Studies within Fragment-Based Drug Discovery: Library Synthesis and Hit-to-Lead Optimisation

This thesis reports two projects aimed at addressing challenges within fragment-based drug discovery. The first project describes efforts towards utilising synthetic methodology to address deficiencies within fragment screening collections. This involved the development of a modular, robust and scalable route to access α,α-disubstituted amino ester building blocks, which in turn were derivatised to allow the rapid assembly of five (a total of eight in collaboration) spirocyclic scaffolds. Importantly, this library was structurally diverse, comprising three (a total of six in collaboration) pharmacophore-like heterocycles and carbocycles. Moreover, numerous three-dimensional exit vectors were incorporated within each core spirocycle, and the ability of these handles to effect a diverse set of chemical modifications was exemplified through the generation of 16 (a total of 21 in collaboration) examples. Computational studies highlighted the excellent physicochemical and 3D properties of the library, as well as the broad coverage of underexplored chemical space that was achieved. This library was also screened for antibacterial activity in a phenotypic assay against the clinically relevant bacterial strains, Pseudomonas aeruginosa and Staphylococcus aureus. The second project examined the inhibition of propionate detoxification mechanisms in bacteria as an attractive strategy for the development of antibacterial agents. A fragment screening campaign against 2-methylcitrate synthase (PrpC) from Pseudomonas aeruginosa identified several hit compounds based on an indole unit. Synthetic efforts were undertaken to elaborate these fragment hits to increase potency. The adopted strategy focused on growing the indole fragment towards the nearby oxaloacetate binding pocket and occupying it with a fragment mimicking its natural substrate. This approach yielded a compound with an in vitro half maximal inhibitory concentration of 130 µM against the enzymatic activity of PrpC

Spirocycles as Rigidified sp3-Rich Scaffolds for a Fragment Collection.

Novel divergent methodology to access sp3-rich spirocyclic fragments is reported. First, a robust modular synthesis of bis-alkene amino ester building blocks was developed. Three different carbocycles and six heterocycles were then constructed to assemble eight spirocycles. Importantly, strategic exit vectors were incorporated within each scaffold to aid fragment growth and were elaborated via chemical modifications. Finally, computational methods demonstrate higher levels of rigidity, three-dimensionality, and structural diversity of the library compared to a commercial collection

Transcriptional Regulation Is a Major Controller of Cell Cycle Transition Dynamics

DNA replication, mitosis and mitotic exit are critical transitions of the cell cycle which normally occur only once per cycle. A universal control mechanism was proposed for the regulation of mitotic entry in which Cdk helps its own activation through two positive feedback loops. Recent discoveries in various organisms showed the importance of positive feedbacks in other transitions as well. Here we investigate if a universal control system with transcriptional regulation(s) and post-translational positive feedback(s) can be proposed for the regulation of all cell cycle transitions. Through computational modeling, we analyze the transition dynamics in all possible combinations of transcriptional and post-translational regulations. We find that some combinations lead to ‘sloppy’ transitions, while others give very precise control. The periodic transcriptional regulation through the activator or the inhibitor leads to radically different dynamics. Experimental evidence shows that in cell cycle transitions of organisms investigated for cell cycle dependent periodic transcription, only the inhibitor OR the activator is under cyclic control and never both of them. Based on these observations, we propose two transcriptional control modes of cell cycle regulation that either STOP or let the cycle GO in case of a transcriptional failure. We discuss the biological relevance of such differences

Synthesis of novel 18-crown-6 type ligands containing a phenothiazine 5,5-dioxide unit

Novel crown ethers containing a phenothiazine 5,5-dioxide unit have been synthesized. Macrocyclization reactions rendering N-tosyl protected crown ethers were performed in the absence of metal ion templates. These new crown ethers are useful precursors of sensor and selector molecules with wide applications

Analysis of a Generic Model of Eukaryotic Cell-Cycle Regulation

We propose a protein interaction network for the regulation of DNA synthesis and mitosis that emphasizes the universality of the regulatory system among eukaryotic cells. The idiosyncrasies of cell cycle regulation in particular organisms can be attributed, we claim, to specific settings of rate constants in the dynamic network of chemical reactions. The values of these rate constants are determined ultimately by the genetic makeup of an organism. To support these claims, we convert the reaction mechanism into a set of governing kinetic equations and provide parameter values (specific to budding yeast, fission yeast, frog eggs, and mammalian cells) that account for many curious features of cell cycle regulation in these organisms. Using one-parameter bifurcation diagrams, we show how overall cell growth drives progression through the cell cycle, how cell-size homeostasis can be achieved by two different strategies, and how mutations remodel bifurcation diagrams and create unusual cell-division phenotypes. The relation between gene dosage and phenotype can be summarized compactly in two-parameter bifurcation diagrams. Our approach provides a theoretical framework in which to understand both the universality and particularity of cell cycle regulation, and to construct, in modular fashion, increasingly complex models of the networks controlling cell growth and division

Contributions of low molecule number and chromosomal positioning to stochastic gene expression

The presence of low-copy-number regulators and switch-like signal propagation in regulatory networks are expected to increase noise in cellular processes. We developed a noise amplifier that detects fluctuations in the level of low-abundance mRNAs in yeast. The observed fluctuations are not due to the low number of molecules expressed from a gene per se but originate in the random, rare events of gene activation. The frequency of these events and the correlation between stochastic expressions of genes in a single cell depend on the positioning of the genes along the chromosomes. Transcriptional regulators produced by such random expression propagate noise to their target genes