microenvironment in the protease is electropositive. The proximity of Trp with His was demonstrated by the sigmoidal shape of the pH-dependent fluorometric titration curve with a pK(F) of 6.1. The vicinity of Trp with Cys was indicated by resonance energy transfer between the intrinsic fluorophore (Trp) and 5-iodoacetamide-fluorescein labeled Cys (extrinsic fluorophore). Our results on the proximity of Trp with essential His and Cys thus confirm the presence of Trp in the active site of the alkaline protease.

Alkaline protease inhibitor: a novel class of antifungal proteins against phytopathogenic fungi.

A Streptomyces sp., which produces an alkaline protease inhibitor (API) exhibiting antifungal activity has been isolated from soil. The protein has been purified to homogeneity. The molecular characterization has revealed that it is a dimer (M(r) 28 kDa) with five disulphide linkages and has a pI of 3.8. API is a competitive type of inhibitor with a K(i) value of 2.5 x 10(-9) M. The inhibitor is stable over a pH range of 6 to 12 and a temperature range of 40 to 95 degrees C. API exhibits antifungal activity (in vitro) against phytopathogenic fungi such as Fusarium, Alternaria, and Rhizoctonia and also against Trichoderma, a saprophytic fungus. The antifungal activity of API appears to be associated with its ability to inhibit the fungal serine alkaline protease(s), which is indispensable for its growth. Retardation of the rate of fungal spore germination, as well as hyphal extention, was observed in the presence of API. Both the protease inhibitory and the antifungal activity were abolished on treatment of API with DTT (5 mM), suggestive of a common site for both the activities. This is the first report on API as a potential biocontrol agent against phytopathogenic fungi.

Molecular and biotechnological aspects of microbial proteases.

Proteases represent the class of enzymes which occupy a pivotal position with respect to their physiological roles as well as their commercial applications. They perform both degradative and synthetic functions. Since they are physiologically necessary for living organisms, proteases occur ubiquitously in a wide diversity of sources such as plants, animals, and microorganisms. Microbes are an attractive source of proteases owing to the limited space required for their cultivation and their ready susceptibility to genetic manipulation. Proteases are divided into exo- and endopeptidases based on their action at or away from the termini, respectively. They are also classified as serine proteases, aspartic proteases, cysteine proteases, and metalloproteases depending on the nature of the functional group at the active site. Proteases play a critical role in many physiological and pathophysiological processes. Based on their classification, four different types of catalytic mechanisms are operative. Proteases find extensive applications in the food and dairy industries. Alkaline proteases hold a great potential for application in the detergent and leather industries due to the increasing trend to develop environmentally friendly technologies. There is a renaissance of interest in using proteolytic enzymes as targets for developing therapeutic agents. Protease genes from several bacteria, fungi, and viruses have been cloned and sequenced with the prime aims of (i) overproduction of the enzyme by gene amplification, (ii) delineation of the role of the enzyme in pathogenecity, and (iii) alteration in enzyme properties to suit its commercial application. Protein engineering techniques have been exploited to obtain proteases which show unique specificity and/or enhanced stability at high temperature or pH or in the presence of detergents and to understand the structure-function relationships of the enzyme. Protein sequences of acidic, alkaline, and neutral proteases from diverse origins have been analyzed with the aim of studying their evolutionary relationships. Despite the extensive research on several aspects of proteases, there is a paucity of knowledge about the roles that govern the diverse specificity of these enzymes. Deciphering these secrets would enable us to exploit proteases for their applications in biotechnology.

Molecular cloning and expression of the glucose/xylose isomerase gene from Streptomyces sp. NCIM 2730 in Escherichia coli.

A partial genomic library of Streptomyces sp. NCIM 2730 was constructed in Escherichia coli using pUC8 vector and screened for the presence of the D-glucose/xylose isomerase (GXI) gene using an 18-mer mixed oligonucleotide probe complementary to a highly conserved six-amino acid sequence of GXI from actinomycetes. Eight clones which hybridized with the radiolabelled oligoprobe showed the ability to complement xylose isomerase-defective E. coli mutants. The restriction map of the insert from one (pMSG27) of the eight GXI-positive clones showing detectable GXI activity was constructed. GXI-deficient strains of E. coli were able to utilize xylose as the sole carbon source for their growth upon transformation with pMSG27. E. coli JM105 (pMSG27) and E. coli JC1553 (pMSG27) were inducible by IPTG suggesting that the expression of the cloned gene was under the control of the lacZ promoter. Western blot analysis revealed that the cloned gene is expressed as a fusion protein of M(r) 110. This is the first report of expression of a catalytically active GXI from Streptomyces in Escherichia coli.

Genome characterization of glucose-isomerase-producingStreptomyces sp. NCIM 2730: detection of sequences homologous to a rice repeat sequence.

Restriction analysis of the genomic DNA from a high glucose/xylose-isomerase-yieldingStreptomyces sp. NCIM 2730 revealed a number of distinct bands on a background smear, indicating the occurrence of repeated DNA sequences in the genome. Optical renaturation analysis indicated that 25% of the genome comprised rapidly reannealing sequences with a copy number of 50 and a kinetic complexity of 3×10(3). Hybridization of theStreptomyces genomic library with theStreptomyces DNA, supported the estimate of the repetitive DNA content derived from the re-association kinetics of the DNA. Hybridization of DNA from three differentStreptomyces species with a rice repetitive DNA probe revealed the presence of homologous sequences, which is a unique finding.

Evidence for specific interaction of guanidine hydrochloride with carboxy groups of enzymes/proteins.

Experimental evidence for the interaction of Gdn.HCl with carboxylate groups of the proteins is presented, for the first time, based on (i) the inhibition by low concentrations of Gdn.HCl of enzymes that are known to require essential carboxyl groups for their catalytic activity unaccompanied by structural changes in the protein and (ii) failure of the carboxyl-specific Woodward's reagent K to react specifically with the carboxyl groups of the proteins/enzymes pretreated with Gdn.HCl. The results demonstrate that the specificity and the reversibility of interaction of Gdn.HCl with carboxylate groups of proteins can be gainfully utilized for probing the functional role of carboxylate residues in the proteins.