Alpha screen – Amplified Luminescent Proximity Homogeneous Assay (ALPHA) is a very sensitive high-throughput screening technology used for detection of binding of biomolecules, for example protein-protein or protein-peptide interactions. Both binding partners are conjugated to fluorescent dye-labeled donor and acceptor beads. Interaction between the biomolecules results in close vicinity and thus a fluorescence signal.
Candidate – Drug candidates are obtained after chemical optimization of lead compounds in terms of increased activity against the target of choice and improved pharmacokinetic parameters, while concomitantly showing reduced activity against unrelated targets. Preclinical development candidates are advanced into PK/PD studies, followed by testing in an animal disease model. Thereafter, they enter clinical trials.
Epigenetic targets – Epigenetics is the summary of all processes that cause changes in gene expression without influencing the underlying DNA sequence. Proteins involved in these mechanisms – called epigenetic targets – function by modifying the genome (e.g. by DNA methylation) or DNA associated proteins, such as histones. One example of epigenetic regulation is the process of cellular differentiation that explains how different cells are able to fulfill specialized functions, despite containing identical DNA information.
FBDD – The identification of small fragments specifically binding to a target protein as part of a drug discovery process is called fragment-based drug discovery (FBDD). Finding fragments in libraries of several hundred up to multiple thousand compounds is performed by various biophysical screening methods including, for example, MST, NMR screening, surface plasmon resonance (SPR) or X-ray crystallography. Identified and validated fragment hits are further optimized by directed growth, combining or linking of different fragments to generate leads and finally a drug candidate with optimized affinity and selectivity.
Fragment – A fragment is a chemical compound of low molecular weight (typically lower than 300 Da) and with a relatively simple structure. Large fragment libraries of (103 – 107 individual compounds) can be used to screen for binders to a specific target. These fragment hits will then enter further steps of FBDD.
Hit – The identification of hits from chemical libraries is the first step in small molecule drug discovery. A hit is a molecule derived from a screen of thousands to millions of compounds, in which it qualified by binding to the target protein.
KD – The KD value or dissociation constant describes the affinity of an interaction between two biomolecules, for example a ligand and a protein. It corresponds to the concentration of molecule A, at which half of the number of molecules B is bound to A. The KD value ranges from sub-picomolar (very strong binding) to molar (very weak binding) concentrations and can be determined by a variety of biophysical methods.
Lead – After the initial library screen, the identified hit molecules that are found to bind to the specific target are re-tested to validate activity. Confirmed hit compounds are then ranked according to the results of the re-screen. To develop lead compounds, the structures of the best hits are further optimized by chemical synthesis to generate novel molecules with optimized binding affinity and selectivity for the target protein.
Molecular replacement – A method for solving the phase problem in X-ray crystallography is molecular replacement. This technique is based on an existing structure of a protein homologous to the protein of interest from which the diffraction data was derived. This known structure can be used as a search model to determine the orientation and position of the molecules within the unit cell to obtain the phases subsequently required to generate the electron density map.
MST – Microscale thermophoresis (MST) is applied for the analysis of biomolecules, including determination of binding affinities, enzyme activity and binding kinetics of a variety of molecular interactions in solution. We have established MST as a screening technique in pharmaceutical research applications for analysis of the interaction of fragments and compounds with protein targets. The method is based on detection of changes in the hydration shell of biomolecules caused by ligand binding. Hallmarks of the technology are an extremely fast assay set-up and a massive reduction of artifacts since the assay is performed in solution at close-to-native conditions.
NMR – Nuclear magnetic resonance (NMR) is a technique for determination of the three-dimensional structure of proteins and other biomolecules based on re-emission of electromagnetic radiation of magnetic nuclei in a magnetic field. Moreover, it can be used as a screening technology in fragment-based drug discovery approaches to identify interactions of small molecule ligands with their respective target proteins. For fragment screening, changes in the NMR parameters occurring upon binding are monitored and analyzed.
Nuclear receptors – Nuclear receptors are intracellular transcription factors that sense different hormones. Upon binding to a specific ligand, they become activated and subsequently regulate gene expression by directly binding to DNA, a property that clearly differentiates them from other types of receptors. They control a wide variety of pathways, including fatty acid metabolism, embryonic development and adult homeostasis.
SAD/MAD – Single/multiple wavelength anomalous dispersion (SAD/MAD) are methods used in X-ray crystallography to solve the so-called phase problem. This is caused by the fact that both amplitude and phase have to be known in order to calculate an electron density map for modeling of the target molecule. Phases, however, cannot be directly measured in an X-ray experiment. In SAD and MAD methods, one or more atoms of the protein have been replaced by heavy atoms (for example, selenium atoms) via incorporation of seleno-methionine to cause significant anomalous scattering from incoming X-rays in a well-characterized way. The anomalous signal is recorded at a single (SAD) or at multiple (MAD) wavelengths of coherent X-ray light to generate experimental phase information.
Serine/threonine kinases – Generally, protein kinases are a family of enzymes that modify the activity of other proteins by phosphorylation, i.e. covalent attachment of a phosphate group coming from a nucleoside triphosphate (usually ATP) onto a free hydroxyl group of the substrate. Serine/threonine kinases specifically phosphorylate serine and/or threonine residues of their protein substrates. They participate in regulating many cellular signaling pathways, including cell proliferation, programmed cell death (apoptosis), cell differentiation and embryonic development.
Thermal melt – For buffer screens or identification of conditions that increase the stability of a protein, thermal melt analysis can be performed. This is acquired by heating a protein sample up to 95°C and measuring unfolding, i.e. exposure of hydrophobic patches, using a fluorescent dye. By adding small molecules or varying the buffer composition, the target protein stability or activity can be analyzed.
TINS – Target immobilized NMR Screening (TINS) is a ligand screening method used in FBDD for hit finding. It is based on the NMR technology generally applied for the analysis of the three-dimensional structure of proteins, and allows rapid screening of drug compound libraries with high sensitivity. In TINS, both the target and a reference protein are immobilized on a solid matrix and binding of a fragment is detected by comparing the NMR spectra of the compounds in presence of the target to the control. The major advantages of TINS are the low amount of protein required and the lack of limitation to a specific target type, allowing also screening of membrane proteins.
Tyrosine kinases – Like serine/threonine kinases, tyrosine kinases catalyze the transfer of a phosphate group from a nucleoside triphosphate donor to an acceptor protein. Tyrosine kinases represent the second large subgroup of kinases. Phosphorylation occurs at tyrosine residues of the substrate, causing changes in the activity of proteins involved, for instance, in signal transduction processes such as cell division.