Siegel Eric L, Rich Connor, Saravanan Sanchana, Pearson Patrick, Xu Guang, Rich Stephen M
Laboratory of Medical Zoology, Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA.
New England Center of Excellence in Vector-Borne Disease, Amherst, MA 01003, USA.
Microorganisms. 2024 Oct 15;12(10):2064. doi: 10.3390/microorganisms12102064.
's inosine-5'-monophosphate dehydrogenase (IMPDH, GuaB encoded by the gene) is a potential therapeutic target. GuaB is necessary for replication in mammalian hosts but not in standard laboratory culture conditions. Therefore, we cannot test novel GuaB inhibitors against without utilizing mammalian infection models. This study aimed to evaluate modifications to a standard growth medium that may mimic mammalian conditions and induce the requirement of GuaB usage for replication. The effects of two GuaB inhibitors (mycophenolic acid, 6-chloropurine riboside at 125 μM and 250 μM) were assessed against (+) grown in standard Barbour-Stoenner-Kelly-II (BSK-II) medium (6% rabbit serum) and BSK-II modified to 60% concentration rabbit serum (BSK-II/60% serum). BSK-II directly supplemented with adenine, hypoxanthine, and nicotinamide (75 μM each, BSK-II/AHN) was also considered as a comparison group. In standard BSK-II, neither mycophenolic acid nor 6-chloropurine riboside affected growth. Based on an ANOVA, a dose-dependent increase in drug effects was observed in the modified growth conditions (F = 4.471, = 0.001). Considering higher drug concentrations at exponential growth, mycophenolic acid at 250 μM reduced spirochete replication by 48% in BSK-II/60% serum and by 50% in BSK-II/AHN ( < 0.001 each). 6-chloropurine riboside was more effective in both mediums than mycophenolic acid, reducing replication by 64% in BSK-II/60% serum and 65% in BSK-II/AHN ( < 0.001 each). These results demonstrate that modifying BSK-II medium with physiologically relevant levels of mammalian serum supports replication and induces the effects of GuaB inhibitors. This represents the first use of GuaB inhibitors against , building on tests against purified GuaB. The strong effects of 6-chloropurine riboside indicate that can salvage and phosphorylate these purine derivative analogs. Therefore, this type of molecule may be considered for future drug development. Optimization of this culture system will allow for better assessment of novel Borrelia-specific GuaB inhibitor molecules for Lyme disease interventions. The use of GuaB inhibitors as broadcast sprays or feed baits should also be evaluated to reduce spirochete load in competent reservoir hosts.
肌苷-5'-单磷酸脱氢酶（IMPDH，由guaB基因编码）是一个潜在的治疗靶点。guaB对于在哺乳动物宿主体内的复制是必需的，但在标准实验室培养条件下并非如此。因此，我们无法在不利用哺乳动物感染模型的情况下测试新型guaB抑制剂对[病原体名称未明确给出]的作用。本研究旨在评估对标准生长培养基的改良，这种改良可能模拟哺乳动物条件并诱导复制对guaB使用的需求。评估了两种guaB抑制剂（125μM和250μM的霉酚酸、6-氯嘌呤核苷）对在标准巴伯-斯托纳-凯利-II（BSK-II）培养基（6%兔血清）和改良为60%浓度兔血清的BSK-II（BSK-II/60%血清）中生长的[病原体名称未明确给出]（+）的影响。直接添加腺嘌呤、次黄嘌呤和烟酰胺（各75μM，BSK-II/AHN）的BSK-II也被视为一个比较组。在标准BSK-II中，霉酚酸和6-氯嘌呤核苷均未影响[病原体名称未明确给出]的生长。基于方差分析，在改良的生长条件下观察到药物作用呈剂量依赖性增加（F = 4.471，P = 0.001）。考虑到指数生长期的较高药物浓度，250μM的霉酚酸在BSK-II/60%血清中使螺旋体复制减少48%，在BSK-II/AHN中减少50%（各P < 0.001）。6-氯嘌呤核苷在两种培养基中均比霉酚酸更有效，在BSK-II/60%血清中使复制减少64%，在BSK-II/AHN中减少65%（各P < 0.001）。这些结果表明，用生理相关水平的哺乳动物血清改良BSK-II培养基可支持[病原体名称未明确给出]的复制并诱导guaB抑制剂的作用。这代表了首次将guaB抑制剂用于针对[病原体名称未明确给出]，是在对纯化的[病原体名称未明确给出]guaB进行测试的基础上。6-氯嘌呤核苷的强效作用表明[病原体名称未明确给出]可以挽救并磷酸化这些嘌呤衍生物类似物。因此，这类分子可考虑用于未来的药物开发。优化这种培养系统将有助于更好地评估用于莱姆病干预的新型疏螺旋体特异性guaB抑制剂分子。还应评估将guaB抑制剂用作喷洒剂或诱饵饲料以降低易感储存宿主中螺旋体载量的效果。
