Reversible male contraception by targeted inhibition of serine/threonine kinase 33

Abstract

Men or mice with homozygous serine/threonine kinase 33 (STK33) mutations are sterile owing to defective sperm morphology and motility. To chemically evaluate STK33 for male contraception with STK33-specific inhibitors, we screened our multibillion-compound collection of DNA-encoded chemical libraries, uncovered potent STK33-specific inhibitors, determined the STK33 kinase domain structure bound with a truncated hit CDD-2211, and generated an optimized hit CDD-2807 that demonstrates nanomolar cellular potency (half-maximal inhibitory concentration = 9.2 nanomolar) and favorable metabolic stability. In mice, CDD-2807 exhibited no toxicity, efficiently crossed the blood-testis barrier, did not accumulate in brain, and induced a reversible contraceptive effect that phenocopied genetic STK33 perturbations without altering testis size. Thus, STK33 is a chemically validated, nonhormonal contraceptive target, and CDD-2807 is an effective tool compound.

Fig. 1.. DEC-Tec selection.

Enrchment profile of Baylor College of Medicine (BCM) DNA-encoded chemical library qDOS28_1 against STK33 at 0.1 µM (x axis, z-score) versus no target control (y axis, z-score). A series of hit compounds were identified with similar building block 1 (BB1 in blue; attached to the DNA), same building block 2 (BB2 in red), and same building block 3 (BB3 in black). The enrichment of each tri-synthon is shown as sequencing counts/z-score at 0.1 µM in the box.

Fig. 2.. STK33 hits, analogs, and biological characteristics.

(A) Chemical structures of CDD-2110, CDD-2211, CDD-2212, and CDD-2807. CDD-2110 is the hit with a short linker enriched in the STK33 selection. Blue, red, and black moieties correspond to BB1, BB2, and BB3, respectively. (B) Chemical properties, biochemical activity, cellular activity, and metabolism data for CDD-2110, CDD-2211, CDD-2212, and CDD-2807. Dissociation constant (K_d) and inhibition constant (K_i) values were calculated from LanthaScreen binding assay and Z’-LYTE assay, respectively. Dose-responses were done with at least five different concentrations in duplicate, and parameters (K_d and K_i) optimized by calculations described in the supplementary materials are given with standard errors. Half-maximal inhibitory concentration (IC₅₀) values were calculated from the NanoBRET assay. Half-life (t_1/2) was measured using either MLM or HLM stability assays; assay data >60 min is an extrapolated estimate. (C) Summary of CDD-2807 fractional occupancy values for kinases with >30% occupancy (red: 30 to 49.9%; yellow, 50 to 79.9%; green: 80 to 100%) from NanoBRET K192 assay performed at 1 µM.

Fig. 3.. Crystal structure of the STK33/CDD-2211 complex.

(A) Overall structure of the STK33/CDD-2211 dimer complex. Chain A is shown with surface and chain B in cartoon representation. N and C termini are labeled. The bound CDD-2211 is shown in sticks with its carbon atoms in yellow, oxygens in red, and nitrogens in blue. The activation loop is colored in red. (B) Fo-Fc omit density for CDD-2211 in the STK33/CDD-2211 complex contoured at 1σ. (C) Electrostatic surface of the STK33 active site with CDD-2211. (D) Detailed interaction between STK33 and CDD-2211. Hinge residue backbone atoms that form hydrogen bonds with the ligand are shown as spheres. Key interacting residues shown are shown as sticks. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; S, Ser; T, Thr; and V, Val.

Fig. 4.. CDD-2807 treatment induces a reversible contraceptive effect.

(A and B) Litter size (mean ± SEM) from the 2-month fertility assessment showed that CDD-2807–treated mice had a significant reduction in fertility in months one and two in protocol 1 [(A); n = 6] and protocol 2 [(B); n = 7]. Fertility was restored to levels equal to those of controls after halting CDD-2807 treatment for 3 weeks in protocol 1; in protocol 2, fertility was restored an average of 53.5 ± 5.1 days after completion of CDD-2807 treatment compared to the controls (3.5 ± 0.2 days). (C) There are no observed morphological differences in testes from control and CDD-2807 treatment in mice from protocol 2 at 63 days; testis size of CDD-2807–treated mice in protocol 2 (87.13 ± 5.87 mg) was not statistically changed versus control males (111.10 ± 6.50 mg). (D to G) CDD-2807–treated mice (n = 3) from protocol 2 at 63 days had a significant decrease in sperm counts (D), motility (E), progressive sperm (F), and hyperactivated sperm (G) compared to control mice (mean ± SEM; n = 3). (H) For SEM quantification, a minimum of 100 sperm per group were counted and determined to be either normal or abnormal (having head or tail defects). Counts were totaled and then turned into a percentage of normal versus abnormal sperm per group. By SEM analysis, 94.7 ± 2.7% of the sperm in the CDD-2807–treated mice (n = 3) in protocol 2 had morphologic defects at 63 days, including head and tail defects as shown, compared to only 10.9 ± 2.7% abnormal sperm for the controls (n = 3). Scale bars: 0.5 cm (C), 5 mm (H). **P < 0.01, ***P < 0.001, ****P < 0.0001.