After spending about a decade in the area of synthetic and mechanistic organic chemistry, Dr Gupta developed a strong interest also in modern biology, especially structural membrane biology, during the period of his post-doctoral training under the supervision of Prof. H.G. Khorana at MIT. During his total scientific career of about 37 years, he has made several significant contributions to a number of areas related to chemistry and biology, viz. new drug design and development, immunomodulation, liposomes as new drug delivery systems, phospholipid and C-nucleoside synthesis, membrane phospholipid organization and dynamics, and role of cytoskeleton - membrane bilayer (or cystosolic protein) associations in regulating the membrane structure, dynamics and functions. These studies have resulted in publication of about 110 original research papers, 10 review articles, 5 book chapters and 5 patents.

I) New Drug Design and Development

Dr Gupta has synthesized a number of novel heterocyclic compounds containing pyrimidine and pyridopyrrole nucleus and got them tested for biological activity. While some of these compounds showed strong CNS/CVS activities, 2-substituted quinazolo-4-ones exhibited very strong antihyperglycemic activity. The latter observation was the first of its kind as it demonstrated for the first time the association of pyrimidine nucleus with antidiabetic activity, and formed the basis of design of newer antihyperglycemic agents, which led to the discovery of a very potent antidiabetic compound, 2-piperizino-quinazolo-4-one ("Centpiperilone"). This compound was more effective than even insulin in lowering down the blood sugar levels in diabetic patients, but it had to be dropped at the final stage due to nausea it caused in the patients. Besides this, he has developed a new and highly economic process for large scale preparation of the antihypertensive drug `Clonidine'. Also, an efficient and totally indigenous process for production of the anti-inflammatory drug `Indomethacin' was developed. These processes were adopted by IDPL and Themis for manufacturing Clonidine and Indomethacine, respectively, in our country.

II) Immunomodulation

With a view to improve the immunogenic response of purified antigens and therapeutic efficacy of anti parasitic drugs, a large number of muramylpeptide, trehalose and tuftsin derivatives were synthesized and evaluated for their immunomodulatory activity. Some of these compounds, especially hydrophobic derivatives of muramylpeptides, strongly boosted the immunogenic response of some model antigens, and also greatly improved the chemotherapeutic efficacy of some antiparasitic drugs, e.g. diethyl carbamazine. Further, prophylactic treatment with some muramylpeptide and tuftsin derivatives was shown to considerably increase the nonspecific host's resistance against a variety of infections. Also, a combination of interferon inducers with these compounds greatly improved their anti-viral activity.

III) Liposomes as New Drug Delivery Systems

Liposomes have been widely considered useful as drug/enzyme vehicles in therapy, and also as adjuvants in increasing the immunogenic potential of a variety of antigens. However, their successful application in therapy was limited by their rapid lysis in blood, major uptake by the reticuloendothelial cells, and lack of availability of simple procedures for directing them to specific cells in vivo. The main emphasis of Dr Gupta has therefore been on sorting out some of the problems associated with the liposomes as drug delivery systems. He has made several significant contributions to this area, some of which are as follows: (i) he has designed and developed liposomes that are considerably more stable and longer living in the blood circulation, as compared to conventional liposomes, which are known to be lysed by the plasma proteins. (ii) He has shown that covalent attachment of anti-erythrocyte F(ab')2 to the liposomes surface enables the liposomes to specifically recognize the erythrocytes in vivo and deliver their contents to these cells. It was further demonstrated that the entrapment of anti-malarial drugs like chloroquine (chq), in the antibody-coated liposomes increases the drug efficacy not only against the chq-sensitive but also against the chq-resistant malarial infections. Encouraged by these results, the liposomes were coated with F(ab')2 fragments of a monoclonal antibody which specifically recognized the malaria-infected erythrocytes. These monoclonal antibody bearing liposomes upon loading with chq were found to be fully effective in treatment of chq-resistant experimental malaria. (iii) He has developed tuftsin bearing liposomes which besides ensuring delivery of the entrapped material to the macrophages, also increased the nonspecific host's resistance against parasitic infections, e.g. malaria, leishmania, filaria and tuberculosis. His further work has demonstrated that these liposomes upon loading with amphotericin B are very effective in treatment of experimental aspergillosis. (iv) He has demonstrated that immunogenic response of antigens is greatly improved by coupling them covalently to the liposomes surface. (v) Finally, he has established fusogenic character of yeast lipids vesicels which upon loading with antigens can generate both antigen-specific CD4+ and CD8+ T-lymphocytes responses. These studies thus established the usefulness of liposomes not only as drug carriers but also as the carriers of antigens and nucleic acids in therapy.

IV) Phospholipid and C-Nucleoside Synthesis

Non-availability of pure and defined chain phospholipids was the major limitation in studies of their structure and dynamics in biological membranes, using biophysical techniques. This problem was largely overcome by Dr Gupta by developing a very efficient and simple procedure for preparation of pure glycerophospholipids. This procedure became very popular in preparation of photosensitive, and 13C and radiolabelled phospholipids. Also, easy procedures were developed for preparation of a number of biologically interesting phosphatidylcholine (PC) analogs, like 1-acyl-2-carbamyloxy PCs, l-ether-2-carbamyloxy PCs, 1,2-diether PCs, 1-ether-2-acyl PCs, 1,2,4-butane triol-based PCs, head-group modified PCs, etc. Besides, highly efficient procedures were developed for preparing some biologically active C-nucleosides.

V) Membrane Phospholipids Organization and Dynamics in Model Membrane Systems

Dr Gupta has been the first one to successfully develop an organochemical approach to study the lipid-lipid and lipid-protein interactions in biological membranes. He synthesized photosensitive phospholipids which were found suitable for use as probes to study the membrane-embedded regions of integral membrane proteins, e.g. glycophorin and cytochrome b5. Also, these lipids served a useful purpose in detection of phase separations in lipid mixtures as well as in defining the phospholipid conformation in lipid bilayers.

Various phospholipids in biological membranes are differentially distributed in two leaflets of the membrane bilayer. To determine the factors that generate this asymmetry in membranes, a number of studies were performed worldwide with pure phospholipid components in small unilamellar vesicles. These studies suggested that the transbilayer phospholipid distribution at least in highly curved membranes is controlled by the phospholipid polar head-group surface area and/or length. To further investigate the structural parameters that control the inside-outside phospholipid distributions in lipid bilayers of highly curved membranes, a number of polar head-group modified phosphatidylcholines and ethanolamines were synthesized by Dr Gupta and their transbilayer distributions studied in small unilamellar vesicles, using phospholipases and amino-group labelling reagents as the external membrane probes. Results of these studies clearly demonstrated that the phospholipid polar head-group effective volume, rather than the head-group surface area or length, is the main parameter that controls the phospholipid distribution in two monolayers of the membrane bilayer. Further studies with purified phospholipid components of mammalian erythrocyte membrane revealed that the above phospholipid distribution principle was valid only in case of highly curved phospholipid vesicles/membranes, and not in case of large size vesicles. In large unilamellar vesicles, phospholipids distributed randomly across the membrane bilayer, irrespective of their polar head-group area or volume. Based on these results it was suggested that the phospholipid-phospholipid interactions have no role to play in generation and maintenance of the asymmetric transbilayer phospholipid distribution normally observed in all types of biological membranes.

The preferred conformation of glycerophospholipids in bilayers is such that their C-2 ester group is aligned at the bilayer interface and the C-1 ester is buried within the hydrophobic matrix. Consequently, the first methylene residue of the alkyl chain attached to the carbon atom of the C-2 ester group becomes exposed to the hydrophilic environment. To examine whether replacement of this methylene group by some polar residue, like NH, would result in stabilization of the above phospholipid conformation in lipid bilayers, a number of PC analogs where the C-2 ester group was replaced by the carbamyloxy function (N-COO) were synthesized, and the resulting carbamyl PCs were studied using a variety of biophysical and biochemical techniques. The results of these studies clearly indicated that carbamyl PCs are much more ordered than their natural counterpart in the lipid bilayers. Also, it showed that inclusion of cholesterol in carbamyl PC bilayers, in contrst to natural PC bilayers, does not significantly affect the phospholipid ordering in the bilayers.

To analyse the factors that determine the above-said preferred conformation of glycerophopsholipids in micelles and bilayers, Dr Gupta and his co-workers synthesized PC analogs wherein the glycerol backbone was replaced by the 1,2,4-butanetriol moiety and studied the resulting PC analogs in both micelles and bilayers using NMR, differential scanning calorimetry and fluorescence spectroscopy. These studies have very clearly demonstrated that preferred conformation of the glycerol backbone (which in turn is known to determine the phospholipid conformation) is primarily determined by the intramolecular stacking of the two acylchains in PCs.

VI) Role of Cytoskeleton - Membrane Bilayer (or Cytosolic Protein) Associations in Regulating the Membrane Structure, Dynamics and Function

Earlier studies in Dr Gupta's laboratory on transbilayer phospholipid organization in membranes of malaria-infected erythrocytes and erythrocytes of humans suffering from chronic myeloid leukemia revealed that this organization becomes abnormal in these cells in the sense that the aminophospholipids, which are almost exclusively present in the inner surface of the normal erythrocyte membrane bilayer, become accessible to membrane impermeable enzymatic and chemical probes. Since this abnormality in these cells was accompanied by abnormalities in the membrane-associated cytoskeleton (Membrane skeleton), it was proposed that the membrane skeleton-bilayer associations could play an important role in generation and maintenance of the phospholipid asymmetry in erythrocyte membrane. To examine the validity of this proposal, the membrane skeleton was structurally modified in intact erythrocytes by loading them with free Ca++ or subjecting them to heating at the helix-to-coil transition temperature of the major skeletal protein, spectrin. While abnormalities introduced in the membrane skeleton of human erythrocytes by loading them with Ca2+ were found to be accompanied with changes in the membrane phospholipid organization, no such correlation was, however, observed in the heat-treated cells; these cells had normal transbilayer phospholipid distributions in spite of the fact that their membrane skeleton was extensively modified by the heat treatment. These results led Dr Gupta to suggest that the membrane skeleton-bilayer interactions, alone, can not control the asymmetric phospholipid distribution across the erythrocyte membrane bilayer. Further studies on these lines finally demonstrated that both the membrane skeleton-bilayer interactions and an ATP-dependent out-to-in aminophospholipid pump are required to generate and maintain the phospholipid asymmetry in native erythrocytes. While the phospholipid pump seems to be the major factor in generation and maintenance of the asymmetry, the membrane skeleton-bilayer interactions are perhaps required for stabilizing the aminophospholipid arrangement in the inner monolayer.

To further anlayse the comparative roles of cytoskeleton-membrane bilayer interactions and aminophospholipid pump in generating and maintaining the membrane phospholipid asymmetry, Dr Gupta selected Saccharomyces cerevisiae as the model cell, since this cell has been well characterized genetically and it is relatively easy to specifically modify the specific components of its cytoskeleton by modifying the corresponding gene. These cells, like higher eucaryotic cells, have been shown by Dr Gupta to possess aminophospholipid translocase. Using, yeast mutants having well defined changes in their cytoskeletal proteins, he has further demonstrated that actin cytoskeleton controls directly (or indirectly) the in-to-out translocation of aminophospholipids in the yeast cell plasma membrane. This apart, his recent studies with Leishmania parasites have established the presence of an aminophospholipid translocation system also in these parasites.

Cytoskeleton-membrane bilayer interactions in cells control not only the membrane structure and dynamics but also the membrane function. Since the anion channel protein in erythrocytes is specifically assocaited with the underlying membrane skeleton, it was considered of interest to analyse the role of membrane skeleton - bilayer associations in regulating the function of the anion channel protein. The membrane skeleton in intact cells was modified by increasing the intracellular free Ca2+ concentration, and the membrane function was studied by measuring the anion exchange and amino acid uptake in Ca2+-loaded cells. Results of these studies revealed that structural alterations in membrane skeletal proteins did affect the anion channel protein function.

Apart from these studies, Dr Gupta has identified in cytosol of mature mammalian erythroytes a 70 kDa protein which was similar to the well known class of 70 kDa heat shock proteins (hsp 70). This protein under normal conditions localized mainly in the cytosol, but it had strong tendency to bind the membrane under heat or pathologic ( e.g. malaria) stress. The binding was almost exclusively restricted to the membrane skeleton, and seemed to primarily involve the hydrophobic interactions. From these results, he has suggested that hsp 70-like proteins in mature mammalian erythrocytes could perhaps play an important role in protecting the cells under stress by stabilizing the membrane skeleton through their interactions with skeletal proteins.

Furthermore, he analysed the role of the host cell cytoskeleton in budding of the extrcellularly enveloped viruses, like influenza virus. Using influenza A virus - infected MDCK cells as the model system, he has shown that this virus puts two of its proteins, viz. matrix protein and nucleoprotein, in the MDCK cell cytoskeleton which perhaps modifies the cytoskeletal dynamics by specifically interacting with the cytoskeletal actin.

VII) Characterization of Actin Network in Kinetoplastid Parasites, Especially Leishmania

Actin is a major cytoskeletal protein which plays a key role in several essential cellular processes, like cell shape maintenance, cell division, endocytosis, organelle movement, vesicle translocation, etc. The protein is abundantly present in most of the eukaryotic cells and its homologs have been reported to exist in bacteria. However, its presence in kinetoplastid parasites, which are causative organisms of dreaded diseases like sleeping sickness and 'kala-azar', could not be established in spite of the presence of actin gene(s) in the genomes of these parasites. This prompted Dr Gupta to undertake detailed studies on structural and functional characterization of actin network in kinetoplastid parasites, especially Leishmania. His current efforts are therefore directed exclusively towards complete structural and functional characterization of actin and actin-binding proteins in Leishmania parasites. To this end, he has already cloned and characterized Leishmania donovani actin, one actin-related protein (ARP), coronin and profilin, and studied the subcellular localization and interactions of Leishmania actin. This actin besides being localized in the subcortical regions and cytoplasm of the parasite, was found to be present also in the nucleus and k-DNA network of the kinetoplast, and to interact with subpellicular microtubules. Also it lacked the ability to form the cables that are commonly seen in mammalian cells, and formed patches, bundles and short and fine filaments in Leishmania promastigotes. However, sufficiently long actin protofilaments have been identified in amastigote forms of these parasites. Further work on these lines is currently in progress.

His current research efforts are exclusively directed towards complete structural and functional characterization of the actin and actin-binding proteins in Leishmania parasites. To this end, he has already cloned and characterized Leishmania donovani actin, an actin-related protein (ARP), coronin and profilin, and studied the subcellular localisation and interactions of Leishmania actin. This protein besides being localised in the subcortical regions and cytoplasm of the parasite, was found to be present also in the nucleus and k-DNA network of the kinetoplast, and to interact with microtubules. Further, it lacked the ability of form the cables that are commonly seen in mammalian cells,a dn formed only patches, bundles and short, fine filaments. Further work in this direction is presently in progress.

VII) Promotion of Excellence in Science and Institution Building

Apart from making some pioneering contributions to the filed of Structural Membrane Biology and Drug Targeting, Dr Gupta played a key role in developing the Institute of Microbial Technology (IMTech), a constituent laboratory of CSIR, as the centre of excellence in the field of Biotechnology, during his tenure (April 6, 1992 to April 19, 1997) as the Director of that Institute. He not only inducted about two dozens of highly talented young scientists who have now been making notable contributions to the area of Modern Biology but also provided sharp focus to overall R&D activities and created very strong research units on "Yeast Genetics" and "Protein Design". As a result, this institute has been the first one in the country to develop and licence to industry the first and second generation streptokinase as drugs for marketing besides being considered as one among the very best institutions in Modern Biology in India. Recognizing his contributions to IMTech, CSIR appointed him as the Director of CDRI and assigned him the task to modernise this Institute as well. During his now more than six years tenure as the Director, CDRI, he has made visible impacts on every aspect related to the science and management in this institute. He not only attracted young talent in large numbers (>30) to CDRI and created most of the modern disciplines (like, Structural Biology, Genomics & Proteomics, Molecular and Cellular Biology of disease processes and pathogens, Combinatorial Chemistry and High throughput Drug Screening) that are essentially required for development of the target-specific, lesser toxic drugs but also strengthened the Ph.D. level training by introducing a national test for selecting students and also by affiliating the CDRI Ph.D. programme with the Jawaharlal Nehru University. In addition, he sharply focussed the R&D activities of the Institute by making a due consideration of the existing capabilities in terms of both the manpower and infrastructure and initiated a new work culture by forming R&D partnership with industry in the very early phase of new drug development. Thus, the R&D collaborations have been firmed up with Novo Nordisk, Dabur and Cadila Pharmaceuticals in the areas of new drug development for treatments of diabetes and dislipidemia, cancer, and peptic ulcers, respectively and negotiations are currently in progress to licence out the area of antitubercular drugs to Lupin. Besides this, several products (viz. Bulaquine, Guglip, CT-1, Centbutindole, Cenpropazine and Consap) have been licensed out to industry for marketing and a number of herbal preparations (e.g. Picroliv, CDR-134, a medication for cerebral stroke) and synthetic products (2 antidiabetics, one antimalarial, 3 antiosteoporosis agents, 3-4 lipid lowering agents, 3 antitubercular compounds) are currently in various phases of new drug development.Further, to promote excellence, he introduced several incentives including cash awards which has now created a fierce but healthy inter group competition. As a result, the number of CDRI publications in SCI journals and average impact factor have steadily increased during the last 5 years. Finally, he successfully convinced the CSIR for creating a state-of-the-art new CDRI so that the institute may continue to make a measurable impact at the international level in future in the area of new drug R&D, and acquired 62 acres of land in the outskirts of Lucknow for construction of new world class laboratories for housing CDRI. Most of the formalities for undertaking the construction have been completed and the construction work should hopefully begin in early or mid 2004.