A phenotypic screening platform utilising human spermatozoa identifies compounds with contraceptive activity

Abstract

There is an urgent need to develop new methods for male contraception, however a major barrier to drug discovery has been the lack of validated targets and the absence of an effective high-throughput phenotypic screening system. To address this deficit, we developed a fully-automated robotic screening platform that provided quantitative evaluation of compound activity against two key attributes of human sperm function: motility and acrosome reaction. In order to accelerate contraceptive development, we screened the comprehensive collection of 12,000 molecules that make up the ReFRAME repurposing library, comprising nearly all the small molecules that have been approved or have undergone clinical development, or have significant preclinical profiling. We identified several compounds that potently inhibit motility representing either novel drug candidates or routes to target identification. This platform will now allow for major drug discovery programmes that address the critical gap in the contraceptive portfolio as well as uncover novel human sperm biology.

Keywords: contraception; developmental biology; human; spermatozoa.

Figure 1.. Phenotypic Assay workflows.

(A) Graphical summary of modular screening workflow where motility measurement is followed by acrosome reaction (AR) measurement allowing a screening throughput of >1400 compounds per donor pool (B) Steps in imaging and analysis: human sperm are recorded with brightfield illumination (first panel) then sperm heads detected using a particle tracking algorithm (second panel) which are then tracked across the timelapse series of images (third panel) and subsequently classified (fourth panel). Each panel contains a zoomed-in subsection of the field. Colour coding for tracking distance and kinetic classification is shown in the panel insets: Tracking panel - rainbow gradient (showing progression over time); Classification panel - red for immotile (IM), yellow for non-progressively motile (NPM) and blue for progressively motile (PM). Scale bars: 100 μm (main images), 5 μm (insets). (C) Sperm counts per well after microscopy and detection shown in a combined violin/box plot. Colours: purple violin outline (probability density of values), yellow dots (outliers of boxplot). (D) Graphical summary of the expected populations determined by flow cytometry based on distribution of cells measured with FL3-A (Pi) vertical axis and FL1-A (PNA-488) horizontal axis: dead cells (upper left, Pi+ PNA-); dead and acrosome-reacted (upper right, Pi+ PNA+); unstained/live/non-reacted (lower left) and live acrosome-reacted (lower right, Pi-PNA+). (E) Example flow cytometry data comparing sperm treated with the Ca²⁺ ionophore (A23187) which induces AR (left panel) with sperm from DMSO-treated well (right panel). Colours indicate event density. (F) Combined violin/box plot data showing flow cytometry event counts per well. Colours and label as in (C).

Figure 1—figure supplement 1.. Further characterisation of phenotypic assays.

(A) Example data for a single sperm track with x and y coordinates being used to calculate standard sperm kinetics. Colour represents the cumulative distance travelled in microns. Acronyms: VCL (curvilinear velocity), ALH (amplitude of lateral head displacement; maximum value), STR (straightness ratio). (B) Explanation of main kinematic parameters (VCL, VSL [straight line velocity], VAP [average path velocity] and ALH). (C) Comparison of data from the standard computer-assisted semen analysis (CASA) with the high-throughput system using a Bland-Altman plot of VAP, VCL and VSL. Colours: turquoise line (mean of differences), dashed purple lines (limit of agreement, mean of differences + /- 1.96 * SD). (D) Effect of DMSO on sperm motility (VCL) relative to untreated wells. Green line represents concentration range used in this study (0.0625–0.1%) (E) Effect of pre-dispense incubation time on sperm motility (% reduction in VCL). Colours: green line (arbitrary 10% cut-off). (F) Plots of FL3-A vs FL1-A for the flow cytometry assay controls using acrosome specific PNA-488 dye (FL-1) and cell viability marker propidium iodine (Pi, FL-3) upon addition of A23187 Ca-ionophore. Panels from left to right: with ionophore and acrosome stain; with ionophore and propidium iodide only, with ionophore but unstained. Colours: red rectangles show population gates; coloured hexagons show event density (bin size = 40).

Figure 1—figure supplement 2.. Screening consistency over time analysis.

(A) Distribution of average path velocities (VAP) for sperm motility classes (IM = immotile; NPM = non progressively motile; PM = progressively motile), using data from two positions within each well (Position 1, Position 2), represented as combined box/violin plot. Each box/violin plot is a summary of a screening day (n = 4, 384-well plates). Colours: each screening day is represented as a different colour; grey = boxplot outliers. (B) Distribution of numbers of tracked sperm per well across screening days, represented as combined box/violin plot. Each box/violin plot is a summary of one screening plate. Colours as in (A). (C) Distribution of events identified in the flow cytometry assay, represented as combined box/violin plots. Each box/violin plot is a summary of a screening plate. Colours as in (A).

Figure 2.. ReFRAME Library Screening: Motility Assay Results.

(A) Primary screening results of the motility assay. Each dot represents a well (either compound or control well) showing % of DMSO control (Curvilinear Velocity: VCL). Positive controls (Pristimerin), negative controls (DMSO) and individual compound datapoints are shown. The dashed line showing the 15% of control (reduction in VCL) – the cut-off for primary hit selection. Total number of compounds = 11,903, excluding wells with auto-focus errors and with ‘sticky’ compounds which have been excluded from analysis. See dataset (http://doi.org/10.5061/dryad.jdfn2z36z) Figure 2—source code 1. (B) Assay robustness: the standard high throughput screening metric, Z' (see Materials and methods; Zhang et al., 1999) was used to determine the performance of the assay for all screening plates. Dashed lines indicate min/max Z' values. (C) Tracking data visualizations of 3 example wells showing sperm tracks of both imaging positions (Position 1 [Pos. 1] and Position 2 [Pos. 2] respectively) within the wells. A DMSO control well (left panels ‘Control’) shows a large number of progressively motile (PM) sperm (yellow) with few non-progressively motile (NPM) sperm (green and very few immotile (IM) sperm (purple) – this is in contrast to the shorter tracks and higher levels of NPM and IM in the middle panel (‘20% reduction’) for a compound that shows 20% inhibition of motility (i.e. 80% of control) and the right hand panel (‘80% reduction’) for a compound showing 80% inhibition (i.e. 20% of control), showing almost all cells are in the IM and NPM classes. (D) Histogram of sperm tracks quantification of the data show in (C). (E) Summary of motility assay hit rate (0.5%) and reconfirmation rate (0.24%). (F-G) Dose response confirmation of two hits. 8-point 3-fold dilution curves are shown with 10 μM as the highest concentration. Two data points per concentration (n = 2, data point is mean ± SD). Each curve is a 4-parameter logistic fit. Each plot shows estimated values Hill Slope and EC₅₀. The chemical structure of the hit compound is shown as well as some annotation and physicochemical properties. Physicochemical properties were calculated using RDKit, Python and KNIME: SlogP = partition coefficient (Wildman and Crippen, 1999); TPSA is the Topological Polar Surface Area (Ertl et al., 2000); MW is the exact Molecular weight; QED = Quantitative Estimate of Drug-likeness (Bickerton et al., 2012).

Figure 2—figure supplement 1.. Disulfiram Washout CASA measurement of percentage total motility in samples: prior to treatment (Squares); 20 mins after treatment with DMSO (triangles) or 10 μM disulfiram (circles); 60 min after washout of compound/DMSO.

Different donor pools analysed on three different days are represented by the three different colours. Data shown are four technical replicates per donor pool for DMSO/Disulfiram and two technical replicates for the untreated samples. For each measurement a minimum of 200 cells were recorded. See Figure 2—figure supplement 1—source data 1 along with Figure 2—figure supplement 1—source code 1 .

Figure 3.. ReFRAME library screening: ar assay results.

(A) Results of primary screening of the library using the acrosome assay (live cells, acrosome reacting, Pi- PNA+ population). Each dot represents a well (either compound or control well). Shown is % Events (number of events in Pi- PNA+ gate relative to total events in the sampled well). Datapoints for compounds, negative controls (DMSO) and positive controls (A23187) are shown. Black dashed line = 15% threshold for primary hits selection. See Figure 3—source data 1 along with Figure 3—source code 1. (B-C) Data from two example wells: a DMSO well (left panel) and a well with 65% Pi- PNA+ population (right panel). (D) Summary of acrosome assay results before and after triage.

Figure 3—figure supplement 1.. Further analysis of AR screening data and triaging strategy.

Results are shown of primary screening data for two of the other populations in the flow cytometry assay: (A) Acrosome reaction positive and propidium iodide positive (PI+ PNA+) events and (B) acrosome reaction negative and propidium positive (Pi+ PNA-) events. Shown is % Events (number of events relative to total events per well). Datapoints for compounds, negative controls (DMSO) and positive controls (A23187) are shown. See Figure 3—source data 1 along with Figure 3—source code 1 . (C) Control experiment showing microscopy images of A23187-induced acrosome-reacted sperm. Shown are individual channels (greyscale) for acrosome signal stained with PNA-Alexa488 (green band in merged image) and DNA stained with Propidium iodide indicating lack of cell viability (purple in merged image). Scale bar = 10 μM. (D) Orthogonal assay to eliminate fluorescent compounds: Two primary hits were analyzed using fluorescence microscopy without addition of staining reagents: top panels show the non-specific fluorescence of Merbromin and in bottom panels fluorescence of Manoalide which stains only significantly in the midpiece/tail. Grey scale images are shown in the left two panels and a merged image on the right. Key: green channel = Ex 488 nm with Em bandpass filter BP 525/50 nm; red channel = Ex 561 nm; Em Bandpass filter BP600/37 nm). Scale bar = 10 μM.