The adenosine receptors are proteins that reside in the extracellular membranes of cells. Activation of adenosine receptors plays a role in many physiological and pathological processes, such as... Show moreThe adenosine receptors are proteins that reside in the extracellular membranes of cells. Activation of adenosine receptors plays a role in many physiological and pathological processes, such as immune responses and cancers. Binding the adenosine receptors by either agonists or antagonists is therefore an interesting strategy currently carried out in multiple drug discovery programs. In order to make such drug discovery programs successful, our general understanding of adenosine receptor functioning should be improved. In that sense, new molecular tools can aid studies towards the adenosine receptors, for example by aiding the detection of receptor proteins or deciphering activation pathways. In this thesis, the development (synthesis, pharmacological evaluation and biological application) of chemical probes as molecular tool compounds for the adenosine receptors is described. This comprises the development of a covalent ligand for the adenosine A2B receptor, affinity-based probes for the adenosine A1 and A3 receptors and a ligand-directed probe for the adenosine A2B receptor. These chemical probes will aid future studies towards the roles of the A2B, A1 and A3 receptors in various conditions. Show less
Protein post-translational modification with ubiquitin (Ub) is a versatile signal regulating almost all aspects of cell biology, and an increasing range of diseases is associated with impaired Ub... Show moreProtein post-translational modification with ubiquitin (Ub) is a versatile signal regulating almost all aspects of cell biology, and an increasing range of diseases is associated with impaired Ub modification. In this light, the Ub system offers an attractive, yet underexplored route to the development of novel targeted treatments. A promising strategy for small molecule intervention is posed by the final components of the enzymatic ubiquitination cascade, E3 ligases, as they determine the specificity of the protein ubiquitination pathway. Here, we present UbSRhodol, an autoimmolative Ub-based probe, which upon E3 processing liberates the pro-fluorescent dye, amenable to profile the E3 transthiolation activity for recombinant and in cell-extract E3 ligases. UbSRhodol enabled detection of changes in transthiolation efficacy evoked by enzyme key point mutations or conformational changes, and offers an excellent assay reagent amenable to a high-throughput screening setup allowing the identification of small molecules modulating E3 activity. Show less
Solute carrier transporters (SLCs) limit receptor activation via uptake of extracellular ligands. Novel concepts are emerging that describe the modulation of intracellular and plasma membrane... Show moreSolute carrier transporters (SLCs) limit receptor activation via uptake of extracellular ligands. Novel concepts are emerging that describe the modulation of intracellular and plasma membrane receptors by ligand influx and efflux via SLCs, respectively. Here, we evaluate recent insights and provide an outlook for developing potential therapeutic strategies. Show less
Liu, X.; Ye, K.; Vlijmen, H.W.T. van; Emmerich, M.T.M.; IJzerman, A.P.; Westen, G.J.P. van 2021
In polypharmacology drugs are required to bind to multiple specific targets, for example to enhance efficacy or to reduce resistance formation. Although deep learning has achieved a breakthrough in... Show moreIn polypharmacology drugs are required to bind to multiple specific targets, for example to enhance efficacy or to reduce resistance formation. Although deep learning has achieved a breakthrough in de novo design in drug discovery, most of its applications only focus on a single drug target to generate drug-like active molecules. However, in reality drug molecules often interact with more than one target which can have desired (polypharmacology) or undesired (toxicity) effects. In a previous study we proposed a new method named DrugEx that integrates an exploration strategy into RNN-based reinforcement learning to improve the diversity of the generated molecules. Here, we extended our DrugEx algorithm with multi-objective optimization to generate drug-like molecules towards multiple targets or one specific target while avoiding off-targets (the two adenosine receptors, A1AR and A2AAR, and the potassium ion channel hERG in this study). In our model, we applied an RNN as the agent and machine learning predictors as the environment. Both the agent and the environment were pre-trained in advance and then interplayed under a reinforcement learning framework. The concept of evolutionary algorithms was merged into our method such that crossover and mutation operations were implemented by the same deep learning model as the agent. During the training loop, the agent generates a batch of SMILES-based molecules. Subsequently scores for all objectives provided by the environment are used to construct Pareto ranks of the generated molecules. For this ranking a non-dominated sorting algorithm and a Tanimoto-based crowding distance algorithm using chemical fingerprints are applied. Here, we adopted GPU acceleration to speed up the process of Pareto optimization. The final reward of each molecule is calculated based on the Pareto ranking with the ranking selection algorithm. The agent is trained under the guidance of the reward to make sure it can generate desired molecules after convergence of the training process. All in all we demonstrate generation of compounds with a diverse predicted selectivity profile towards multiple targets, offering the potential of high efficacy and low toxicity. Show less
Drug development is a time- and resource-consuming process that starts with the discovery and validation of a (protein) target that contributes to pathogenesis or disease progression. One of the... Show moreDrug development is a time- and resource-consuming process that starts with the discovery and validation of a (protein) target that contributes to pathogenesis or disease progression. One of the essential steps in this process is to validate that pharmacological modulation (e.g. inhibition) of the target leads to the desired phenotype, a process which is collectively referred to as target validation. Target validation heavily relies on the availability of suitable chemical tools to study engagement of the compound to the intended biological target. The development of selective chemical tools can be challenging to achieve due to the off-target activity towards structurally and/or functionally related homologs, e.g. other members within the same protein class. The field of chemical genetics combines the specificity of genetics with benefits of acute, pharmacological modulation by small molecules. This thesis describes chemical genetic approaches that can be used for target engagement and target validation studies of two different enzyme classes: kinases and serine hydrolases. Show less
The world continues to be plagued by numerous diseases and health complications. Unfortunately, current drug discovery efforts are lagging behind compared to the pace of emergence of new diseases... Show moreThe world continues to be plagued by numerous diseases and health complications. Unfortunately, current drug discovery efforts are lagging behind compared to the pace of emergence of new diseases or the development of resistance to current therapies. Medicinal plants especially for people in developing countries are sometimes the only remedies available for the prevention and treatment of diseases. Modern science has also come to recognize the importance of medicinal plants in drug discovery and development given the contributions that this sector has made to drug discovery. As such, several pharmacognostic/natural products chemistry techniques have been developed to aid research on the medicinal properties of plants. The application of these techniques is very useful in two main areas in that they facilitate the validation of the medicinal properties of folk remedies and in the discovery of new remedies from natural products. This thesis focused on the evaluation of the medicinal properties of Vernonia guineensis Benth. (Asteraceae), which is commonly called Ginseng in Cameroon. The species was found to possess antimicrobial, antiplasmodial, antiproliferative/anticancer and anthelmintic effects. Results obtained and reported in this thesis will form a useful foundation for further research into the medicinal potential of this plant Show less
Receptors tyrosine kinases or RTKs are cell surface receptors that regulate numerous cellular processes, but also have a critical role in the development and progression of many types of cancer.... Show moreReceptors tyrosine kinases or RTKs are cell surface receptors that regulate numerous cellular processes, but also have a critical role in the development and progression of many types of cancer. The overexpression of EphA4, a member of the RTK family, has been observed in a variety of malignant carcinomas. The aim of the research project associated with this thesis was to develop high affinity inhibitors of the tyrosine kinase EphA4. Ligand discovery was based on two complementary approaches, a computational screen and an NMR based screen using Target Immobilized NMR Screening (TINS). In addition, orthogonal biophysical methods including Surface Plasmon Resonance (SPR) and protein observed NMR were employed to analyse fragment binding. The crystal structure of the EphA4 kinase domain was solved and the structure of the kinase domain in complex with dasatinib, a well-known kinase inhibitor, was also elucidated. The in silico approach discovered a potent inhibitor of EphA4 for which the binding mode was elucidated via X-ray crystallography. Moreover, the TINS approach identified two compounds that may constitute starting points for the generation of more potent EphA4 inhibitors. Show less
Membrane proteins are an interesting class due to the variety of cellular functions and their importance as pharmaceutical targets, but they pose significant challenges for fragment-based drug... Show moreMembrane proteins are an interesting class due to the variety of cellular functions and their importance as pharmaceutical targets, but they pose significant challenges for fragment-based drug discovery approaches. Here we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. Using the recently developed Target Immobilized NMR Screening (TINS) approach, we screened 1,200 fragments for binding to the enzyme Disulphide bond forming protein B. Biochemical and biophysical validation of the 8 most potent hits revealed an IC50 range of 7 to 200 uM, which could be categorized as cofactor binding inhibitors or mixed model inhibitors of both cofactor and substrate protein interaction. Our results establish the utility of fragment-based methods in the development of inhibitors of membrane proteins, making a wide variety3of important membrane bound pharmaceutical targets amenable to such an approach. We first tested the immobilization procedure on G protein coupled receptors and ion channels. Furthermore, we used Nanodiscs, an alternative solubilization strategy, to solubilize teh protein without detergents. This resulted in less broad NMR signals, less non-specific binding issues, and identification of the binders from the original screen, proving that the nanodisc solubilization technique is compatible with TINS. Show less
