Protein kinases: Structure modeling, inhibition, and protein-protein interactions

Human protein kinases belong to a large and diverse enzyme family that contains more than 500 members. Deregulation of protein kinases is associated with many disorders, and this is why protein kinases are attractive targets for drug discovery. Due to the high conservation of the ATP binding pocket among this family, designing specific and/or selective inhibitors against certain member(s) is challenging. Several studies have been conducted on protein kinases to validate them as suitable drug targets. Although there are numerous target-validated protein kinases, the efforts to develop small molecule inhibitors have so far led to only a limited number of therapeutic agents and drug candidates. In our studies, we tried to understand the basic structural features of protein kinases using available computational tools. There are wide structural variations between different states of the same protein kinase that affect the enzyme specificity and inhibition. Many protein kinases do not yet have an available X-ray crystal structure and have not yet been validated to be drug targets. For these reasons, we developed a new homology modeling approach to facilitate modeling non-crystallized protein kinases and protein kinase states. Our homology modeling approach was able to model proteins having long amino acid sequences and multiple protein domains with reliable model quality and a manageable amount of computational time. Then, we checked the applicability of different docking algorithms (the routinely used computational methodology in virtual screening) in protein kinase studies. After performing the basic study of kinase structure modeling, we focused our research on cyclin dependent kinase 2 (CDK2) and glycogen synthase kinase-3β (GSK-3β). We prepared a non-redundant database from 303 available CDK2 PDB structures. We removed all structural anomalies and proceeded to use the CDK2 database in studying CDK2 structure in its different states, upon ATP, ligand and cyclin binding. We clustered the database based on our findings, and the CDK2 clusters were used to generate protein ligand interaction fingerprints (PLIF). We generated a PLIF-based pharmacophore model which is highly selective for CDK2 ligands. A virtual screening workflow was developed making use of the PLIF-based pharmacophore model, ligand fitting into the CDK2 active site and selective CDK2 shape scoring. We studied the structural basis for selective inhibition of CDK2 and GSK-3β. We compared the amino acid sequence, the 3D features, the binding pockets, contact maps, structural geometry, and Sphoxel maps. From this study we found 1) the ligand structural features that are required for the selective inhibition of CDK2 and GSK-3β, and 2) the amino acid residues which are essential for ligand binding and selective inhibition. We used the findings of this study to design a virtual screening workflow to search for selective inhibitors for CDK2 and GSK-3β. Because protein–protein interactions are essential in the function of protein kinases, and in particular of CDK2, we used protein–protein docking knowledge and binding energy calculations to examine CDK2 and cyclin binding. We applied this study to the voltage dependent calcium channel 1 (VDAC1) binding to Bax. We were able to provide important data relevant to future experimental researchers such as on the possibility of Bax to cross biological membranes and the most relevant amino acid residues in VDAC1 that interact with Bax

Mechanisms of Porphyrinoid and Carotenoid Spectral Tuning Revealed with Quantum Chemistry

Continued advances in a myriad of biomedical and technological fields require the rational design of molecules or supramolecular architectures with specific photophysical properties. Central to this endeavor is a mechanistic understanding of optical property modulation as a function of molecular structure, conformation, and environment. Natural pigments and protein-pigment complexes constitute a ‘solutions manual’ to challenges in electronic (optical) engineering that has been refined over a few billion years of evolution, and from which design principles can be deduced. In this thesis, unique mechanisms for modulating the optical properties of natural or synthetic porphyrinoid and carotenoid pigments are elucidated with quantum chemical methods. Our investigations add a new conformational mechanism, as well as design principles for regioisomer-dependent electronic substituent effects to the cannon of structural tools for regulating the optical properties of pyrrole-modified porphyrins. The lessons learned provide insight into analogous spectral tuning mechanisms found in nature. We also delineate the molecular factors optimally regulating light harvesting in a natural photosynthetic antenna complex. These discoveries have advanced the fundamental understanding and practical utilization of structure-optical property modulation mechanisms, and may aid the design of next-generation photonic-based technologies

Synthesis and applications of novel fluorescent and colorimetric coumarin-based sensors towards analyte sensing in aqueous systems

The continuous growth of mankind has not been considerate to the environment. The release of millions of tonnes of toxic heavy metal cations and anionic species through industrial, mining, agricultural, and electronic dumping has led to disease and, in many instances, death. This is usually suffered by low-income informal populations residing in third world countries. Moreover, many unnecessary deaths of children are becoming more prevalent because of consumption and contact with contaminated water, agricultural, and animal sources. Bioaccumulation of these toxic species in fish, plants, and animals, inevitably make their way back to the unaware general population. As growth by mining, agriculture, and electronics are indeed vital aspects of human development, the negative side effects of these activities usually continue unregulated. Therefore, as these processes are set to continue until more stringent regulatory processes are put into legislature; low-cost, sensitive, selective organic based sensors are a step in the right direction towards highlighting the need for environmental restoration and remediation; whilst also aiming to preventing unnecessary disease and death in the process. Herein, coumarin derived small-molecule fluorescent and colorimetric sensors for the quantitative and qualitative assessment of cationic and anionic species in aqueous and organic media are described. Ten fluorescent sensors supporting 1,4-disubstituted triazolyl moieties were synthesized according to Cu(I)-catalyzed azide-alkyne cycloaddition “click” reactions. These sensors were screened for their cationic and anionic affinities in a variety of solvent systems. Majority of the sensors responded well towards Fe3+, characterized by a strong fluorescent quenching response with a good degree of sensitivity and selectivity. Selected sensors were further investigated for their affinities towards anionic species; however, they did not display the same degree of selectivity or sensitivity towards these chosen anions. Titration studies of selected sensors with Fe3+ were able to be used towards determining the modes of fluorescent quenching; the photophysical mechanisms by which quenching occurs; stoichiometric binding ratios, association constants, and the number of coordination sites present between the sensors and Fe3+. Reversibility studies of the sensor-metal complex was investigated with EDTA. Partial reversibility was achieved for the chosen sensors with Fe3+. Hydrogen potential studies further described the application of these sensors over a good pH range. The binding site between the sensors and Fe3+ was investigated by NMR studies.Thesis (PhD) -- Faculty of Science, School of Biomecular and Chemical Sciences, 202

Synthesis and applications of novel fluorescent and colorimetric coumarin-based sensors towards analyte sensing in aqueous systems

The continuous growth of mankind has not been considerate to the environment. The release of millions of tonnes of toxic heavy metal cations and anionic species through industrial, mining, agricultural, and electronic dumping has led to disease and, in many instances, death. This is usually suffered by low-income informal populations residing in third world countries. Moreover, many unnecessary deaths of children are becoming more prevalent because of consumption and contact with contaminated water, agricultural, and animal sources. Bioaccumulation of these toxic species in fish, plants, and animals, inevitably make their way back to the unaware general population. As growth by mining, agriculture, and electronics are indeed vital aspects of human development, the negative side effects of these activities usually continue unregulated. Therefore, as these processes are set to continue until more stringent regulatory processes are put into legislature; low-cost, sensitive, selective organic based sensors are a step in the right direction towards highlighting the need for environmental restoration and remediation; whilst also aiming to preventing unnecessary disease and death in the process. Herein, coumarin derived small-molecule fluorescent and colorimetric sensors for the quantitative and qualitative assessment of cationic and anionic species in aqueous and organic media are described. Ten fluorescent sensors supporting 1,4-disubstituted triazolyl moieties were synthesized according to Cu(I)-catalyzed azide-alkyne cycloaddition “click” reactions. These sensors were screened for their cationic and anionic affinities in a variety of solvent systems. Majority of the sensors responded well towards Fe3+, characterized by a strong fluorescent quenching response with a good degree of sensitivity and selectivity. Selected sensors were further investigated for their affinities towards anionic species; however, they did not display the same degree of selectivity or sensitivity towards these chosen anions. Titration studies of selected sensors with Fe3+ were able to be used towards determining the modes of fluorescent quenching; the photophysical mechanisms by which quenching occurs; stoichiometric binding ratios, association constants, and the number of coordination sites present between the sensors and Fe3+. Reversibility studies of the sensor-metal complex was investigated with EDTA. Partial reversibility was achieved for the chosen sensors with Fe3+. Hydrogen potential studies further described the application of these sensors over a good pH range. The binding site between the sensors and Fe3+ was investigated by NMR studies.Thesis (PhD) -- Faculty of Science, School of Biomecular and Chemical Sciences, 202

Metal Complexes of Dioxolene and Iminonitroxyl Radical Ligands

This thesis focuses on the design, synthesis and analysis of multinuclear mixed-valent dioxolene complexes and Cu(II) nitroxyl complexes. Chapter 1 contains a review of the relevant literature for the field of mixed-valent dioxolene complexes and molecular magnetism in nitroxyl complexes. Chapter 2 reports the synthesis and crystallographic analysis of a series of novel highly porous solvent-supported supramolecular assemblies of triptycene derivatives. Chapter 3 reports the complexation and characterisation of triptycene derivatives and related multi-dioxolene ligands with Pt(II). Chapter 4 reports the synthesis, complexation and characterisation of a rigid tris(dioxolene) CTC derivative with Pt(II). It also describes the attempted synthesis of coordination polymers containing multi-dioxolene ligands. Chapter 5 reports the synthesis and characterisation of a series of Cu(II) complexes containing the biradical ligand bisimpy. Chapter 6 contains a description of all synthetic procedures undertaken during this work

Towards novel Au (III) porphyrins : synthesis and characterization of a range of mesotetraalkylporphyrins.

Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.The principal goal of this work was to synthesize and fully characterize a range of fre