Evaluation of Site-Diversified, Fully Functionalized Diazirine Probes for Chemical Proteomic Applications

Photoaffinity probes combined with the chemical proteomic platform have emerged as versatile tools for ligand and target discovery. However, photoaffinity probes with retained activity cannot always label the known target, indicating that it is challenging to profile a ligand’s targets based on its photoaffinity probe modified at a single site. Herein, we construct a series of site-diversified probes (P1-P6) of 4-anilinoquinazoline, a scaffold shared by several marketed EGFR-targeted drugs, via attaching a “fully functionalized” diazirine tag to six different sites, respectively. Chemical proteomic analysis revealed that these probes show different proteome-wide profiles and distinct competition patterns by erlotinib. Remarkably, low activity P4 towards EGFR inhibition has better EGFR labelling efficiency than the higher one, P5, which highlights the dominance of labelling accessibility of diazirine over probe affinity. In addition, the integrated analysis of protein targets of site-diversified probes can also help distinguish false positive targets. We anticipate that site-diversification of the probes of a given scaffold is an indispensable strategy to truly harness the power of photoaffinity-based chemoproteomics in drug discovery.

Evaluation of Site-Diversified, Fully Functionalized Diazirine Probes for Chemical Proteomic Applications

Photoaffinity probes combined with the chemical proteomic platform have emerged as versatile tools for ligand and target discovery. However, photoaffinity probes with retained activity cannot always label the known target, indicating that it is challenging to profile a ligand’s targets based on its photoaffinity probe modified at a single site. Herein, we construct a series of site-diversified probes (P1-P6) of 4-anilinoquinazoline, a scaffold shared by several marketed EGFR-targeted drugs, via attaching a “fully functionalized” diazirine tag to six different sites, respectively. Chemical proteomic analysis revealed that these probes show different proteome-wide profiles and distinct competition patterns by erlotinib. Remarkably, low activity P4 towards EGFR inhibition has better EGFR labelling efficiency than the higher one, P5, which highlights the dominance of labelling accessibility of diazirine over probe affinity. In addition, the integrated analysis of protein targets of site-diversified probes can also help distinguish false positive targets. We anticipate that site-diversification of the probes of a given scaffold is an indispensable strategy to truly harness the power of photoaffinity-based chemoproteomics in drug discovery.

Optimisation of the Synthesis and Cell Labelling Conditions for [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS: a Direct In Vitro Comparison in Cell Types with Distinct Therapeutic Applications

Background There is a need to better characterise cell-based therapies in preclinical models to help facilitate their translation to humans. Long-term high-resolution tracking of the cells in vivo is often impossible due to unreliable methods. Radiolabelling of cells has the advantage of being able to reveal cellular kinetics in vivo over time. This study aimed to optimise the synthesis of the radiotracers [89Zr]Zr-oxine (8-hydroxyquinoline) and [89Zr]Zr-DFO-NCS (p-SCN-Bn-Deferoxamine) and to perform a direct comparison of the cell labelling efficiency using these radiotracers. Procedures Several parameters, such as buffers, pH, labelling time and temperature, were investigated to optimise the synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS in order to reach a radiochemical conversion (RCC) of >95 % without purification. Radio-instant thin-layer chromatography (iTLC) and radio high-performance liquid chromatography (radio-HPLC) were used to determine the RCC. Cells were labelled with [89Zr]Zr-oxine or [89Zr]Zr-DFO-NCS. The cellular retention of 89Zr and the labelling impact was determined by analysing the cellular functions, such as viability, proliferation, phagocytotic ability and phenotypic immunostaining. Results The optimised synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS resulted in straightforward protocols not requiring additional purification. [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS were synthesised with an average RCC of 98.4 % (n = 16) and 98.0 % (n = 13), respectively. Cell labelling efficiencies were 63.9 % (n = 35) and 70.2 % (n = 30), respectively. 89Zr labelling neither significantly affected the cell viability (cell viability loss was in the range of 1–8 % compared to its corresponding non-labelled cells, P value > 0.05) nor the cells’ proliferation rate. The phenotype of human decidual stromal cells (hDSC) and phagocytic function of rat bone-marrow-derived macrophages (rMac) was somewhat affected by radiolabelling. Conclusions Our study demonstrates that [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS are equally effective in cell labelling. However, [89Zr]Zr-oxine was superior to [89Zr]Zr-DFO-NCS with regard to long-term stability, cellular retention, minimal variation between cell types and cell labelling efficiency.

At the molecular resolution with MINFLUX?

Gwosch et al. (2020) and Balzarotti et al. (2017) purport MINFLUX as the next revolutionary fluorescence microscopy technique claiming a spatial resolution in the range of 1-3 nm in fixed and living cells. Though the claim of molecular resolution is attractive, I am concerned whether true 1 nm resolution has been attained. Here, I compare the performance with other super-resolution methods focussing particularly on spatial resolution claims, atypical image rendering, visualisation enhancement, subjective filtering of localizations, detection vs labelling efficiency and the possible limitations when imaging biological samples containing densely labelled structures. I hope the analysis and evaluation parameters presented here are not only useful for future research directions but also microscope users, developers and core facility managers when deciding on an investment for the next 'state-of-the-art' instrument.

A General Model to Optimise Copper(II) Labelling Efficiency of Double-Histidine Motifs for Pulse Dipolar EPR Applications

<div> <p>Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to studies of biomolecules underpinning health and disease by providing highly accurate and precise geometric constraints. Combining double-histidine (dH) motifs with Cu^II spin labels shows promise for further increasing the precision of distance measurements, and for investigating subtle conformational changes. However, non-covalent coordination-based spin labelling is vulnerable to low binding affinity. Dissociation constants of dH motifs for Cu^II-nitrilotriacetic acid were previously investigated <i>via </i>relaxation induced dipolar modulation enhancement (RIDME), and demonstrated the feasibility of exploiting the double histidine motif for EPR applications at sub-μM protein concentrations. Herein, the feasibility of using modulation depth quantitation in Cu^II-Cu^IIRIDME to simultaneously estimate a pair of non-identical independent <i>K_D</i> values in such a tetra-histidine model protein is addressed. Furthermore, we develop a general speciation model to optimise Cu^IIlabelling efficiency, in dependence of pairs of identical or disparate <i>K_D</i> values and total Cu^II label concentration. We find the dissociation constant estimates are in excellent agreement with previously determined values, and empirical modulation depths support the proposed model. </p> </div> <br>

A general model to optimise CuII labelling efficiency of double-histidine motifs for pulse dipolar EPR applications

A multi-site speciation model facilitates double-histidine motif labelling efficiency optimisation for pulse dipolar EPR measurements. Results suggest affinities differing by a factor of 10 between an α-helical and a β-sheet double-histidine motif.

A General Model to Optimise Copper(II) Labelling Efficiency of Double-Histidine Motifs for Pulse Dipolar EPR Applications

<div> <p>Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to studies of biomolecules underpinning health and disease by providing highly accurate and precise geometric constraints. Combining double-histidine (dH) motifs with Cu^II spin labels shows promise for further increasing the precision of distance measurements, and for investigating subtle conformational changes. However, non-covalent coordination-based spin labelling is vulnerable to low binding affinity. Dissociation constants of dH motifs for Cu^II-nitrilotriacetic acid were previously investigated <i>via </i>relaxation induced dipolar modulation enhancement (RIDME), and demonstrated the feasibility of exploiting the double histidine motif for EPR applications at sub-μM protein concentrations. Herein, the feasibility of using modulation depth quantitation in Cu^II-Cu^IIRIDME to simultaneously estimate a pair of non-identical independent <i>K_D</i> values in such a tetra-histidine model protein is addressed. Furthermore, we develop a general speciation model to optimise Cu^IIlabelling efficiency, in dependence of pairs of identical or disparate <i>K_D</i> values and total Cu^II label concentration. We find the dissociation constant estimates are in excellent agreement with previously determined values, and empirical modulation depths support the proposed model. </p> </div> <br>

A General Model to Optimise Copper(II) Labelling Efficiency of Double-Histidine Motifs for Pulse Dipolar EPR Applications

<div> <p>Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to studies of biomolecules underpinning health and disease by providing highly accurate and precise geometric constraints. Combining double-histidine (dH) motifs with Cu^II spin labels shows promise for further increasing the precision of distance measurements, and for investigating subtle conformational changes. However, non-covalent coordination-based spin labelling is vulnerable to low binding affinity. Dissociation constants of dH motifs for Cu^II-nitrilotriacetic acid were previously investigated <i>via </i>relaxation induced dipolar modulation enhancement (RIDME), and demonstrated the feasibility of exploiting the double histidine motif for EPR applications at sub-μM protein concentrations. Herein, the feasibility of using modulation depth quantitation in Cu^II-Cu^IIRIDME to simultaneously estimate a pair of non-identical independent <i>K_D</i> values in such a tetra-histidine model protein is addressed. Furthermore, we develop a general speciation model to optimise Cu^IIlabelling efficiency, in dependence of pairs of identical or disparate <i>K_D</i> values and total Cu^II label concentration. We find the dissociation constant estimates are in excellent agreement with previously determined values, and empirical modulation depths support the proposed model. </p> </div> <br>

A General Model to Optimise Copper(II) Labelling Efficiency of Double-Histidine Motifs for Pulse Dipolar EPR Applications

<div> <p>Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to studies of biomolecules underpinning health and disease by providing highly accurate and precise geometric constraints. Combining double-histidine (dH) motifs with Cu^II spin labels shows promise for further increasing the precision of distance measurements, and for investigating subtle conformational changes. However, non-covalent coordination-based spin labelling is vulnerable to low binding affinity. Dissociation constants of dH motifs for Cu^II-nitrilotriacetic acid were previously investigated <i>via </i>relaxation induced dipolar modulation enhancement (RIDME), and demonstrated the feasibility of exploiting the double histidine motif for EPR applications at sub-μM protein concentrations. Herein, the feasibility of using modulation depth quantitation in Cu^II-Cu^IIRIDME to simultaneously estimate a pair of non-identical independent <i>K_D</i> values in such a tetra-histidine model protein is addressed. Furthermore, we develop a general speciation model to optimise Cu^IIlabelling efficiency, in dependence of pairs of identical or disparate <i>K_D</i> values and total Cu^II label concentration. We find the dissociation constant estimates are in excellent agreement with previously determined values, and empirical modulation depths support the proposed model. </p> </div> <br>

Cysteine specific bioconjugation with benzyl isothiocyanates

We present herein the development of a new fluorescent dye equipped with a benzyl isothiocyanate warhead, which resulted improved photophysical properties and enhanced labelling efficiency on the Fab antibody subunit and the trastuzumab antibody.