Identification of Novel Broad Spectrum Influenza Virus Inhibitors

描述（由申请人提供）：我们的目标是开发靶向流感进入的小分子抑制剂。在第一阶段，我们确定了几种化合物，选择性地抑制血凝素（HA）介导的病毒进入过程。基于其抗病毒效力、选择性和化学易处理性，选择了三种HA特异性进入抑制剂（MBX 494、MBX 994和MBX 726）用于进一步研究和开发。这些流感抑制剂显示出针对广谱流感病毒株的高效力（IC 50 = 0.3-11 μ M）和有利的选择性指数值（SI>20-200），包括高致病性禽流感（HPAI）A/H5 N1、2009年大流行性流感A/H1N1和奥司他韦耐药性A/H1N1毒株。这些抑制剂还显示出与神经氨酸酶抑制剂奥司他韦的协同作用。MBX 494、MBX 994和MBX 726以组特异性方式抑制组1 HA的H1和H5亚型，但不抑制组2 HA的H3或H7亚型。初步的作用机制（MOA）研究表明，化合物在感染早期起作用，并抑制HA介导的病毒-细胞膜融合过程。抑制剂未能阻断具有组2 HA、VSV和LASV的流感病毒，进一步表明它们不作用于细胞因子（例如，内体pH或唾液酸残基或病毒/细胞膜）。MBX 494、MBX 994和MBX 726分别具有氨基烷基酚醚、氨基乙酰胺磺酰胺和简单磺酰胺骨架。初步的化学优化已经产生了新的活性类似物，建立了这些化学支架优化的适用性。在第二阶段，我们将设计，合成和评估类似物作为HA特异性抑制剂。抑制剂设计将由效力、靶标选择性、流感谱、与奥司他韦的协同活性和最小细胞毒性驱动。将进一步评价最有希望的先导抑制剂的有利的（i）体外ADME性质，包括稳定性，以及（ii）体内药代动力学（PK）和药效学（PD）性质，（iii）毒性，和（iv）在动物模型中的功效，以选择最终的先导抑制剂。 临床前候选人。我们有五个具体目标。在目标1中，我们将设计和合成命中系列的类似物，以建立SAR并提高效力。我们还将使用基于结构的药物设计（SBDD）来增加抗流感效力（IC 50 <0.1 μ M）并最小化细胞毒性（CC 50>100 μ M）。在目标2中，我们将在体外评估类似物的效力和细胞毒性，以产生10-20个优先的先导化合物。在目标3中，我们将通过评估体外“药物样”ADME特性来进一步优先考虑抑制剂。在目的4中，我们将分析抗流感活性的MOA。在目标5中，我们将在小鼠模型中验证优先的先导抑制剂的疗效和毒性，以确定最佳的临床前开发候选药物。目的1、2、3和5构成了临床前候选药物优化过程，因为它们共同提供了结构和活性、体外ADME和体内安全性和有效性之间的关系。了解新合成的类似物的MOA将确保类似物保持所需的机制，而不会获得额外的或不同的非特异性机制。

DESCRIPTION (provided by applicant): Our goal is to develop small molecule inhibitors that target influenza entry. In Phase I, we identified several compounds that selectively inhibit the hemagglutinin (HA) mediated virus entry process. Three HA specific entry inhibitors (MBX494, MBX994, and MBX726) were selected for further research and development based on their antiviral potency, selectivity and chemical tractability. These influenza inhibitors display high potency (IC50 = 0.3-11 ¿M) and favorable selectivity index values (SI>20-200) against a wide spectrum of influenza virus strains, including the high pathogenic avian influenza (HPAI) A/H5N1, 2009 pandemic influenza A/H1N1 and an oseltamivir resistant A/H1N1 strain. The inhibitors also displayed synergy with the neuraminidase inhibitor, oseltamivir. MBX494, MBX994, and MBX726 act in a group-specific manner to inhibit the H1 and H5 subtypes of group 1 HA but not the H3 or H7 subtypes of group 2 HA. Preliminary mechanism of action (MOA) studies suggest that the compounds act early during infection and inhibit the HA-mediated virus-cell membrane fusion process. Failure of the inhibitors to block influenza viruses with group 2 HA, VSV and LASV, further suggests that they do not act on cellular factors (e.g., endosomal pH, or sialic acid residues or viral/cell membrane). MBX494, MBX994, and MBX726 have aminoalkyl phenol ether, aminoacetamide sulfonamide and simple sulfonamide scaffolds respectively. Preliminary chemical optimization has already generated new active analogs, establishing the suitability of these chemical scaffolds for optimization. In Phase II, we will design, synthesize, and evaluate analogs as HA specific inhibitors. Inhibitor design will be driven by potency, target selectivity, influenza spectrum, synergistic activity with oseltamivir, ad minimal cytotoxicity. The most promising lead inhibitors will be evaluated further for favorable (i in vitro ADME properties, including stability, as well as (ii) in vivo pharmacokinetic (PK) and pharmacodynamic (PD) properties, (iii) toxicity, and (iv) efficacy in animal models to select final pre-clinical candidates. We have five specific aims. In Aim 1, we will design and synthesize analogs of the hit series to establish the SAR and improve potency. We will also use structure based drug design (SBDD) to increase the anti-influenza potency (IC50 <0.1 ¿M) and minimize cytotoxicity (CC50 >100 ¿M). In Aim 2, we will evaluate the analogs for potency and cytotoxicity in vitro to generate 10-20 prioritized leads. In Aim 3, we will further prioritize inhibitors by evaluating in vitro "drug-like" ADME properties. In Aim 4, we will analyze the MOA of anti-influenza activity. In Aim 5, we will validate the prioritized lead inhibitors for efficacy and toxcity in murine models to identify the best preclinical development candidate. Aims 1, 2, 3 and 5 constitute the preclinical candidate optimization process since together they provide the relationships between structure and activity, in vitro ADME and in vivo safety and efficacy in vivo. Understanding the MOA of the newly synthesized analogs will ensure that analogs maintain the desired mechanism and do not acquire additional or different non-specific mechanisms.