Optimization of modulators of Gbg-SNARE interaction

描述（由申请人提供）：通过调节胞吐作用释放化学递质是许多形式的细胞间通讯的基础，包括激素释放和突触传递。胞吐作用受到复杂的调节，并涉及蛋白质-蛋白质相互作用和膜重塑事件的网络。G蛋白偶联受体（GPCR）在协调这种复杂的调节中发挥核心作用，并且众所周知，Gi/o偶联的GPCR通过释放G蛋白βγ亚基来抑制神经分泌细胞释放递质。这种深刻的抑制有可能有助于突触前整合和突触可塑性。这种抑制作用的最佳研究机制是调节Ca 2+通道的电压敏感性。 然而，Gβγ也可以通过与可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白以及组装的SNARE复合物结合，直接抑制Ca 2+进入远端点的神经递质释放。我们已经表明，SNAP 25的C-末端对于抑制递质释放的能力至关重要。我们设计了一种 Gβγ-SNAP 25相互作用，并筛选了一个小的化合物库，这些化合物是基于已知的调节蛋白质-蛋白质相互作用（PPI）的抑制剂和增强剂的化学型设计的。在这份协议中，我们将 优化Gβγ-SNAP 25相互作用的抑制剂和增强剂在目的1中，我们将对两类PPI化合物进行药物化学优化，以提高其效力、选择性和生物利用度。在目标2中，我们将确定与其他Gβγ相互作用蛋白相比，化合物对Gβγ-SNAP 25相互作用的选择性。在目标3中，我们将确定优化的分子对培养的海马神经元的神经递质释放的影响，并确定它们是否与突触前Gi/o偶联GPCR的激动剂和拮抗剂协同作用。这些化合物应该使我们能够评估Gβγ-SNARE相互作用对GPCR调节胞吐作用的重要性。它们可以与突触前GPCR的激动剂或拮抗剂平行工作，从而与突触前功能协同作用，选择性地影响神经递质的突触前而非突触后作用。我们提出的研究解决了神经科学中的一个基本问题，即神经递质控制胞吐作用的分子机制。这些研究可能为开发新疗法确定目标，这些新疗法可能揭示与分泌和神经调节相关的疾病的病理基础。

DESCRIPTION (provided by applicant): Release of chemical transmitters by regulated exocytosis underlies many forms of intercellular communication, including hormone release and synaptic transmission. Exocytosis is subject to complex modulation and involves a web of protein-protein interactions and membrane remodeling events. G protein-coupled receptors (GPCRs) play a central role in orchestrating this complex regulation, and Gi/o- coupled GPCRs are well known to inhibit transmitter release from neurosecretory cells by release of G protein βγ subunits. This profound inhibition has the potential to contribute to presynaptic integration and synaptic plasticity. The best-studied mechanism for this inhibition is modulation of the voltage sensitivity of Ca2+ channels. However, Gβγ can also directly inhibit neurotransmitter release at a point distal to Ca2+ entry by binding to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins as well as the assembled SNARE complex. We have shown that the C-terminus of SNAP25 is critical for the ability to inhibit transmitter release. We have devised an assay of Gβγ-SNAP25 interaction using the AlphaScreen, and have screened a small library of compounds that were designed based on known chemotypes which modulate protein-protein interaction (PPI) for both inhibitors and enhancers of this. In this grant, we will optimize both inhibitors and enhancers of Gβγ-SNAP25 interaction In Aim 1, we will carry out medicinal chemical optimization of both classes of PPI compounds to increase their potency, selectivity, and bioavailability. In Aim 2, we will determine the selectivity of the compounds for Gβγ-SNAP25 interaction compared to other Gβγ-interacting proteins. In Aim 3, we will determine the effects of the optimized molecules on neurotransmitter release from hippocampal neurons in culture, and determine whether they synergize with the agonists and antagonists of presynaptic Gi/o-coupled GPCRs. These compounds should allow us to evaluate the importance of Gβγ-SNARE interaction for GPCR modulation of exocytosis. They may work in parallel with agonists or antagonists of presynaptic GPCRs, and thus synergize with presynaptic functions, selectively affecting presynaptic but not postsynaptic actions of neurotransmitters. The investigations we propose address a fundamental problem in neuroscience, the molecular mechanisms by which neurotransmitters control exocytosis. These studies may define targets for the development of new therapies that may shed light on the pathological basis of diseases related to secretion and neuromodulation.