TRPtracker: a community database for monitoring praziquantel sensitivity at TRPMPZQ variants

Abstract

The anthelmintic praziquantel (PZQ) has been used for decades as the clinical therapy for schistosomiasis, and remains the only available drug. As a cheap and effective drug therapy for all human disease-causing Schistosoma species, usage of PZQ underpins mass drug administration strategies aimed at eliminating schistosomiasis as a public health problem by 2030. Concern over the potential emergence of resistance to PZQ is therefore warranted, as it would constitute a major threat to this approach. In terms of molecular adaptations conferring PZQ resistance, variation in the sequence and/or expression of the drug target is an obvious mechanism and should be a priority for surveillance efforts. The target of PZQ is a transient receptor potential ion channel, TRPM_PZQ, which is established as a locus that regulates schistosome sensitivity to PZQ. Here, we describe the establishment of a community resource, 'TRPtracker', which coalesces data on TRPM_PZQ natural variants together with measurements of individual variant sensitivity to PZQ. A compendium of laboratory-generated mutants in TRPM_PZQ is also compiled in TRPtracker to map regions within TRPM_PZQ critical for PZQ sensitivity. Aggregation of data from multiple research groups into TRPtracker permits rapid community-wide exchange of data, cataloguing which TRPM_PZQ variants have been functionally profiled, where geographically these variants have been found, their frequency within populations and their potential impact on PZQ sensitivity.

Keywords: database; resistance; schistosomiasis; transient receptor potential channel.

Figure 1.. Schematic overview of TRPtracker and functional profiling of TRPM_PZQ.

(A) Schematic overview of profiling workflow for lab-generated mutants and natural variants, which can be profiled functionally and in terms of expression levels. These data and sample metadata are compiled in the TRPtracker resource. (B) Example concentration response curves to illustrate impact of different TRPM_PZQ variants on the relative activity (RA) of PZQ. Increases in potency yield higher RA values (blue), decreases in potency or decreases in efficacy yield lower RA values (orange) relative to the wild type TRPM_PZQ concentration response curve (green). (C) Relative activity plot of Sm.TRPM_PZQ variants from the TRPtracker dataset, stratified into different function categories: null (RA = 0, red), decreased sensitivity (RA = 0.6 – 1, orange), wild type (RA = 0.6 – 1.4, green) and increased sensitivity (RA> 1.4, blue).

Figure 2.. Functional effects of various TRPM_PZQ mutants.

(A) Distribution of functionally ‘null’ laboratory mutants (red circles) along the length of the Sm.TRPM_PZQ coding sequence. (B) Representations from the AlphaFold protein structure database [56, 57] for a monomer of Sm.TRPM_PZQ (left), Hs.TRPM2 (O94759, middle) and Hs.TRPM8 (Q7Z2W7,right). The Sm.TRPM_PZQ prediction shows several predicted structural domains [58] corresponding to the voltage-sensing like domain (VSLD, S1-S4, magenta), pore domains (S5-S6, green), MHR3/4 (orange), MHR1/2 (turquoise), nudix hydrolase domain (yellow). Overall, Sm.TRPM_PZQ presents with homology to the vertebrate TRPM8-like menthol binding pocket found in the VSLD (S1-S4) within a broader protein structure with cytoplasmic domain homology to Hs.TRPM2 [16]. The monomers of Hs.TRPM2 and Hs.TRPM8 are colored in terms of predicted effect on protein ‘pathogenicity’ function using AlphaMissense [29], with benign variation colored blue and deleterious mutants scored with increasing warm coloration toward red. (C) Functional profiling of Sm.TRPM_PZQ mutants in response to PZQ. These include null mutants (Sm.TRPM_PZQ [F107A] and Sm.TRPM_PZQ[G108A]) as well sensitizing mutants (Sm.TRPM_PZQ [F1521A] and Sm.TRPM_PZQ[W1667A]). (D) Interrogation of a null mutant ‘hot-spot’ in the NH₂-terminal MHR1/2 domain of TRPM_PZQ. The null mutants found in Sm.TRPM_PZQ profiled in (C) localize to the end of a β-strand (F107 in red, G108 in cyan, top) which shows structural and sequence conservation with the β5-strand in Hs.TRPM2 (T174 in red, G175 in cyan; bottom, [59]). This region of TRPM2 is implicated in the binding of ADP-ribose [59], shown in white (PDB, 8E6V). (E) Projection of location of the sensitizing mutants Sm.TRPM_PZQ [F1521A] (magenta, S4-S5 linker) and Sm.TRPM_PZQ[W1667A] (green, TRP domain) relative to the PZQ (yellow) in the VSLD binding pocket.

Figure 3.. Profiling function and expression of various natural TRPM_PZQ variants.

(A) Distribution of RA values for natural TRPM_PZQ variants that have been functionally profiled in in the database. Data is presented as a half-box plot, with data on the left and the standard deviation (box), range (vertical line) and mean (black square) of this population shown on the right. (B) Collection sites where the natural TRPM_PZQ variants that have been functionally profiled in this study were sampled. (C) Distribution of normal sensitivity (green), decreased sensitivity (orange) and functionally ‘null’ natural variants of Sm.TRPM_PZQ (red circles) identified by various groups along the length of the Sm.TRPM_PZQ. (D) representative Western blot for expression of indicated TRPM_PZQ variants. (E) densitometric measurements from independent blots (n=3) quantified for these same TRPM_PZQ variants.