On-demand male contraception via acute inhibition of soluble adenylyl cyclase

Abstract

Nearly half of all pregnancies are unintended; thus, existing family planning options are inadequate. For men, the only choices are condoms and vasectomy, and most current efforts to develop new contraceptives for men impact sperm development, meaning that contraception requires months of continuous pretreatment. Here, we provide proof-of-concept for an innovative strategy for on-demand contraception, where a man would take a birth control pill shortly before sex, only as needed. Soluble adenylyl cyclase (sAC) is essential for sperm motility and maturation. We show a single dose of a safe, acutely-acting sAC inhibitor with long residence time renders male mice temporarily infertile. Mice exhibit normal mating behavior, and full fertility returns the next day. These studies define sAC inhibitors as leads for on-demand contraceptives for men, and they provide in vivo proof-of-concept for previously untested paradigms in contraception; on-demand contraception after just a single dose and pharmacological contraception for men.

Fig. 1. A potent sAC inhibitor with longer residence time.

a Molecular structure of TDI-10229 and TDI-11861. b Overlay of the sAC complexes with TDI-10229 (cyan sticks) and TDI-11861 (magenta sticks). sAC, shown contoured as a molecular surface colored according to atom type, is from the TDI-11861 complex. c Concentration-response curves of TDI-10229 (teal) (IC₅₀ = 160 nM) and TDI-11861 (blue) (IC₅₀ = 3 nM) on in vitro adenylyl cyclase activity of purified recombinant human sAC protein in the presence of 1 mM ATP, 2 mM Ca²⁺, 4 mM Mg²⁺, and 40 mM HCO₃⁻, normalized to the respective DMSO-treated control; mean ± SEM (n = 6 with individual replicates indicated as symbols). d Concentration-response curves of TDI-10229 (teal) (IC₅₀ = 102 nM) and TDI-11861 (blue) (IC₅₀ = 7 nM) on sAC-dependent cAMP accumulation in sAC-overexpressing rat 4-4 cells grown in media containing 10% FBS treated with 500 μM IBMX for 5 min, normalized to the respective DMSO-treated control; mean ± SEM (TDI-10229: n = 13, TDI-11871: n = 8 with individual replicates indicated as symbols). e, f Sensorgrams of e TDI-10229 (teal) or f TDI-11861 (blue) binding to immobilized human sAC protein measured using surface plasmon resonance. Representative traces of experiments repeated at least three times showing binding kinetics of different concentrations of inhibitor (colored lines) along with best fits using a 1:1 binding model (black lines). TDI-10229: k_on = 2.3 × 10⁵/ms, K_D = 176 nM, k_off = 55.8 × 10⁻³/s; TDI-11861: k_on = 2.1 × 10⁵/ms, K_D = 1.4 nM, k_off = 0.3 × 10^-3/s. Source data are provided in the Source Data file, n = biological replicates.

Fig. 2. TDI-11861 inhibits essential functions required for fertilization in mouse and human sperm.

a, c Intracellular cAMP levels in a mouse and c human sperm incubated in non-capacitating (striped bars) or capacitating media (solid bars) in the absence (grey) or presence of 5 μM TDI-10229 (teal) or 10 nM TDI-11861 (blue). Shown are cAMP levels measured after 12 (mouse) or 30 minutes (human); mean + SEM (mouse: vehicle n = 20, TDI-10229 n = 10, TDI-11861 n = 12; human: vehicle n = 18, TDI-10229 n = 8, TDI-11861 n = 9 with individual replicates indicated as symbols). b, d Intracellular cAMP levels in b mouse and d human sperm following dilution into inhibitor-free media. After preincubation (5 min) in 5 μM TDI-10229 (teal) or 10 nM TDI-11861 (blue), sperm were diluted (1:100) in inhibitor-free non-capacitating (striped bars) or capacitating media (solid bars). Shown are cAMP levels measured 12 (mouse) or 30 (human) minutes after dilution; mean + SEM (mouse: TDI-10229 n = 7, TDI-11861 n = 12; human: TDI-10229 n = 5, TDI-11861 n = 10 with individual replicates indicated as symbols). e, f Mean flagellar beat frequency along the length of the tail (arc length, µm) of e mouse and f human sperm in the absence (grey) or presence of 5 μM TDI-10229 (teal) or 10 nM TDI-11861 (blue) before and after stimulation with 25 mM NaHCO₃. Solid lines indicate the time-averaged values, dotted lines the SEM, n = 3, ≥60 individual sperm from three different mice or three different human donors. g, h Acrosome reaction in g mouse sperm evoked by 50 heat-solubilized zona pellucidae (striped bars) and h human sperm evoked by 10 μM progesterone (striped bars) after incubation for 90 min(mouse) or 180 min(human) in capacitating media in the absence (grey) or presence of 5 μM TDI-10229 (teal) or 10 nM TDI-11861 (blue) in the absence or presence of 5 mM db-cAMP (dibutyrl-cAMP)/500 μM IBMX (isobutylmethylxanthine); mean + SEM (mouse: vehicle n = 10, TDI-10229 n = 5, TDI-11861 n = 6; human: vehicle n = 14, TDI-10229 n = 7, TDI-11861 n = 6 with individual replicates indicated as symbols). ZP = zona pellucidae. Differences between conditions were analyzed using one-way ANOVA compared to the DMSO-treated capacitated control *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided in the Source Data file, n = biological replicates.

Fig. 3. A single dose of systemically delivered sAC inhibitors block essential mouse sperm functions ex vivo.

a, c Relative cAMP increase due to incubation in capacitating conditions of epididymal mouse sperm isolated at the indicated times following (a) oral administration (gavage) or (c) injection (i.p.) with vehicle (DMSO:PEG 400:PBS 1:4:5) (grey), 50 mg/kg TDI-10229 (teal) or 50 mg/kg TDI-11861 (blue). Isolated sperm were measured directly (solid bars) or after 100-fold dilution (striped bars) into inhibitor-free non-capacitating or capacitating media, and cAMP was measured after incubation for 12 minutes. Values shown are cAMP levels in capacitating sperm relative to cAMP levels in non-capacitated sperm from the same mouse; mean + SEM (oral: vehicle n = 9, TDI-10229 n = 8, TDI-11861 n = 9; systemic: vehicle n = 12, TDI-10229 n = 11, TDI-11861 n = 11 with individual replicates indicated as symbols). b, d Percentage of motile epididymal mouse sperm isolated 1 hour post (b) oral administration (gavage) or (d) injection (i.p.) with vehicle (grey), 50 mg/kg TDI-10229 (teal) or 50 mg/kg TDI-11861 (blue). Isolated sperm were diluted 1:100 in inhibitor-free non-capacitating media, and percent motility was assessed by computer-assisted sperm analysis (CASA). For sperm isolated from TDI-11861-injected males, motility was also assessed in the presence of 5 mM db-cAMP/500 μM IBMX (striped bars); mean + SEM (oral: vehicle n = 7, TDI-10229 n = 11, TDI-11861 n = 11, TDI-11861+cAMP = 9; systemic: vehicle n = 15, TDI-10229 n = 9, TDI-11861 n = 16, TDI-11861+cAMP = 8 with individual replicates indicated as symbols). Differences between conditions were analyzed using one-way ANOVA compared to sperm isolated from vehicle-injected mice, *P < 0.05, **P < 0.01, ****P < 0.0001. Source data are provided in the Source Data file, n = biological replicates.

Fig. 4. Systemically delivered TDI-11861 blocks motility and capacitation of ejaculated mouse sperm recovered post-coitally from the uterus.

a Intracellular cAMP levels in ejaculated uterine mouse sperm isolated 2 hours following injection (i.p.) with vehicle (grey) or 50 mg/kg TDI-11861 (blue) incubated in non-capacitating (striped bars) or capacitating media (solid bars); mean + SEM (vehicle n = 5, TDI-11861 n = 8 with individual replicates indicated as symbols). b Representative motility tracks of ejaculated uterine mouse sperm isolated 2 hours following injection (i.p.) with vehicle or 50 mg/kg TDI-11861. Scale bar = 300 μM. Track´s color code: motile (green), progressive (turquoise), hyperactivated (purple), slow (pink), static (red), not counted due to late entry (yellow). c, d Percentage of (c) progressively motile and (d) hyperactivated epididymal (solid bars) and ejaculated uterine (striped bars) mouse sperm isolated two-hour post injection (i.p.) with vehicle (grey) or 50 mg/kg TDI-11861 (blue); mean + SEM (progressive: vehicle epididymal n = 8, TDI-11861 epididymal n = 7, vehicle ejaculated n = 9, TDI-11861 ejaculated n = 8, hyperactivated: vehicle epididymal n = 8, TDI-11861 epididymal n = 9, vehicle ejaculated n = 7, TDI-11861 ejaculated n = 7 with individual replicates indicated as symbols). Differences between vehicle-injected and TDI-11861-injected sperm were analyzed using two-tailed, unpaired t test, **P < 0.01. Source data are provided in the Source Data file, n = biological replicates.

Fig. 5. TDI-11861 inhibition of sperm motility correlates with pharmacokinetics.

Percentage of motile epididymal mouse sperm (left axis and bars; mean + SEM; vehicle (grey) n = 15, TDI-11861 (blue) 0.25h n = 10, 0.5h 1.5h 2h 2.5h 3.5h 4h n = 6, 1h n = 16, 3h n = 5, 4.5 h n = 9, 6h 9h 24h n = 8 with individual replicates indicated as symbols) and TDI-11861 blood concentration (right axis and blue squares; mean ± SEM; n = 5) quantified at the indicated time points post injection (i.p.) with vehicle or 50 mg/kg TDI-11861. Source data are provided in the Source Data file, n = biological replicates.