Sialic acid analogs against multidrug-resistant gonorrhea

摘要 淋病是全球一个主要的公共卫生问题。全球约有8700万新发淋病病例 每年。2018年，美国报告了583，405例病例，自2018年以来发病率增加了82.6%。 2009年的历史低点。同时感染HIV和淋病可增加HIV传播率5- 折阴道微生物组的生态失调（即，细菌性阴道病（BV））会增加淋病的风险， 艾滋病毒的获得和传播。女性淋病的严重后遗症包括不孕，异位妊娠， 妊娠和慢性盆腔疼痛。淋病奈瑟菌（Ng）对几乎所有抗生素都具有耐药性 在临床上使用。几乎每个大陆都有对头孢曲松和阿奇霉素耐药的报告， 无法治愈的淋病时代开发针对Ng的新疗法是全球公共卫生的优先事项。 淋球菌部署了独特的免疫逃避策略，其中它通过一种免疫抑制剂将其脂寡糖（LOS）封端。 表面LOS唾液酸转移酶（Lst），使用宿主衍生的CMP-Neu 5Ac。LOS唾液酸化使Ng能够逃避 宿主免疫的几个方面，包括补体和阳离子抗菌肽（CAMP）。我们 发现用CMP-壬酮糖酸盐（CMP-NulO），如CMP-军团胺酸（CMP- Leg 5，7Ac 2）和CMP-酮脱氧壬酮糖酸（CMP-Kdn）用这些NulO限制它们的LOS。掺入 NulO转化为Ng LOS使得细菌对补体和CAMP敏感。重要的是，CMP-NULO 显著缩短小鼠中Ng阴道定殖的持续时间和负荷。Cathelicidin（Cathelicidin） CAMP家族）在CMP-NulO的MOA中起关键作用。我们的有效性、安全性和稳定性研究 建立了CMP-Leg 5，7Ac 2和CMP-Kdn作为阴道内分娩的有希望的候选物，以预防Ng 采集在目标1的第1-2年，NRC将为本提案中的所有工作制定CMP-NulO，执行 药物经济学分析和评估放大。工艺开发、质量放行试验、开发 在3-5年内，将对已确定的铅进行效价测定和在模拟生殖道液中的稳定性研究。目的 2将阐明可能影响CMP-NulO安全性有效性的因素。这些包括CMP-NulO对 有利或不利的阴道细菌的生长和活力，BV- 相关病原体和天然存在的抗NulO抗体对CMP-NulO体外功效的影响， 在体内，检查宫颈阴道分泌物的CMP-NulO水解酶活性，并测试 结合含NulO的聚糖以改进CMP-NulO针对Ng的MOA。目标3将制定 将CMP-NulO植入阴道环（IVR）中，在体外和绵羊模型中进行PK研究，并检查 IVRs上阴道微生物群的生物膜形成。目标1-3第1-2年的工作将确定一个牵头项目; 第三至第五年的工作将侧重于进一步的产品开发和国家自主研发扶持活动。目标4将验证 前导CMP-NulO对遗传和地理上不同的Ng分离株的活性。目标5包括 所有目的的监管指导和监督，最终将与FDA举行IND前会议。

ABSTRACT Gonorrhea is a major public health problem globally. About 87 million new cases of gonorrhea occur worldwide annually. In 2018, 583,405 cases were reported in the U.S, an 82.6% increase in disease incidence since the historic low in 2009. Concomitant infection with HIV and gonorrhea can increase rates of HIV transmission 5- fold. Dysbiosis of the vaginal microbiome (i.e., bacterial vaginosis (BV)) increases the risk of gonorrhea and HIV acquisition and transmission. Serious sequelae of gonorrhea in women include infertility, ectopic pregnancy and chronic pelvic pain. Neisseria gonorrhoeae (Ng) has become resistant to almost every antibiotic in clinical use. Reports of resistance to ceftriaxone and azithromycin from almost every continent portends an era of untreatable gonorrhea. Development of novel therapies against Ng is a global public health priority. Gonococci deploy a unique immune evasion strategy wherein it caps its lipooligosaccharide (LOS) by a surface LOS sialyltransferase (Lst) using host-derived CMP-Neu5Ac. LOS sialylation enables Ng to evade several aspects of host immunity, including complement and cationic antimicrobial peptides (CAMPs). We discovered that gonococci fed with CMP-nonulosonates (CMP-NulOs), such as CMP-legionaminic acid (CMP- Leg5,7Ac2) and CMP-ketodeoxynonulosonate (CMP-Kdn) cap their LOS with these NulOs. Incorporation of NulOs into Ng LOS renders bacteria susceptible to complement and CAMPs. Importantly, CMP-NulOs significantly shorten the duration and burden of Ng vaginal colonization in mice. Cathelicidin (a member of the CAMP family) played a key role in the MOA of CMP-NulOs. Our efficacy, safety and stability studies have established CMP-Leg5,7Ac2 and CMP-Kdn as promising candidates for intravaginal delivery to prevent Ng acquisition. In years 1-2 of Aim 1, the NRC will produce CMP-NulOs for all work in this proposal, perform a pharmacoeconomic analysis and assess scale-up. Process development, quality release assays, development of potency assays and stability in simulated genital tract fluids of the identified lead will follow in years 3-5. Aim 2 will elucidate factors that may affect safety efficacy of CMP-NulOs. These include effects of CMP-NulOs on growth and viability of favorable or unfavorable vaginal bacteria, the effects of sialidases elaborated by BV- associated pathogens and naturally-occurring anti-NulO antibodies on the efficacy of CMP-NulOs in vitro and in vivo, examining cervicovaginal secretions for CMP-NulO hydrolase activity and testing the specificity of CAMPs to bind NulO-containing glycans to refine the MOA of CMP-NulO against Ng. Aim 3 will formulate the CMP-NulOs into intravaginal rings (IVRs), perform PK studies in vitro and in a sheep model, and examine biofilm formation by vaginal microbiota on IVRs. Work in years 1-2 of the Aims 1-3 will identify a single lead; works in years 3-5 will focus on further product development and IND-enabling activities. Aim 4 will verify activity of the lead CMP-NulO against genetically and geographically diverse Ng isolates. Aim 5 includes regulatory guidance and oversight for all Aims that will culminate in a pre-IND meeting with the FDA.