Establishing Trypanosoma cruzi farnesyl pyrophosphate synthase as a viable target for biosensor driven fragment‐based lead discovery

Abstract

Procedures for producing and exploring Trypanosoma cruzi farnesyl pyrophosphate synthase (tcFPPS) for surface plasmon resonance (SPR) biosensor‐driven fragment‐based discovery have been established. The method requires functional sensor surfaces with high sensitivity for extended times and appropriate controls. Initial problems with protein stability and lack of useful reference compounds motivated optimization of experimental procedures and conditions. The improved methods enabled the production of pure, folded and dimeric protein, and identified procedures for storage and handling. A new coupled enzymatic assay, using luciferase for detection of pyrophosphate, was developed and used to confirm that the purified enzyme was active after purification and storage. It also confirmed that sensor surfaces prepared with structurally intact protein was active. An SPR‐biosensor assay for fragment library screening and hit confirmation was developed. A thermal shift assay was used in parallel. A library of 90 fragments was efficiently screened by both assays at a single concentration in the presence and absence of the catalytic cofactor Mg2+. Hits were selected on the basis of response levels or ΔT m > 1°C and selectivity for tcFPPS in the presence of Mg2+. Characterization of hits by SPR showed that all had low affinities and the relationships between steady‐state responses and concentrations were not sufficiently hyperbolic for determination of KD‐values. Instead, ranking could be performed from the slope of the linear relationship at low concentrations. This pilot screen confirms that the procedures developed herein enables SPR‐biosensor driven fragment‐based discovery of leads targeting tcFPPS, despite the lack of a reference compound. Significance Statement To enable the discovery of drugs, it is essential to have access to relevant forms of the target protein and valid biochemical methods for studying the protein and effects of compounds that may be evolved into drugs. We have established methods for the discovery of drugs for treatment of American Trypanosomiasis (Chagas disease), using farnesyl pyrophosphate synthase from Trypanosoma cruzi as a target.

Multiplexed experimental strategies for fragment library screening using SPR biosensors

“…4b). 18 The reliability of this approach was supported by the observation that a larger number of fragments interacted with the surface in the absence of Mg 2+ , indicative of non-specific binding to unfolded regions.…”

“…Fragments previously identified to interact with tcFPPS were identified as hits also in these experiments. 18 Due to very low affinities, saturation of the binding was not seen with the highest concentrations in hit validation step using concentration series (Fig. 5m-u).…”

“…Pre-screening was performed when using FL1056 but not FL90 since suitable screening conditions had already been identified for FPPS and FL90. 18 The advantage of pre-screening is illustrated with a representative dataset for FL1056 and AChBP (Fig. 4a).…”

“…d , T m for hits determined by DSF, *is data from. 18 e, Crystal structure of AChBP with fragment bound at Cys-loop interface between each monomer of pentameric structure.…”

“…Production of N-terminally His-tagged farnesyl pyrophosphate synthase from Trypanosoma cruzi (tcFPPS), Trypanosoma brucei (tbFPPS) and human (hFPPS) were carried out as previously described for tcFPPS. 18 Proteins were concentrated via centrifugation using a filter with a 30 kDa cut-off (Amicon Ultra-15), and the buffer was exchanged to storage buffer using PD10 columns (Cytiva). The storage buffer was 25 mM HEPES, 5 mM MgCl2 and 1 mM TCEP pH 6.5, supplemented with 100 mM NaCl for hFPPS, 25 mM NaCl for tbFPPS and 200 mM NaCl for tcFPPS.…”