Drug discovery for male subfertility using high-throughput screening: a new approach to an unsolved problem

Sarah J. Martins da Silva*, Sean G. Brown, Keith Sutton, Louise V. King, Halil Ruso, David W. Gray, Paul G. Wyatt, Mark C. Kelly, Christopher L. R. Barratt, Anthony G. Hope

*Corresponding author for this work

    Research output: Contribution to journalArticle

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    Abstract

    STUDY QUESTION

    Can pharma drug discovery approaches be utilized to transform investigation into novel therapeutics for male infertility?

    SUMMARY ANSWER

    High-throughput screening (HTS) is a viable approach to much-needed drug discovery for male factor infertility.

    WHAT IS KNOWN ALREADY

    There is both huge demand and a genuine clinical need for new treatment options for infertile men. However, the time, effort and resources required for drug discovery are currently exorbitant, due to the unique challenges of the cellular, physical and functional properties of human spermatozoa and a lack of appropriate assay platform.

    STUDY DESIGN, SIZE, DURATION

    Spermatozoa were obtained from healthy volunteer research donors and subfertile patients undergoing IVF/ICSI at a hospital-assisted reproductive techniques clinic between January 2012 and November 2016.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    A HTS assay was developed and validated using intracellular calcium ([Ca2+]i) as a surrogate for motility in human spermatozoa. Calcium fluorescence was detected using a Flexstation microplate reader (384-well platform) and compared with responses evoked by progesterone, a compound known to modify a number of biologically relevant behaviours in human spermatozoa. Hit compounds identified following single point drug screen (10 μM) of an ion channel-focussed library assembled by the University of Dundee Drug Discovery Unit were rescreened to ensure potency using standard 10 point half-logarithm concentration curves, and tested for purity and integrity using liquid chromatography and mass spectrometry. Hit compounds were grouped by structure activity relationships and five representative compounds then further investigated for direct effects on spermatozoa, using computer-assisted sperm assessment, sperm penetration assay and whole-cell patch clamping.

    MAIN RESULTS AND THE ROLE OF CHANCE

    Of the 3242 ion channel library ligands screened, 384 compounds (11.8%) elicited a statistically significant increase in calcium fluorescence, with greater than 3× median absolute deviation above the baseline. Seventy-four compounds eliciting ≥50% increase in fluorescence in the primary screen were rescreened and evaluated further, resulting in 48 hit compounds that produced a concentration-dependent increase in [Ca2+]i. Sperm penetration studies confirmed in vitro exposure to two hit compounds (A and B) resulted in significant improvement in functional motility in spermatozoa from healthy volunteer donors (A: 1 cm penetration index 2.54, 2 cm penetration index 2.49; P < 0.005 and B: 1 cm penetration index 2.1, 2 cm penetration index 2.6; P < 0.005), but crucially, also in patient samples from those undergoing fertility treatment (A: 1 cm penetration index 2.4; P = 0.009, 2 cm penetration index 3.6; P = 0.02 and B: 1 cm penetration index 2.2; P = 0.0004, 2 cm penetration index 3.6; P = 0.002). This was primarily as a result of direct or indirect CatSper channel action, supported by evidence from electrophysiology studies of individual sperm.

    LIMITATIONS, REASONS FOR CAUTION

    Increase and fluxes in [Ca2+]i are fundamental to the regulation of sperm motility and function, including acrosome reaction. The use of calcium signalling as a surrogate for sperm motility is acknowledged as a potential limitation in this study.

    WIDER IMPLICATIONS OF THE FINDINGS

    We conclude that HTS can robustly, efficiently, identify novel compounds that increase [Ca2+]i in human spermatozoa and functionally modify motility, and propose its use as a cornerstone to build and transform much-needed drug discovery for male infertility.

    Original languageEnglish
    Pages (from-to)974-984
    Number of pages11
    JournalHuman Reproduction
    Volume32
    Issue number5
    Early online date16 Mar 2017
    DOIs
    Publication statusPublished - 1 May 2017

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    Martins da Silva, S. J., Brown, S. G., Sutton, K., King, L. V., Ruso, H., Gray, D. W., Wyatt, P. G., Kelly, M. C., Barratt, C. L. R., & Hope, A. G. (2017). Drug discovery for male subfertility using high-throughput screening: a new approach to an unsolved problem. Human Reproduction, 32(5), 974-984. https://doi.org/10.1093/humrep/dex055