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ZoBio

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ZoBio

ZoBio is an established, market leading supplier of an array of biophysical techniques to support modern drug discovery efforts.

ZoBio has established and demonstrated the efficacy of its proprietary Target Immobilized NMR Screening (TINS) technology for the discovery of high quality, diverse fragment ligands for a wide array of challenging targets including membrane bound proteins, such as GPCRs.

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More recently, we have added orthogonal validation technologies such as SPR and protein observed NMR. To further support Fragment- and Structure-based drug discovery, ZoBio has introduced highly innovative, NMR-based methods to rapidly elucidate the structure of target-small molecule complexes.

ZoBio has integrated these technologies into an efficient pipeline to support hit to clinical candidate programs on targets that no other CRO can address.

Fragment screening

ZoBio offers its proprietary TINS technology for ligand screening studies on our own, highly validated fragment library or the customer’s. TINS uses a sample of the target and a reference protein that have been immobilized on sepharose based resin. Binding is detected by a reduction in the height of the NMR signals of a compound that specifically binds to the target. The screen is carried out by repeated cycles of fragment application, assaying for binding and fragment removal.

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TINS is a powerful approach to hit discovery that has been validated on a wide range of targets including viral proteins, proteases, kinases, epigenetic targets, Protein-Protein Interaction targets, molecular chaperones and membrane proteins including GPCRs and ion channels. A typical TINS hit discovery project is completed in 1-2 months.

How does TINS work: http://www.zobio.com/index.php/technology

SPR & NMR based screen follow up

Hits from a TINS screen can be validated and characterized in a number of ways using TINS in competition mode, other NMR or SPR approaches. SPR is a powerful, industry accepted ligand discovery technology that is orthogonal to NMR. In particular, TINS and SPR are highly complementary since both require target immobilization while TINS provides extraordinary sensitivity to weak interactions and SPR is highly quantitative.

We have recently shown that a powerful work flow for a fragment discovery program is TINS screening followed by SPR fragment characterization (Kobayashi et al, J. Biomol. Screening, 2010, 15, p.978). ZoBio has a unique, highly integrated platform of TINS and SPR that allows us to further leverage our unparalleled expertise in protein immobilization for the benefit of our customers.

NMR-based structural biology

While crystallography is a powerful method to obtain high resolution structure information, it is not always successful. ZoBio has extensive experience in the elucidation of the 3D structure of target-ligand complexes using NMR. Starting with simple but efficient ligand based methods when protein observed methods are not applicable. If a protein can be isotope labelled and is of a moderate size (< 40 kDa) then the full range of NMR tools are available including binding site mapping and resonance assignment and structure elucidation via NOE.

For many proteins a structure may already be available. In such cases an alternative approach is available that is both efficient and accurate. Here, only the CH3 groups of I, L & V residues (depicted as balls in the graph) are isotope labelled. The red and blue methyl groups are sufficiently close (~8 Å) to 1H’s of the ligand to give rise to NOEs (schematically shown on the left). The intermolecular NOEs can be used to calculate the structure of the complex even when the target is 100 kDa or more.

Contact details

ZoBio BV
PO Box 9502
2300 RA Leiden
The Netherlands
T +31715274543
E-mail: info@zobio.com
URL: http://www.zobio.com

Fragment Screening

ZoBio offers its proprietary TINS technology for ligand screening studies on our own fragment library or the customer’s. TINS is a powerful approach to hit discovery that has been validated on a wide range of pharmaceutically relevant targets.

TINS uses a sample of the target protein and a reference protein that have been immobilized on sepharose based resin. Binding is detected by a reduction in the height of the NMR signals of a compound that specifically binds to the target. The screen is carried out by repeated cycles of fragment application, assaying for…

Hit Validation: Competition Binding

When known ligands (inhibitors) for the target are available, competition binding studies can be readily performed using TINS. Note that the ligands do not have to be small molecules. Competition binding studies can be carried out using antibodies or other protein (or DNA) ligands. The figure shows two examples carried out in the NMR spectrometer.

Here the target has been immobilized and placed in both cells of the dual-cell sample holder. At the top, a substrate for the immobilized enzyme has been injected into one cell and the substrate plus a potent inhibitor into the other. In the absence of the inhibitor, the substrate is quantitatively converted into product, while…

Hit Validation: Orthogonal NMR Methods

Fragment Hit validation via Protein Observed NMR (HSQC). When the target can be expressed in bacteria, stable NMR visible isotopes can be incorporated. This allows efficient acquisition of NMR spectra (HSQC, see figure upper left) of the protein that are extremely sensitive to ligand binding.

This orthogonal technique can be used to validate and quantify binding (upper right) as well as to define a low resolution binding site on the target. At bottom left, we noted a good correlation between binding detected by TINS (T/R ratio, y axis) and the HSQC experiment (average CSP, x axis) for 86 fragment hits…

Hit Validation: Surface Plasmon Resonance (Biacore)

SPR is a powerful, industry accepted ligand discovery technology that is orthogonal to NMR. In particular, TINS and SPR are highly complementary since both require target immobilization, while TINS provides extraordinary sensitivity to weak interactions and SPR is highly quantitative. We have shown that the combination of TINS screening with Biacore fragment characterization forms a powerful workflow for drug discovery (Kobayashi et al, J. Biomol. Screening, 2010, 15, p.978). ZoBio has a unique, highly integrated platform of TINS and SPR (Biacore T200) that allows us to further leverage our unparalleled expertise in protein immobilization for the benefit of our customers.

The sensorgrams on the left show the titrations of three fairly potent HSP90 fragment hits that were identified by TINS screening. The KD determined by Biacore, varying from double digit µM to a few hundred µM, is in extremely good correspondence with the values determined via an orthogonal method, protein observed NMR using HSQC experiment

Large Scale Druggability Screening

We and others (e.g. Hajduk et al, 2005, J. Med. Chem, 48, p. 2518; Edfeldt et al., 2011 Drug Discovery Today, 16(7), p. 284) have noted a strong correlation between the hit rate (and hit diversity) in NMR fragment screening and the “druggability” of a target. ZoBio has developed its own correlation between TINS and druggability as shown in the figure on our website. In TINS, binding is best represented by the ratio of the heights of each peak of a compound in the presence of the reference and the target (the T/R ratio). Each of these graphs presents the number of compounds with a T/R in each of the bins presented on the x-axis. This gives a profile of the screen that is unique for each target. The figure demonstrates how the profile varies with the known druggability of three different targets.

The scheme presented will allow large scale (15-20 targets/yr), efficient fragment screening on targets at a very early stage in the drug discovery process (e.g. before HTS and even before a purification strategy has been developed). In this way early prioritization based on data can be made, HTS results can be more intelligently interpreted and…

NMR-Based Structural Biology

While crystallography is a powerful method to obtain high resolution structure information, it is not always successful. ZoBio has extensive experience in the elucidation of the 3D structure of target-ligand complexes using NMR. Starting with simple but efficient ligand based methods when protein observed methods are not applicable. If a protein can be isotope labelled and is of a moderate size (< 40 kDa) then the full range of NMR tools are available including binding site mapping, resonance assignment and structure elucidation via NOE. For many proteins a structure may already be available.

In such cases an alternative approach is available that is both efficient and accurate. Here, only the CH3 groups of I, L & V residues (depicted as balls) are isotope labelled. The red and blue methyl groups are sufficiently close (~8 Å) to 1H's of the ligand to give rise to NOEs (schematically shown on…
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