Samir Kumar Dutta

REFINED CRYSTAL STRUCTURE (2.3 A) OF A DOUBLE-HEADED WINGED BEAN ALPHA -CHYMOTRYPSIN INHIBITOR AND LOCATION OF ITS SECOND REACTIVE SITE

The crystal structure of a double‐headed α‐chymotrypsin inhibitor, WCI, from winged bean seeds has now been refined at 2.3 Å resolution to an R‐factor of 18.7% for 9,897 reflections. The crystals belong to the hexagonal space group P6122 with cell parameters a = b = 61.8 Å and c = 212.8 Å. The final model has a good stereochemistry and a root mean square deviation of 0.011 Å and 1.14° from ideality for bond length and bond angles, respectively. A total of 109 ordered solvent molecules were localized in the structure. This improved structure at 2.3 Å led to an understanding of the mechanism of inhibition of the protein against α‐chymotrypsin. An analysis of this higher resolution structure also helped us to predict the location of the second reactive site of the protein, about which no previous biochemical information was available. The inhibitor structure is spherical and has twelve anti‐parallel β‐strands with connecting loops arranged in a characteristic β‐trefoil fold common to other homologous serine protease inhibitors in the Kunitz (STI) family as well as to some non homologous functionally unrelated proteins. A wide variation in the surface loop regions is seen in the latter ones. Proteins 1999;35:321–331.

Design and synthesis of mannose analogues as inhibitors of α-mannosidase

Abstract A series of N-, C- and S- mannopyranosyl derivatives (4,9-16) have been synthesised and their inhibitory activity tested towards jackbean α-mannosidase (EC 3.2.1.24). These compounds are of mechanistic and synthetic interest in the design of new α-mannosidase inhibitors.

A potent tumoricidal co-drug ‘Bet-CA’ - an ester derivative of betulinic acid and dichloroacetate selectively and synergistically kills cancer cells

Selective targeting of cancer cells employing multiple combinations as co-drug holds promise for new generation therapeutics. Betulinic acid (BA), a plant secondary metabolite kills cancer cells and Dichloroacetate (DCA) is capable of reversing the Warburg phenotype by inhibiting pyruvate dehydrogenase kinase (PDK). Here, we report synthesis, characterization and tumoricidal potential of a co-drug Bet-CA, where a DCA molecule has been appended on C-3 hydroxyl group of BA to generate an ester derivative for increased solubility and subsequent cleavage by internal esterase(s) to release one unit each of BA and DCA. In vitro studies revealed pronounced synergistic cytotoxicity of Bet-CA against a broad spectrum of cancer cells and it selectively killed them when co-cultured with human fibroblasts. Bet-CA treatment increased reactive oxygen species (ROS) production, significantly altered mitochondrial membrane potential gradient (ΔΨm); followed by the release of cytochrome c (Cyt c) which prompted cells to undergo mitochondria mediated apoptosis. In vivo experimentation expectedly exhibited tumor inhibitory potential of Bet-CA and clinically achievable doses did not produce any apparent toxicity. Taken together, results suggestively raise an important corollary hypothesis stating that Bet-CA selectively and synergistically combats cancer without producing toxic manifestations and emerges to be the prospect for the new generation therapeutics.

Crystallization and preliminary X-ray studies of psophocarpin B1, a chymotrypsin inhibitor from winged bean seeds.

Psophocarpin B1 is a 20,000 Mr protein of winged bean (Psophocarpus tetragonolobus) seeds having chymotrypsin inhibitory activity. Single crystals of this protein suitable for X-ray crystallographic studies have been obtained by the vapour diffusion method using ammonium sulphate. The crystals are hexagonal, space group P6(4)22 or P6(2)22, cell dimensions a = b = 61 A, c = 210 A. They are stable to irradiation with X-rays and diffract to at least 2.6 A resolution.

Genomic and cDNA Cloning, Expression, Purification, and Characterization of Chymotrypsin-Trypsin Inhibitor from Winged Bean Seeds

A 516-bp winged bean chymotrypsin-trypsin inhibitor (WbCTI) gene was amplified from genomic DNA and cDNA isolated from winged bean using a pair of degenerate primers designed on the basis of the amino acid sequences of WbCTI. The amplified PCR products were cloned and sequenced to confirm their authenticity. DNA sequence analysis of the genomic and cDNA clones of WbCTI revealed the same nucleotide sequence in the coding region and showed WbCTI to be an intron less gene. WbCTI was subcloned into pTrc99A and expressed in Escherichia coli to yield a recombinant protein (rWbCTI). rWbCTI was purified by a rapid and single step immunoaffinity chromatography method, with an overall yield of 1.1 mg/g of wet cells. The homogeneity of the purified protein was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed the presence of a single protein band. Functionally rWbCTI is indistinguishable from WbCTI, since both inhibit bovine trypsin and chymotrypsin in a 1:1 molar ratio. FPLC binding studies also confirmed that rWbCTI binds the proteases in a 1:1 molar ratio.

Role of metabolic modulator Bet-CA in altering mitochondrial hyperpolarization to suppress cancer associated angiogenesis and metastasis.

Solid tumors characteristically reflect a metabolic switching from glucose oxidation to glycolysis that plays a fundamental role in angiogenesis and metastasis to facilitate aggressive tumor outcomes. Hyperpolarized mitochondrial membrane potential is a manifestation of malignant cells that compromise the intrinsic pathways of apoptosis and confer a suitable niche to promote the cancer associated hallmark traits. We have previously reported that co-drug Bet-CA selectively targets cancer cells by inducing metabolic catastrophe without a manifest in toxicity. Here we report that the same molecule at a relatively lower concentration deregulates the cardinal phenotypes associated with angiogenesis and metastasis. In mice syngeneic 4T1 breast cancer model, Bet-CA exhibited effective abrogation of angiogenesis and concomitantly obliterated lung metastasis consistent with altered mitochondrial bioenergetics. Furthermore, Bet-CA significantly lowered vascular endothelial growth factor (VEGF) levels and obviated matrix metalloproteases (MMP-2/9) production directly to the criterion where abrogation of autocrine VEGF/VEGFR2 signalling loop was documented. In vitro studies anticipatedly documented the role of Bet-CA in inhibiting actin remodeling, lamellipodia formation and cell membrane ruffling to constitutively suppress cell motility and invasion. Results comprehensively postulate that Bet-CA, a mitochondria targeting metabolic modulator may serve as an excellent candidate for combating angiogenesis and metastasis.

Structure-function relationship of a bio-pesticidal trypsin/chymotrypsin inhibitor from winged bean

Protease inhibitors are essential bio-molecules that serve as a model system for the study of protein structure and protease-protease inhibitor interaction. We here report a bi-functional serine protease inhibitor from winged bean (WBCTI) that completely retains its inhibitory property against trypsin and chymotrypsin even after heating at 70°C. Detailed circular dichroism and fluorescence studies at different temperatures, 30-90°C, have been performed to understand the reason behind thermal stability of the protein. On the basis of our results it appears that WBCTI maintains its canonical structure up to 70°C. Above that the heat induced conformational change becomes irreversible which causes aggregation followed by precipitation of the protein. Moreover, the activity and stability of the secondary structure are found to decrease drastically in presence of dithiothreitol indicating that the protein acquires additional stability for the occurrence of two disulfide bonds. In addition to the structural characterization, an important property of WBCTI against the polyphagous pest Helicoverpa armigera has been explored in present study. WBCTI has showed reasonable inhibition of the mid-gut proteases of H. armigera. In artificial feeding trial through addition of WBCTI in diet resulted in significant growth retardation, delayed pupae formation and higher mortality of H. armigera larvae.

Site-Directed Mutagenesis to Identify Key Residues in Structure-Function Relationship of Winged Bean Chymotrypsin-Trypsin Inhibitor and 3-D Structure Prediction

Winged bean chymotrypsin trypsin inhibitor (WbCTI) is a Kunitz type serine protease inhibitor that inhibits both trypsin and chymotrypsin at 1:1 molar ratio. Site-directed mutagenesis study was employed to generate two mutants of WbCTI, with an aim to explore its dual inhibitory properties against the proteases. The mutants were expressed in Escherichia coli and, were purified to homogeneity using a single step immunoaffinity column. The two mutants, each containing a single mutation at the amino acid sequence positions of 63 and 64, were named as L63A and R64A, respectively. Purified L63A-WbCTI exhibited anti-trypsin activity with no anti-chymotrypsin activity whereas R64A-WbCTI could inhibit chymotrypsin but not trypsin. To investigate the binding interactions between the mutated forms of WbCTI with the putative proteases, binding studies were carried out using gel filtration chromatography which further confirmed the formation of enzyme-inhibitor complexes. Finally, 3D model structure of WbCTI was designed using computer simulations which further emphasize the roles of L63 and R64 residues for dual inhibitory characteristics of WbCTI.

Synergism of metabolic modulators Bet-CA and LDCA: a rational combinatorial approach to selectively combat cancer associated hallmark traits

Malignant cells exhibit metabolic alterations contrastingly from normal cells and hence, targeting cancer metabolism is being considered as a key strategy for fabricating combinatorial therapeutic regimens that would not only exclude the adverse side-effects of conventional chemotherapy, but also bypass the phenomenon of chemoresistance. Herein, Bet-CA, a co-drug that hampers mitochondrial membrane potential to counter apoptosis resistance, angiogenesis and metastasis was used in combination with a ‘dual-hit’ metabolic modulator LDCA that inhibits lactate dehydrogenase A (LDH-A) enzyme activity to rigorously alter mitochondrial bioenergetics and concomitantly promote apoptosis in cancer cells. Combination of metabolic modulators Bet-CA and LDCA demonstrated synergistic growth inhibitory mechanisms, specifically in melanoma cells. Furthermore, combination synergistically and selectively depleted mitochondrial membrane potential and exerted reactive oxygen species (ROS) generation to consequently promote caspase mediated cell death. Importantly, in a preclinical model of melanoma, combination stringently limited tumor progression without significant toxic manifestations. Thus, with a clear safety profile, our studies present a comprehensive rationale for the efficacy and prospectus of using combinatorial therapy of metabolic modulators and offer a one of a kind solution to counter the enduring challenge of malignancy.

The dual-hit metabolic modulator LDCA synergistically potentiates doxorubicin to selectively combat cancer-associated hallmarks

Conventional chemotherapy induces significant toxicity, leading to serious side effects; thus, an effective strategy to counter this would be to use a combination of drugs that have unrelated mechanisms of action and drug resistance. Doxorubicin, a widely used chemotherapeutic drug, has adverse side effects; thus, it is crucial to reduce its toxicity to improve its therapeutic regimen. Cancer cells vitally differ from normal cells with respect to their metabolism, and the use of metabolic modulators is expected to proximally compensate the various manifestations of cancer. In this study, doxorubicin was used in combination with a dual-hit metabolic modulator, LDCA, with the postulation that this adroit targeting of cancer cells would exert potent therapeutic effects. The results demonstrated that this combination synergistically enhanced the growth inhibition and induced mitochondria-mediated apoptosis by recruiting the caspase cascade, restricting migration, and obviating the clonogenic outgrowth potential of melanoma cells. Interestingly, the combination specifically dampened melanoma cell viability, but spared the normal population; this suggested its low toxicity profile. Furthermore, in the preclinical model of murine melanoma, combination treatment thwarted tumor growth kinetics to restrain oncogenic progression, thus accentuating survival. Comprehensively, we have demonstrated the promising therapeutic potential of the combination of doxorubicin and LDCA in the expanding chapter of combinatorial therapy.