Development of allosteric HIPK4 inhibitors as non-hormonal male contraceptives

安全、有效和可逆的避孕方法对于解决8500万人是必要的 世界各地每年发生的计划外怀孕。除了这些意外的负面影响外， 怀孕对全球可持续性的影响，其中近五分之一的病例是通过不安全的堕胎终止的 对女性的健康有重大风险。自20世纪50年代发明“避孕药”以来，绝大多数人出生时 控制选择一直是女性导向的，包括雌激素或孕激素治疗，屏障方法， 宫内节育器和输卵管结扎术。相比之下，男性仍然仅限于避孕套和输精管结扎术，这对 高故障率和不完全可逆性。 男性避孕的药理学策略将有助于实现与目前女性导向的平等 选择。然而，基于激素的治疗可能会导致代谢紊乱、情绪变化、血栓形成、痤疮、 和睾丸退行性变。正在开发的非激素药物，如维甲酸信号拮抗剂， 氯硝胺衍生物和溴域睾丸特异性蛋白抑制剂也可能具有不受欢迎的靶点。 副作用。确定精子发育和功能的新调节者对于弥合这一差距是必要的， 而可下药的睾丸特异蛋白是特别吸引人的目标。我们的项目专注于一个信令 体现这一范式的蛋白质：HIPK4，同源结构域相互作用蛋白激酶家族的成员 这在发育中的精子中表现出来。我们观察到缺乏HIPK4功能的雄性小鼠是不育的，但 否则看起来有正常的发育、生理和行为。HIPK4缺陷小鼠表现出 与少弱畸形精子症相一致的生精缺陷及其畸形精子是 不能进行体外受精。我们的研究进一步表明，HIPK4调节肌动蛋白驱动的头部 精子细胞伸长过程中的形状。 我们的发现强调了小分子HIPK4抑制剂作为非荷尔蒙男性的潜力 避孕药，特别是靶向保守的ATP结合口袋外区域的拮抗剂。 为了实现这一目标，我们将开发变构HIPK4抑制剂，并评估它们对精子发生和发育的影响。 动物模型中的雄性生育能力。该项目的R61阶段将侧重于为以下项目建立工作流程 鉴定和表征变构HIPK4拮抗剂，包括初级蛋白热移(PTS) 缓蚀剂效力和选择性的二次/三次测定(R61目标2)。我们还将 用于研究HIPK4抑制的结构基础的硅学和结晶学方案的发展(R61目标3)。 在完成这些里程碑之后，我们将继续这个项目的R33阶段，其中包括一个大规模的， 高通量PTS筛选变构HIPK4抑制剂(R33AIM 1)和通过以下途径进行点击到领先的优化 药物化学和基于结构的设计(R33目标2)。然后我们将评估HIPK4拮抗剂在 动物模型，以确定其作为男性避孕药的安全性、有效性和可逆性(R33目标3)。

Safe, effective, and reversible methods for contraception are necessary to address the 85 million unplanned pregnancies that occur worldwide each year. In addition to the negative impact of these unintended pregnancies on global sustainability, nearly one-fifth of these cases are terminated through unsafe abortions with significant risks to women’s health. Since the invention of “the Pill” in the 1950s, the vast majority of birth control options have been female-directed, including estrogen or progestin treatments, barrier methods, intrauterine devices, and tubal ligation. In contrast, men remain limited to condoms and vasectomy, which have high failure rates and incomplete reversibility, respectively. Pharmacological strategies for male contraception would help achieve parity with current female-directed options. However, hormone-based therapies can lead to metabolic disorders, mood changes, thrombosis, acne, and testicular degeneration. Non-hormonal agents in development such as retinoic acid signaling antagonists, lonidamine derivatives, and bromodomain testis-specific protein inhibitors can also have undesirable on-target side effects. Identifying new regulators of sperm development and function will be necessary to bridge this gap, and druggable testis-specific proteins are especially attractive targets. Our project focuses on one signaling protein that exemplifies this paradigm: HIPK4, a member of the homeodomain-interacting protein kinase family that is expressed in developing sperm. We observe that male mice lacking HIPK4 function are infertile but otherwise appear to have normal development, physiology, and behavior. HIPK4-deficient mice exhibit spermatogenic defects that are consistent with oligoasthenoteratozoospermia, and their misshapen sperm are incompetent for in vitro fertilization. Our investigations further indicate that HIPK4 regulates actin-driven head shaping during spermatid elongation. Our findings underscore the potential of small-molecule HIPK4 inhibitors as non-hormonal male contraceptives, particularly antagonists that target regions outside of the conserved ATP-binding pocket. Toward this goal, we will develop allosteric HIPK4 inhibitors and evaluate their effects on spermiogenesis and male fertility in animal models. The R61 phase of this project will focus on establishing a workflow for identifying and characterizing allosteric HIPK4 antagonists, including a primary protein thermal shift (PTS) assay (R61 Aim 1) and secondary/tertiary assays of inhibitor potency and selectivity (R61 Aim 2). We will also develop in silico and crystallographic protocols for studying the structural basis of HIPK4 inhibition (R61 Aim 3). After completing these milestones, we will pursue the R33 phase of this project, which includes a large-scale, high-throughput PTS screen for allosteric HIPK4 inhibitors (R33 Aim 1) and hit-to-lead optimization through medicinal chemistry and structure-based design (R33 Aim 2). We will then evaluate HIPK4 antagonists in animal models to determine their safety, efficacy, and reversibility as male contraceptives (R33 Aim 3).