给老药接一条尾巴：吡嗪酰胺衍生物的 5-烷基改造
下划线 = 中文容易“变味”或丢信息的词句：红线带编号 = 14 条注释卡（悬停看、点击跳转）。这篇走的是另一条路：手上有一个用了七十年的老药骨架，给它接一条越来越长的碳链，看活性怎么变、机制是不是还一样。英文原文附于文末
作者：Michal Juhás、Galina Bouz、Luping Pang、Stephen D. Weeks、Ondřej Jand́ourek、Kristýna Konečná、Pavla Paterová、Pavel Bárta、Marcela Halířová、Marta Kučerová-Chlupáčová、Martin Doležal、Jan Zitko（共 12 人；通讯作者为末位 Jan Zitko，捷克 Hradec Králové）
出处：ACS Omega 2026;11(3):3937–3948
DOI：10.1021/acsomega.5c07249 PMID：41626510
数据：全部合成化合物（37 个）的核磁、红外、元素分析与纯度图谱见原文 Supporting Information
摘要（全译）
由于耐药性的快速出现，结核病（TB）仍是重大的全球健康挑战。尽管抗结核药物的研发取得了可观进展，有效的治疗选择依然有限。本研究报道了一类经 5-烷基与 5-烷酰胺基修饰的吡嗪酰胺（PZA）衍生物的合成与生物学评价，设计目的是通过提高亲脂性、改善对富含脂质的分枝杆菌细胞壁的穿透来增强抗分枝杆菌活性。我们观察到 5-烷基链长度与抗分枝杆菌活性之间存在正相关，庚基取代时活性最强（化合物 4：5-庚基吡嗪-2-甲酰胺，对 Mtb H37Rv 的 MIC = 3.13 μg/mL）。在 C-2 甲酰胺上带苯基取代的 C 系列中，苯环上的各种简单取代基都可被容忍（给电子与吸电子、亲脂与亲水皆可），烷基链长度才是抗分枝杆菌活性的主要决定因素。化合物 23（5-己基-N-(3-三氟甲基苯基)吡嗪-2-甲酰胺）的 MIC = 3.13 μg/mL，选择性指数（SI，对 HepG2 细胞）>25。值得注意的是，所测化合物对耐多药（MDR）结核分枝杆菌菌株表现出显著活性，同时保持较好的选择性谱与低细胞毒性。相反，5-烷酰胺基衍生物（B、D 系列）没有抗分枝杆菌活性。机制研究显示，与 PZA 不同，5-烷基吡嗪酰胺衍生物不被分枝杆菌吡嗪酰胺酶（PncA）水解，提示其作用方式与 PZA 不同。虽然分子建模最初提示烯酰-ACP 还原酶（InhA）可能是 C 系列的潜在靶点，但后续实验验证否定了这一假设；因此，确切的作用机制仍有待阐明。
引言：吡嗪酰胺（PZA）为什么是结核治疗里的特殊角色
〔第 1 段·精译〕结核病主要是一种由结核分枝杆菌（Mtb）引起的肺部感染。TB 感染可以潜伏多年，潜伏期看起来没什么问题；而一旦进入活动期，它既危害患者本人也危害社会，因为 TB 高度传染。按世界卫生组织（WHO）最新数据，TB 仍是重大全球健康威胁：仅 2023 年，就有约 1080 万人发生活动性 TB，125 万人死亡，其中 16 万人合并 HIV 感染；此外，每年还有近 50 万例新发耐多药 TB（MDR-TB）。MDR-TB 属于 WHO 列出的、抗菌研究必须优先应对的病原名单。
〔第 2 段·精译〕目前的 TB 治疗方案很复杂：典型方案是四种抗结核药联合、疗程很长；到了耐药病例，方案更复杂，常常要七种药、超过一年。这种组合方案推高了肝毒性和/或神经毒性风险；而这些缺点又导致依从性差，依从性差又催生更多耐药。当下的抗分枝杆菌研究开始引入新的亚细胞靶点，其中包括与病原体防御宿主免疫系统有关的那些。
〔第 3 段·精译〕吡嗪酰胺（PZA）是一线抗结核药，自 1950 年代起用于临床。即便如此，它的作用机制至今仍未完全搞清楚。除了纯粹的非特异性理论——活性代谢物吡嗪酸（POA）作为质子载体发挥作用——还有若干与 PZA 或其简单衍生物作用相关的具体亚细胞靶点。比较重要的例子包括：5-Cl-PZA 是分枝杆菌脂肪酸合酶 I（FAS I）复合物的抑制剂；而 POA 与 6-Cl-POA 抑制天冬氨酸脱羧酶（PanD）的活性、或促其降解。根据我们通过 SciFinder^n 做的 CAS 数据库检索（2025 年 11 月），已有 962 个 PZA 简单衍生物被作为抗分枝杆菌化合物研究过，其结构改动集中在甲酰胺部分的取代、和/或吡嗪环上的简单取代基。最终只有一个 morphazinamide（又名 morinamide，N-(吗啉甲基)吡嗪-2-甲酰胺）注册上市，但几乎没有临床使用。
〔第 4 段·精译〕我们课题组的方向是做 PZA 衍生物，希望拿到性质更好、可能具有全新作用机制的新活性化合物，以应对耐药。分枝杆菌有一层厚实的亲脂细胞壁，富含分枝菌酸（mycolic acids）——一类烷基链极长的脂肪酸（每个分子 60–90 个碳）。因此，我们决定研究在 PZA 的吡嗪核上接一条烷基链的影响，试图模拟分枝菌酸的结构、增强这些分子进入细菌的能力。此前我们报道过在 PZA 吡嗪核 5 位与 6 位引入 N-烷基氨基取代的衍生物的设计、合成与抗菌评价；在很多同系物里我们观察到，提高衍生物的亲脂性往往有利于其全细胞抗分枝杆菌活性。
读到这儿，你应该能看出 PZA 在结核化疗里有多“别扭”：它是一线药，却是前药——必须靠细菌自己的吡嗪酰胺酶（PncA）转成 POA 才有主要活性；它的活性又强烈依赖环境 pH（酸性条件下才强）；而它的靶点清单至今没吵完：非特异的质子穿梭、FAS I、PanD，各有一批证据，谁也说服不了谁。换句话说，这是抗结核药里少见的“机制、靶点、构效关系三样都不清楚”的一线药。
还有一层现实动机值得点出来：PZA 的敏感性主要由 PncA 的活性决定，而 PncA 失活突变是临床上 PZA 耐药的主要来源之一。那么一个很自然的问题就是——如果我把 PZA 的结构改一改，让它不再需要 PncA 活化，是不是就能绕开这类耐药？这个问题的答案，就是本页后半段（结果 4）要讲的东西。
设计思路：把 PZA 变“更亲脂”
PZA 的计算 logP 是 −1.31，非常亲水。而它要杀的分枝杆菌，外面裹着一层富含分枝菌酸（C60–C90 的超长链脂肪酸）的细胞壁，本质上是脂质屏障。于是一个朴素的设计假设就成立了：把分子做脂一点，也许能更容易穿过这层壁。这个假设好不好，全看你怎么验证它——本书这一页的整个实验设计，就是围绕“怎么验证一条碳链的作用”展开的。
作者做了四个系列，编号 1–37，外加四个游离酸：
A 系列（1–4）：5-烷基吡嗪-2-甲酰胺，烷基从正丁基（Bu）做到正庚基（Hp）。这是最贴近“原始骨架 + 一条尾巴”的系列。
B 系列（9–13）：5-烷酰胺基吡嗪-2-甲酰胺，即在烷链和吡嗪环之间插入一个酰胺键。
C 系列（14–32）：在 A 系列基础上，把 C-2 的甲酰胺换成 N-苯基甲酰胺（苯胺化物），苯环上再挂 H、2-Cl、3-CF3、4-CH3、4-OH 五种简单取代基。
D 系列（33–37）：B 系列的苯胺化物，即“间隔酰胺 + 苯环”双改。
游离酸（5–8）：A 系列 1–4 的羧酸形式，用来对照“酰胺到底重不重要”。
这套设计的巧妙处在于它把两个变量拆开了：A vs B 拆的是“链直接接环上、还是中间插一个酰胺”；A vs C 拆的是“C-2 接不接苯环”；而每个系列内部，烷基从 3 个碳加到 7 个碳，是一组标准的同系物梯度（Pr/Bu/Pe/Hx/Hp，即丙/丁/戊/己/庚）。你以后自己做构效关系（SAR），也应该先问一句：我这一组化合物，到底只变了一个变量，还是同时变了三个？
合成路线不复杂，但有两步值得注意。第一步是 Minisci 自由基烷基化：在水里加热，用银盐/过硫酸铵体系产生烷基自由基，把烷基直接接到吡嗪-2-甲腈的 5 位。有意思的细节是，烷基自由基的来源是“比目标烷基多一个碳的烷酸”——自由基脱羧时丢掉一个碳。收率中等（18–48%），产物是黏稠的无色到浅黄色液体。第二步是严格控 pH = 9 的部分水解：过氧化氢 + 氢氧化钠把腈水解成甲酰胺（1–4），收率 82–92%；再彻底碱解就得到羧酸（5–8），收率 75–91%。C 系列则由 5–8 经 CDI 偶联相应的苯胺得到，收率 20–62%；B、D 系列则从 5-Cl-PZA 出发，先用微波（140 °C、密闭瓶、约 7–8 bar）把氯换成氨基，再酰化。
这里要留一个心眼，也是这篇文章后面所有解释的软肋：logP 是用 ChemDraw 算出来的，不是实测的。计算 logP 在预测趋势上还行，但同系物每加一个碳，logP 大约涨 0.4，而分子的体积、形状、构象自由度也在同时变。所以当后面说“活性与 logP 相关”时，你没法分清到底是“更脂”在起作用，还是“更长/更大”在起作用。这不是这篇独有的问题，而是所有“加碳链”研究的通病——记住这一点，你读同类文章时会立刻多一个判断维度。
结果 1：5-烷基系列——链越长越强
先看 A 系列，也就是最“干净”的那一组：吡嗪环 5 位直接挂烷基，C-2 保持原来的甲酰胺。抗 Mtb H37Rv 的 MIC 依次是：丁基 50、戊基 12.5、己基 6.25、庚基 3.13 μg/mL。四个碳、四个数据点，每加一个碳活性大约翻倍——这是教科书式的同系物梯度。
与之同步变化的是计算 logP：0.71 → 1.13 → 1.55 → 1.97。作者据此给了一个经验阈值：A 系列要 logP > 1.1 才有明显活性；而到 C 系列（带苯环）这个门槛抬到了 logP > 3.5。换句话说，这个骨架对“够不够脂”有一个硬门槛，苯环把分子整体的脂性抬上去，门槛也跟着抬。
| 系列 | 编号 | 烷基 | logP | Mtb H37Rv MIC，μg/mL（μM） | HepG2 IC50（μM） | SI |
|---|---|---|---|---|---|---|
| A | 1 | 丁基 | 0.71 | 50 | n.d. | — |
| A | 2 | 戊基 | 1.13 | 12.5（64.7） | >1000 | >15.5 |
| A | 3 | 己基 | 1.55 | 6.25（30.2） | >250 | >8.3 |
| A | 4 | 庚基 | 1.97 | 3.13（14.1） | >50 | >3.5 |
| 游离酸 | 5 / 6 / 7 | 丁 / 戊 / 己 | 1.37 / 1.78 / 2.20 | >100 / >100 / >100 | n.d. | — |
| 游离酸 | 8 | 庚基 | 2.62 | 25 | n.d. | — |
| B | 9 / 10 | 丙 / 丁 | −0.62 / −0.20 | >100 / >100 | n.d. | — |
| B | 11 | 戊基 | 0.22 | 50 | n.d. | — |
| B | 12 / 13 | 己 / 庚 | 0.64 / 1.05 | >100 / >100 | n.d. | — |
| 对照 | PZA | — | −1.31 | >100 | n.d. | — |
| 对照 | INH | — | −0.64 | 0.2 | n.d. | — |
表 1 A、B 系列与游离酸的活性（数据自原文表 1；MIC 以 μg/mL 为主、括号内为 μM；IC50 仅对 MIC < 25 μg/mL 的化合物测定，“n.d.” 表示未测；SI = IC50(μM)/MIC(μM)）
三行对照特别值得看。第一，游离酸 5–7 全部失活，只有庚基的 8 号有 25 μg/mL——作者认为这可能是非特异效应（长链的表面活性），并明确说 25 就是本文设定的“有活性”门槛。第二，B 系列几乎全灭（只有戊基的 11 号到 50），作者给了两个解释：一是插一个酰胺键会把亲脂性拉回去（13 号的 logP = 1.05，才勉强追上 2 号的 1.13），二是酰胺键可能在化学或酶学上被水解，变成活性很弱的 5-氨基吡嗪酸（MIC = 0.8 mM）。第三，PZA 自己在他们的测定里 MIC > 100 μg/mL，而 INH 是 0.2——这个“PZA 看起来无效”的结果后面会变成一个必须解释的问题。
还有一个容易被忽略的细节：化合物 1（丁基）此前有人报过 MIC = 25 μg/mL（对 H37Rv），本文测到 50；对 M. avium 和 M. kansasii 两篇都是 >100，一致。同一个化合物、不同实验室、差两倍——MIC 这种东西，跨实验室比较时差 2–4 倍是常态，别拿小数点后一位吵架。
结果 2：C-2 苯环取代的容忍度
C 系列是把 C-2 的甲酰胺换成 N-苯基甲酰胺，再在苯环上挂五种取代基（H、2-Cl、3-CF3、4-CH3、4-OH），每种配 3–4 个烷基长度，共 19 个化合物。活性矩阵如下：
| 苯环取代基 | 丁基（C4） | 戊基（C5） | 己基（C6） | 庚基（C7） |
|---|---|---|---|---|
| H（14–17） | 25 | 25 | 6.25 | 6.25 |
| 2-Cl（18–21） | 100 | >100 | 12.5 | 6.25 |
| 3-CF3（22–25） | 25 | 3.13 | 12.5 | 6.25 |
| 4-CH3（26–28） | 25 | 6.25 | —（未合成） | 25 |
| 4-OH（29–32） | 100 | 6.25 | 6.25 | 3.13 |
表 2 C 系列对 Mtb H37Rv 的 MIC（μg/mL）矩阵（数据自原文表 1；“—”表示该组合未合成。加粗为全文最强的两个数）
读这张表，你会先看到两件事。第一，苯环上的取代基几乎随便换：给电子的（CH3、OH）行，吸电子的（Cl、CF3）也行，亲水的（OH）行，亲脂的（CF3、Cl）也行。第二，决定活性的是烷基链长度：每一行里，活性最好的基本都出现在 6–7 个碳。作者把这一点总结成“烷基链长度是抗分枝杆菌活性的主要决定因素”，这也是摘要里那句“不同简单取代基都可被容忍”的来历。
但“宽容”不等于“无差别”，表里有两个例外值得你盯住：3-CF3 这一行最强的不是庚基，而是戊基（23 号，3.13 μg/mL，SI 25.7），到己基反而掉回 12.5；4-CH3 这一行庚基（28 号）又掉到 25，比戊基（27 号，6.25）差。也就是说，“链越长越强”是趋势，不是定律——到了 6–7 个碳，链长、体积、溶解度、蛋白结合开始互相拉扯，最优值会来回摆。这类“趋势里的小反转”才是构效关系里真正有信息量的部分，它们往往提示你：这个位置不是简单地“越深越好”，而是有一个形状匹配的口袋。
顺便指出原文的一处数字矛盾，你读的时候别被绕进去：摘要说化合物 23 是“5-己基-N-(3-三氟甲基苯基)吡嗪-2-甲酰胺”，但表 1 里 23 号是戊基（MIC 3.13、SI 25.7 与摘要一致，只有烷基标签对不上；己基的 3-CF3 是 24 号，MIC 12.5）。以表为准，摘要这里大概率是笔误。这种错误不影响结论，但说明一件事：摘要里的数字和标签，也要回到正文表里去核对。
再往下，作者做了两组“跟自己的旧工作比”的图（原文图 3、图 4）：同样是 5 位取代，直接接烷基（本文 A 系列）在所有链长上都优于插入酰胺间隔基的（B 系列），也优于他们之前报道的 N-烷基氨基系列；在苯胺化物里，5-烷基（C 系列）保留了活性，5-烷酰胺基（D 系列）则完全没活性。结论可以压缩成一句话：烷基要直接贴在环上，中间插任何东西（酰胺、氨基）都会掉活性。这句话背后其实是骨架层面的信息——吡嗪环 5 位是一个“贴得住”的疏水口袋，任何延长链与环之间距离的改动，都会让链端离开那个口袋。
结果 3：对耐药株有效、细胞毒性低
活性最好的分子，必须回答一个临床问题：对耐药菌还有效吗？作者挑了两个代表——A 系列的 4 号（庚基 PZA，代号 HpPZA，因为它本身就是“PZA 加一条庚基尾巴”）和 C 系列的 24 号（己基-3-CF3-苯胺化物，选它是因为广谱活性最好）——去测两株 MDR 临床分离株（IZAK、MATI，均对链霉素、异烟肼、利福平、吡嗪酰胺耐药）。
| 菌株 | 4（HpPZA） | 24（Hx-3-CF3-苯胺化物） | INH | CIP | EMB |
|---|---|---|---|---|---|
| Mtb H37Rv（参考株） | ≤1.56 | 12.5 | 0.39 | 0.2 | 0.39 |
| Mtb IZAK（MDR） | 6.25 | 25 | 12.5 R | 0.2 | 1.56 |
| Mtb MATI（MDR） | ≤1.56 | 6.25 | 12.5 R | 0.1 | 1.56 |
表 3 对 MDR 临床分离株的活性（MIC，μg/mL）（数据自原文表 2；“R”表示耐药；CIP = 环丙沙星，EMB = 乙胺丁醇）
两个数字要记住：4 号对两株 MDR 的 MIC 是 6.25 和 ≤1.56，其中 ≤1.56 已经和一线药乙胺丁醇（EMB，1.56）持平；而 24 号是 25 和 6.25，差一档。这里的关键不在“谁更强”，而在“耐药株还敏感”这件事本身：这两株菌对 PZA 也耐药（大概率是 pncA 失活），可 PZA 的 5-庚基衍生物照样有效——这就给了“我的分子不依赖 PncA”一个强烈的暗示。这条线索在下一页（结果 4）被直接验证。
细胞毒性方面：对 HepG2 的 IC50 普遍 >50 到 >1000 μM，最好的是 23 号（IC50 238.3 μM，MIC 9.3 μM，SI 25.7）和 2 号（IC50 >1000 μM，MIC 64.7 μM，SI >15.5）。选择性指数 25 意味着“杀死细菌所需的浓度，比伤到肝细胞低 25 倍”——这个窗口对先导化合物来说算体面，但离成药还远：SI 只是在单一细胞系上的粗筛，真正的肝毒性要看动物、看长期给药、看代谢产物。
另一组数据是“选择性”的旁证：C 系列化合物对 4 株革兰阳性菌、4 株革兰阴性菌、8 株真菌都没有活性（在溶解度允许的最高浓度下，多数到 500 μM）。对非结核分枝杆菌也基本无效，只有零星几个：11、12、34–36 对 M. kansasii 的 MIC = 6.25，23 号对 M. kansasii 12.5，24 号对 M. smegmatis 3.91。“只打结核、不打别人”——这既说明选择性好（不会把肠道菌群一起扫了），也说明它的作用机制很可能是分枝杆菌特有的（比如细胞壁合成或某种独特的代谢通路），而不是通用的毒性机制。
结果 4：不被 PncA 水解 → 机制与 PZA 不同
这一节是全篇最“较真”的部分，作者追了两条线索，一条被证伪，一条被证实。
线索一：InhA。C 系列的结构里含有一个二苯醚类 InhA 抑制剂的药效团（作者拿 triclosan 和它的烷基化衍生物 PT70 叠上去给你看）。InhA 是分枝杆菌脂肪酸合成 II（FAS II）通路里的烯酰-ACP 还原酶，负责合成分枝菌酸，也是异烟肼（INH）的原发靶点——所以这个猜测非常合理。分子对接也“给面子”：预测酰胺羰基氧作为氢键受体，接受 Tyr158 和 NAD+ 核糖 2′-OH 的氢键，烷链接入脂肪酸入口隧道。可是酶学实验一做，所有化合物在 100 μM 内都不抑制 InhA。对接阳性、实验阴性，这个假设当场作废。
线索二：PncA。PZA 是一个前药，它的多个靶点机制（包括抑制 PanD）都要求先由分枝杆菌吡嗪酰胺酶（PncA）把它转成 POA。那么问题来了：5-烷基衍生物是不是也走这条路？作者拿重组的 Mtb-PncA 去孵育 BuPZA、PePZA 和 PePOA，用改良 Wayne 法检测水解产物（产物与 Fe2+ 形成有色络合物，λ = 458 nm）：PZA 被正常水解成 POA，两个 5-烷基甲酰胺却基本不水解。
为了排除“是不是酶没工作”这种可能，作者又做了抑制实验：把 5-烷基吡嗪-2-甲腈（1:100）和 PncA 预孵育。逻辑是——如果这些腈能正确进入 PncA 活性位点，就应该像未取代的吡嗪-2-甲腈（已知的共价抑制剂）那样把酶不可逆地抑制掉。结果是：未取代的腈有效，5-烷基腈完全无效。也就是说，5-烷基取代的分子连活性位点都进不去，遑论被水解。计算建模给出了原因：在 Mtb-PncA 的晶体结构（PDB 3PL1）里，吡嗪环深埋于活性口袋，5 位的氢正对着蛋白壁（具体是 Trp68）——那个位置放不下任何更大的取代基。
于是“机制与 PZA 不同”这句话就有了具体的含义：PZA 依赖 PncA 活化，5-烷基衍生物不依赖。这既是好消息（绕开了临床最常见的 PZA 耐药机制），也是坏消息——因为你必须重新找一个靶点，而作者在这一页结束时也没找到。他们给了一个有根据的猜想：如果分子以非水解形式起效，机制可能类似 5-Cl-PZA（抑制 FAS I 复合物）；但原文自己也承认，“5-Cl-PZA 与 FAS I 的分子层面相互作用从未被描述过”。换句话说，那也是一个黑箱。
讨论：老药骨架的第二次生命
把全文的结论收拢，其实就三句话：把烷基直接接到吡嗪环 5 位、链加到 6–7 个碳，抗结核活性最好；中间插酰胺间隔基就失效；苯环上换什么取代基都能容忍，链长才是主因。外加强度上的两个亮点：4 号对 MDR 菌株的活性与乙胺丁醇相当；23 号的选择性指数 25.7，肝细胞毒性低。
但讨论里最诚实的一段，是关于“为什么有效”的。庚基这个长度很关键：它让游离酸 8 号也出现了 25 μg/mL 的弱活性，而短链的酸全部失活。作者对此的解释是——长链增强了细胞壁穿透，让基于“质子穿梭 + 胞质酸化”的非特异机制更有效。这跟 PZA 的经典非特异理论是接得上的：POA 是弱酸，能带着质子穿过膜、把胞质酸化；加上一条长脂链，它更容易挤进富含脂质的细胞壁。如果这个解释成立，那么这一整类分子的活性可能根本不需要一个“漂亮的靶点”，而是物理化学层面的膜效应——这也解释了为什么苯环上换什么取代基都行：只要脂性够、链够长、能进去，就能抑菌。
这个解释的好处是自洽，坏处是很难证伪，也难指导优化（你没法“设计”一个更特异的膜效应）。所以作者提出的下一步是往 6 位做：他们的计算显示，PncA 的小口袋容不下 5 位取代，但 6 位在催化位点做小幅构象调整的情况下可能被容忍——这既是一个新的化学空间，也是一种“让分子重新变成 PncA 底物”的可能性。注意这里作者用的是“might be tolerated”，是假说，不是结论。
局限也得摆出来，因为有些是结构性的：第一，机制未明，这是最大的洞；第二，logP 全是计算值，没有实测的脂水分配系数，也没有溶解度数据，而这类长链化合物的溶解度往往是真问题；第三，细胞毒只测了一个 HepG2，且只测了 MIC < 25 的化合物；第四，活性测定的 pH 对 PZA 不公平——他们用 pH 6.6 的 7H9，PZA 自己测出来 >100 μg/mL，却在文中拿文献里“酸性 pH 下 6.25–12.5 μg/mL”的 PZA 值来对比，说自己的化合物“与 PZA 相当”。严格说，这个比较应该在同一 pH 下做，或者干脆只跟 INH 比。读到这里你该有的反应是：“与 PZA 相当”这句话要打问号，“比 INH 差一个数量级”这句话是真的。
这篇在轨迹里的位置
六篇按时间排：2021 年 IJMS 做人源 ProRS 抑制剂 → 2022 年 Communications Biology 做 LeuRS 机制 → 2022 年 IJMS 做 MshC 结构 → 2023 年 STTT 写 CLK 家族综述 → 2026 年 JBC 做脓肿分枝杆菌 MshC 抑制剂（上一页）→ 同年这篇 ACS Omega。前四篇是“机制 + 结构 + 综述”的积累，后两篇同年落地，恰好代表抗菌药物研发的两条经典路径。
跟上一页对照着看，这两条路的差别就出来了。05 是靶点驱动：先证明基因必需（CRISPRi + 斑马鱼），再筛分子（虚拟筛选），再用抗性突变把结合位点钉在催化口袋（D87/W227），最后测组合用药。06 是化合物驱动：手上已经有一个用了七十年的骨架，改它的烷基链、改它的苯环，用 MIC 读数当指南针，再用机制实验（InhA、PncA）去排除可能性。05 的风险是“筛出来的分子进不了细菌”，06 的风险是“分子有效但不知道为什么有效”——你看，两篇文章的局限恰好互补。
而两条路最后撞在同一堵墙上：分子能不能进细菌、能不能穿过那层富含分枝菌酸的细胞壁。05 里三个命中分子都是亲脂性的（激酶抑制剂在 DrugBank 里普遍脂性偏高，这也是它们被筛出来的原因之一），其中 SB-224289 水溶性差到加不进鱼缸；06 里整篇文章就是在调亲脂性，长链带来活性，也带来溶解度的隐忧。做抗菌药物化学，绕不开这个物理化学参数——它既是钥匙，也是天花板。
还有一件小事：这篇有 12 位作者，横跨有机合成、酶学、临床微生物和细胞毒评价，是典型的“化学 + 生物学”合作论文；庞露苹排在第三位，夹在化学作者与生物学作者之间。你以后看作者列表，可以顺着这个位置猜分工——不过更可靠的办法还是翻 CRediT。
给刚进组的你
一、同系物梯度是最便宜的信息。不要一上来就设计十个花哨的取代基。先做一个系列、四个碳数（比如丁/戊/己/庚），看 MIC 怎么走。如果四个点连成一条单调的线，你就知道这个位置“吃长度”；如果中间有个拐点，那个拐点本身就是故事。这篇文章的 A 系列只用了 4 个化合物，就撑起了半篇的结论。
二、设计对照时，一次只动一个变量。A 系列告诉你“链长”的作用，B 系列告诉你“插一个酰胺”的代价，C 系列告诉你“加一个苯环”的代价，游离酸告诉你“酰胺本身”重不重要。四个系列拼起来，才是一个完整的构效关系。你自己设计化合物时，先在纸上画一张“变量表”，确保每一行只差一个东西。
三、MIC 的单位和条件会骗你。μg/mL 与 μM 差着一个分子量；pH 6.6 与 pH 5.5 能让 PZA 的 MIC 差十倍；接种量、培养基、判读方法（浊度 vs 刃天青 vs 菌落计数）都能改结果。这篇里 PZA 自己测出 >100 μg/mL，就是一个活教材：一个“公认有效”的药，在你的实验体系里可能看起来无效，先怀疑体系，别急着怀疑药。
四、阴性结果要写，而且要写清楚。InhA 那一段，作者大可以删掉（没人知道他们试过），但他们写了：对接阳性、酶学阴性、假设作废。这一段的学术价值不比阳性结果低——它替后来的人省了几个月。你以后做课题，遇到“漂亮的假设被自己的数据打脸”，先别沮丧，那是你文章里最可信的一段。
五、老药改造的“合法性”来自安全性，不来自活性。PZA 的骨架之所以值得改，是因为它在人体里已经用了七十年，代谢、毒性、制剂都清楚；你改出来的分子哪怕活性只跟它持平，只要机制不同、能绕开耐药，就有临床价值。反过来，一个全新骨架哪怕活性高十倍，也要从头做安全性——这就是为什么全世界的抗结核药物化学家都在改老药。
▲ 本页翻译难点（对照学习用）
附录 · 英文原文
Targeting Mycobacterium tuberculosis: The Role of Alkyl Substitution in Pyrazinamide Derivatives
Abstract
Tuberculosis (TB) remains a significant global health challenge due to the rapid emergence of drug resistance. Despite substantial progress in anti-TB drug development, effective treatment options are limited. In this study, we report the synthesis and biological evaluation of pyrazinamide (PZA) derivatives with 5-alkyl and 5-alkanamido modifications, designed to enhance antimycobacterial activity by increasing lipophilicity and improving penetration of the lipid-rich mycobacterial cell wall. A positive correlation between the length of the 5-alkyl chain and antimycobacterial activity was observed, with maximal potency achieved with the heptyl substituent (4: 5-heptylpyrazine-2-carboxamide, MIC_M. tuberculosis H37Rv = 3.13 μg/mL). In series C with phenyl substitution on the C-2 carboxamide, different simple substituents were tolerated on the benzene ring (both electron-donating and electron-withdrawing, both lipophilic and hydrophilic), and the length of the alkyl chain was the main determinant of the antimycobacterial activity. Compound 23 (5-hexyl-N-(3-trifluoromethylphenyl)pyrazine-2-carboxamide) exerted MIC = 3.13 μg/mL and selectivity index (SI, compared to HepG2 cells) >25. Notably, the tested compounds exhibited significant activity against multidrug-resistant (MDR) Mycobacterium tuberculosis strains while maintaining favorable selectivity profiles and low cytotoxicity. In contrast, 5-alkanamido derivatives (series B and D) were devoid of antimycobacterial activity. Mechanistic investigations revealed that unlike PZA, the 5-alkyl pyrazinamide derivatives are not hydrolyzed by mycobacterial pyrazinamidase (PncA), indicating a distinct mode of action. While molecular modeling initially suggested enoyl-ACP reductase (InhA) as a potential target of series C, subsequent experimental validation disproved this hypothesis; thus, the precise mechanism of action remains to be elucidated.
Introduction and Design Rationale
Tuberculosis (TB) is a lung infection predominantly caused by Mycobacterium tuberculosis (Mtb). TB infection can stay dormant for years, and the latent infection poses no apparent issues, while the active state of the disease endangers both affected individuals and society, as TB is highly contagious. Based on the most recent data from the World Health Organization (WHO), TB remains a significant global health threat. In 2023 alone, around 10.8 million people developed active TB, and 1.25 million people died from the infection, including 160,000 people with HIV coinfection. In addition, nearly half a million new cases of multidrug-resistant TB (MDR-TB) occur annually. MDR-TB is on the WHO list of priority pathogens, which should be addressed by antimicrobial research.
The current treatment regimen for TB is complex, typically consisting of a combination of four anti-TB drugs for an extended period of time. In drug-resistant cases, the regimen is, however, much more complicated, often combining up to seven drugs for a duration exceeding a year. This combinatorial treatment regimen increases the risk of developing hepatotoxicity and/or neurotoxicity. The negatives of the TB treatment regimen result in low adherence and subsequently an increased emergence of resistance. The current antimycobacterial research employs new subcellular targets, including those associated with the pathogen’s defense against the host’s immune system.
Pyrazinamide (PZA), a first-line antitubercular drug, has been in clinical practice since the 1950s. Despite this, its mechanism of action is still not fully understood. Next to purely nonspecific theories of action where the active metabolite pyrazinoic acid (POA) works as a protonophore,, there are several specific subcellular targets associated with the action of PZA or its simple derivatives. As significant examples, 5-Cl-PZA is an inhibitor of mycobacterial Fatty Acid Synthase I complex,, while POA and 6-Cl-POA inhibit the activity or trigger the degradation of mycobacterial aspartate decarboxylase (PanD).− According to our CAS database search via SciFindern (November 2025, see Supporting Information, Section 1.7 for the query details), 962 simple derivatives of PZA were studied as antimycobacterial compounds, with structural changes focusing on substitution of the carboxamidic moiety and/or simple substituents on the pyrazine ring. Only morphazinamide (aka morinamide, N-(morpholinomethyl)pyrazine-2-carboxamide) was registered but remained with little to no clinical usage.
In our research, we focus on developing derivatives of PZA to achieve novel active compounds with improved properties and potentially a new mechanism of action, addressing resistance. Mycobacteria possess a thick lipophilic cell wall rich in mycolic acids, which are fatty acids with extremely long alkyl chains (60–90 carbon atoms per molecule). Hence, we decided to investigate the influence of attaching an alkyl chain to the pyrazine core of PZA as an attempt to resemble the mycolic acid structure and enhance the penetration of such molecules into mycobacteria. Previously, we reported on the design, synthesis, and antimicrobial evaluation of derivatives with N-alkylamino substitution in positions 5 and 6 of the pyrazine core of PZA.− In many of these homologues, we observed that increasing the lipophilicity of the derivatives often favors their whole-cell antimycobacterial activity.
In this paper, we report on the continuation of our efforts in preparing 5-alkyl- (series A) and 5-alkanamidopyrazine-2-carboxamides (series B) and their corresponding N-phenyl derivatives (series C and D, respectively). For proper evaluation of the structure–activity relationships, we also tested the corresponding free carboxylic acids of series A. For structures, refer to. The terminal alkyl chain ranged from n-propyl to n-heptyl (in the paper abbreviated as Pr, Bu, Pe, Hx, and Hp). All prepared compounds were evaluated in vitro for their antimycobacterial activity against M. tuberculosis H37Rv, nontuberculous mycobacteria M. kansasii and M. avium, and Mycolicibacterium smegmatis and M. aurum (commonly applied surrogate model organisms in antimycobacterial research). Compounds that showed in vitro activity (here defined as MIC <25 μg/mL against Mtb H37Rv) were advanced into cytotoxicity screening in the HepG2 liver cancer cell line. Enoyl reductase InhA was speculated and experimentally tested as a possible mycobacterial molecular target for the title compounds. We also addressed the question of whether the derivatives with unsubstituted carboxamide can be substrates of mycobacterial pyrazinamidase (PncA), whose activity is a determinant of PZA susceptibility in mycobacteria. The obtained results were compared to previous works in order to draw structure–activity relationships (SAR) and recommendations that will direct future synthetic efforts.
[1] Chemical structures of the title compounds: (A) series A5-alkyl derivatives 1–4; (B) series B5-alkanamido derivatives 9–13; (C) series C5-alkyl-N-phenyl derivatives 14–32; and (D) series D5-alkanamido-N-phenyl derivatives 33–37. Compounds 5–8 are the corresponding carboxylic acids of compounds 1–4.
Results and Discussion
Chemistry
5-Alkylpyrazine-2-carbonitriles (BuCN, PeCN, HxCN, and HpCN) were prepared with Minisci radical alkylation of pyrazine-2-carbonitrile according to a previously published procedure., The reaction was performed in water upon heating, the source of the alkyl radical was the corresponding alkanoic acid (one carbon longer than the intended alkyl to be introduced), and the radicals were produced using a silver nitrate/ammonium peroxydisulfate system (, a). The 5-alkylpyrazine-2-carbonitriles were isolated as viscous, colorless to slightly yellow liquids in moderate yields of 18–48%. The carbonitriles underwent partial hydrolysis (, b) using hydrogen peroxide and NaOH under strictly controlled pH = 9. The resulting 5-alkylpyrazine-2-carboxamides (1–4) were isolated as white solids in high yields of 82–92%. Finally, the corresponding 5-alkylpyrazine-2-carboxylic acids (5–8) were prepared with complete basic hydrolysis of the carboxamides (, c) and, after acidification, isolated as low-melting white solids in yields of 75–91%.
[1] Synthesis of Simple Alkylated Derivatives
5-Chloropyrazine-2-carboxamide (5-Cl-PZA,, I) was prepared from commercially available 5-hydroxypyrazinoic acid (5-OH-POA) following the previously published procedure. Briefly, 5-OH-POA was activated with thionyl chloride (SOCl2) with a catalytic amount of N,N-dimethylformamide (DMF) in anhydrous toluene. The solvents were evaporated, and repeated evaporation with toluene was used to remove excess SOCl2. The crude acyl chloride was dissolved in anhydrous dichloromethane (DCM), followed by the addition of aqueous ammonia (, a). 5-Cl-PZA precipitated from the reaction, and after a simple washing with cold water, it was isolated in 77% yield. The anilide of 5-chloropyrazinoic acid (5-Cl-POA-anilide,, II) was prepared analogously, following a previously described procedure, and was isolated in 70% yield.
[2] Synthesis of Alkanamido Derivatives
The substitution of the chlorine for the amino group (, b) was achieved using nucleophilic substitution of the corresponding chloro derivative (I or II) upon treatment with aqueous ammonia (25%) in MeOH (cosolvent) under microwave-assisted heating. The reaction was performed at 140 °C in a closed-vial system. The generated pressure (dependent on the volume of solvents) was approximately 7–8 bar. The closed system setup was convenient for keeping the volatile ammonia in the reaction. Earlier reported attempts to perform the reaction in boiling EtOH in an open atmosphere did not lead to satisfactory yields even with reaction times of several hours. The final alkanamido derivatives of PZA 9–13 and POA-anilide 33–37 were obtained using acylation with aliphatic acyl chlorides in yields of 32–58% and 42–80%, respectively.
Anilides of 5-alkylpyrazine-2-carboxylic acids (14–32) were prepared from 5-alkylpyrazinoic acids (5–8) using a standard 1,1′-carbonyldiimidazole (CDI) coupling reagent () and isolated as solids with a typical yield 20–62%.
[3] Synthesis of Anilides of 5-Alkylpyrazine-2-carboxylic acids (14–32) via CDI-Mediated Coupling
In Vitro Antimycobacterial Activity
All synthesized compounds, including isolated intermediates and the free acids (compounds 5–8), were evaluated for in vitro antimycobacterial activity against Mtb H37Rv, M. kansasii, M. avium, M. smegmatis, and M. aurum using the Microplate Alamar Blue Assay. The antimycobacterial activity results were expressed as minimum inhibitory concentrations (MIC) in μg/mL; isoniazid (INH) and pyrazinamide (PZA) were used as standards. The results for Mtb H37Rv are presented in, along with the determined cytotoxicity for the human hepatocellular carcinoma cell line (HepG2) for the active compounds (defined as MIC < 25 μg/mL). TB treatment regimen is known to carry a significant risk of hepatotoxicity, and therefore HepG2 cell linebesides being the standard cell line for cytotoxicity evaluationis of great value in our case.
[1] Prepared Compounds with Their Calculated Lipophilicity (log P), Antimycobacterial Activity against Mtb H37Rv Expressed as Minimum Inhibitory Concentration (MIC in μg/mL) Converted to μM in Parentheses, HepG2 Cytotoxicity Expressed as Half Maximal Inhibitory Concentration (IC50 in μM), and Selectivity Index (SI)
| Mtb H37Rv | HepG2 | ||||||
|---|---|---|---|---|---|---|---|
| Series | Code | Alkyl | R | Log P | MIC [μg/mL] (μM) | IC50 [μM] | SI |
| A | 1 | Butyl | - | 0.71 | 50 | n.d. | |
| A | 2 | Pentyl | - | 1.13 | 12.5 (64.7) | >1000 | >15.5 |
| A | 3 | Hexyl | - | 1.55 | 6.25 (30.2) | >250 | >8.3 |
| A | 4 | Heptyl | - | 1.97 | 3.13 (14.1) | >50 | >3.5 |
| 5 | Butyl | - | 1.37 | >100 | n.d. | ||
| 6 | Pentyl | - | 1.78 | >100 | n.d. | ||
| 7 | Hexyl | - | 2.2 | >100 | n.d. | ||
| 8 | Heptyl | - | 2.62 | 25 | n.d. | ||
| B | 9 | Propyl | - | –0.62 | >100 | n.d. | |
| B | 10 | Butyl | - | –0.2 | >100 | n.d. | |
| B | 11 | Pentyl | - | 0.22 | 50 | n.d. | |
| B | 12 | Hexyl | - | 0.64 | >100 | n.d. | |
| B | 13 | Heptyl | - | 1.05 | >100 | n.d. | |
| C | 14 | Butyl | H | 2.61 | 25 | n.d. | |
| C | 15 | Pentyl | H | 3.03 | 25 | n.d. | |
| C | 16 | Hexyl | H | 3.45 | 6.25 (22.1) | >50 | >2.3 |
| C | 17 | Heptyl | H | 3.87 | 6.25 (21.0) | >25 | >1.2 |
| C | 18 | Butyl | 2-Cl | 3.17 | 100 | n.d. | |
| C | 19 | Pentyl | 2-Cl | 3.59 | >100 | >100 | |
| C | 20 | Hexyl | 2-Cl | 4.01 | 12.5 (39.3) | 313.6 | 8 |
| C | 21 | Heptyl | 2-Cl | 4.42 | 6.25 (18.8) | >50 | >2.7 |
| C | 22 | Butyl | 3-CF3 | 3.53 | 25 | n.d. | |
| C | 23 | Pentyl | 3-CF3 | 3.95 | 3.13 (9.3) | 238.3 | 25.7 |
| C | 24 | Hexyl | 3-CF3 | 4.37 | 12.5 (35.6) | >100 | >2.8 |
| C | 25 | Heptyl | 3-CF3 | 4.79 | 6.25 (17.1) | >100 | >5.8 |
| C | 26 | Butyl | 4-CH3 | 3.1 | 25 | n.d. | |
| C | 27 | Pentyl | 4-CH3 | 3.52 | 6.25 (22.1) | >100 | >4.5 |
| C | 28 | Heptyl | 4-CH3 | 4.35 | 25 | n.d. | |
| C | 29 | Butyl | 4-OH | 2.22 | 100 | n.d. | |
| C | 30 | Pentyl | 4-OH | 2.64 | 6.25 (21.9) | >100 | >4.6 |
| C | 31 | Hexyl | 4-OH | 3.06 | 6.25 (20.9) | >25 | >1.2 |
| C | 32 | Heptyl | 4-OH | 3.48 | 3.13 (10.0) | >50 | >5.0 |
| D | 33 | Propyl | - | 1.28 | >100 | n.d. | |
| D | 34 | Butyl | - | 1.7 | >100 | n.d. | |
| D | 35 | Pentyl | - | 2.12 | >100 | n.d. | |
| D | 36 | Hexyl | - | 2.54 | >100 | n.d. | |
| D | 37 | Heptyl | - | 2.95 | >100 | n.d. | |
| PZA | - | - | –1.31 | >100 | n.d. | ||
| INH | - | - | –0.64 | 0.2 | n.d. |
Regarding antimycobacterial activity against Mtb H37Rv, the main observation is the superior activity of derivatives with direct alkyl chain attachment (series A: 1–4 and series C: 14–32) over the alkanamido derivatives (series B and D), which were all inactive (mostly MIC >100 μg/mL). The inactivity might be explained, especially in compounds with unsubstituted carboxamide at C2, by the generally lowered lipophilicity (as seen in the predicted log P) of the alkanamido derivatives in comparison to alkyl derivatives. For example, the highest homologue from group B, compound 13 (log P = 1.05), barely leveled the lipophilicity of the alkyl derivative 2 (log P = 1.13), which was the first homologue to have significant activity. Another explanation might relate to the possibility of hydrolysis (chemical or enzymatic) of the amidic bond in the carboxamido linker, resulting in only weakly active 5-aminopyrazinoic acid (MIC = 0.8 mM). For one and only compound 1, the antimycobacterial activity had been published previously, i.e., MIC = 25 μg/mL against Mtb H37Rv and MIC > 200 μg/mL against both M. avium and M. kansasii,, which is in accordance with our findings, i.e., MIC = 50 μg/mL against Mtb H37Rv and MIC > 100 μg/mL against both M. avium and M. kansasii.
5-Alkylpyrazinoic acids (5–8) derived from group A carboxamides were inactive. The MIC = 25 μg/mL (which is the activity threshold set in this paper) of the highest homologue 8 might be attributed to nonspecific effects, possibly connected with the expected surface activity.
For series A and C, the activity is positively correlated with the length of the alkyl chain, with butyl being the least favorable, as seen in compounds 1 (MIC = 50 μg/mL) from series A, and 14 (R = H; MIC = 25 μg/mL), 18 (R = 2-Cl; MIC = 100 μg/mL), 22 (R = 3-CF3; MIC = 25 μg/mL), 26 (R = 4-CH3; MIC = 25 μg/mL), and 29 (R = 4-OH; MIC = 100 μg/mL) from series C. Besides compounds with the butyl side chain, other compounds of series A and C were active. For series A, the optimum log P value appears to be higher than 1.1, while in the C series, the activity is observed in derivatives with log P values above 3.5. In series C, the activity was present in derivatives with both electron-donating and electron-withdrawing substituents, and both lipophilic and hydrophilic substituents on the benzene ring. The most active compounds inhibiting the growth of Mtb H37Rv at MIC = 3.13–6.25 μg/mL showed biological activity comparable to that of the standard PZA (MIC = 6.25–12.5 μg/mL) at acidic pH. However, the compounds did not reach the activity of the other standard isoniazid (MIC = 0.2 μg/mL).
For the purpose of demonstrating the selectivity of the most active compounds, the selectivity index (SI) was calculated as the ratio of micromolar IC50 toward the cell line HepG2 to micromolar MIC against Mtb H37Rv. In the case of two compounds, the SI was higher than 15 (SI > 15.5 for compound 2 and SI = 25.7 for compound 23), which is beneficial from the point of potential drug safety and especially low hepatotoxicity.
None of the title compounds exerted significant antimycobacterial activity (MIC ≤ 25 μg/mL) against any of the other tested (nontuberculous) mycobacterial strains, except for compounds 11, 12, 34–36 that were active against M. kansasii (MIC = 6.25 μg/mL), compound 23 was active against M. kansasii (MIC = 12.5 μg/mL), and compound 24 was active against M. smegmatis (MIC = 3.91 μg/mL). The full results of these screenings are shown in Table S4 in the Supporting Information.
Compounds 4 (HpPZA) and 24 (Hx-3-CF3-anilide) were selected as representatives of active compounds from their structural groups and tested for the inhibition of MDR clinical isolates of Mtb. Compound 4 was selected as the most active derivative in its structural group, while the selection of anilide 24 was motivated by its broad-spectrum antimycobacterial activity (see Supporting Information, Table S4). The MDR strains were resistant to streptomycin, isoniazid, rifampicin, and pyrazinamide; see Supporting Information Table S1 for the susceptibility profiles. Both compounds showed potent activity against MDR clinical isolates of Mtb, with compound 4 overshadowing compound 24, showing excellent activity with MIC ≤1.56 μg/mL comparable to the first-line anti-TB drug ethambutol. The activity was preserved despite resistance to other first-line anti-TB drugs, pointing to a different mechanism of action or drug target. For results, see and Supporting Information Figure S14.
[2] Inhibitory Activities of Compounds 4 and 24 against Virulent Reference Strain Mtb H37Rv and MDR Clinical Isolates of Mtb Expressed as Minimum Inhibitory Concentration (MIC in μg/mL)
| Strain | 4 (HpPZA) | 24 (Hx-3-CF3-anilide) | INH | CIP | EMB |
|---|---|---|---|---|---|
| Mtb H37Rv | ≤1.56 | 12.5 | 0.39 | 0.2 | 0.39 |
| Mtb IZAK | 6.25 | 25 | 12.5 R | 0.2 | 1.56 |
| Mtb MATI | ≤1.56 | 6.25 | 12.5 R | 0.1 | 1.56 |
In Vitro Antibacterial and Antifungal Activity
As a complementary test, final compounds of series C were screened for their antibacterial and antifungal activity. The panel involved four Gram-positive and four Gram-negative bacterial strains and eight fungal strains of clinical significance. None of the tested compounds showed antibacterial or antifungal activity up to the highest tested concentration governed by compounds’ solubility (500 μM for most compounds). For detailed results, see Table S5 and Table S6 in Supporting Information.
Comparison to Previous Work
Results in provide a direct comparison of the antimycobacterial activity against Mtb H37Rv of the title derivatives with unsubstituted carboxamide at C2 (alkyl derivatives of series A and alkanamido derivatives of series B) and the 5-alkylamino derivatives previously published by our group. 5-Alkylpyrazine-2-carboxamides (series A) reported in our current study were of superior activity compared to the other structures at all alkyl chain lengths. Direct attachment of the alkyl substituent was favorable, while inserting an amidic or amino spacer led to a decrease in the activity.
[3] Comparison of Antimycobacterial Activity against Mtb H37Rv of Simple 5-Substituted Pyrazine-2-carboxamides
In an analogical comparison for N-phenylpyrazine-2-carboxamides (), we noticed the significant activity of previously published 5-alkylamino derivatives, the preserved activity of 5-alkyl derivatives (structural type C), and again the lack of activity of 5-alkanamido compounds (structural type D).
[4] Comparison of Antimycobacterial Activity against Mtb H37Rv of 5-Substituted N-Phenylpyrazine-2-carboxamides
Investigation of the Mechanism of Action
Inhibition of Enoyl-ACP Reductase (InhA)
Selected 5-alkyl-N-phenylpyrazine-2-carboxamides (structural type C) were evaluated for their potential to inhibit mycobacterial enoyl-[acyl-carrier-protein] reductase (enoyl-ACP reductase, InhA, UniProt P9WGR1). InhA belongs to the Fatty Acid Synthase II (FAS II) pathway and is responsible for the synthesis of mycolic acids. InhA is a clinically validated antimycobacterial target and is the primary target of the first-line anti-TB drug isoniazid (INH). The rationale for the testing is the presence of the InhA inhibitor pharmacophore () in our title compounds. The pharmacophore is visualized on two confirmed diphenyl ether class inhibitors, triclosan (TCL) and its alkylated derivative PT70 (N.B. the corresponding position of the terminal alkyl relative to the linker).
[2] Enoyl-ACP-reductase inhibitor pharmacophore, depicted on confirmed inhibitors triclosan (TCL) and PT70, projected on the title compounds of series C.
Preliminary docking studies (see Supporting Information, Figures S16–S17) indeed predicted that our title compounds could bind to the active site of InhA in a manner similar to that of the confirmed inhibitors. The carbonyl oxygen of the amidic linker acted as an HBA (accepting an H-bond from Tyr158 and an H-bond from 2′–OH of the ribose of the NAD+ cofactor), and the alkyl chain was oriented into the entry tunnel, which, in a normal situation, is occupied by the alkyl chain of the growing fatty acid intermediate. Nevertheless, in the enzymatic assay, all tested compounds failed to inhibit InhA up to the highest tested concentration of 100 μM. Therefore, InhA is not the molecular target for the title compounds. For results, see Table S7 in the Supporting Information.
Enzymatic Hydrolysis via Mycobacterial Pyrazinamidase Mtb-PncA
The first-line anti-TB drug pyrazinamide (PZA) is a multitarget inhibitor, but the most significant targets/mechanisms of action, including the inhibition of mycobacterial aspartate decarboxylase PanD,, require the conversion of the prodrug PZA to active pyrazinoic acid (POA). The activation happens through the action of mycobacterial pyrazinamidase (PncA). To test whether the same activation pathway is possible for the title 5-alkylcarboxamides of series A, selected compounds (BuPZA, PePZA, and PePOA) were incubated with PncA from M. tuberculosis (Mtb-PncA, UniProt I6XD65). The hydrolysis product 5-alkylpyrazinoic acid was detected via the formation of the colored Fe2+-complex both visually and spectrophotometrically at λ = 458 nm (modified Wayne assay). The extent of hydrolysis was compared to that of the natural substrate PZA. The Mtb-PncA enzyme worked correctly and hydrolyzed PZA to POA, but neither of the two tested 5-alkylcarboxamides (BuPZA and PePZA) was significantly hydrolyzed in comparison to PZA ().
[3] Hydrolysis by pyrazinamidase (PncA). The absorbance corresponds to the concentration of the hydrolytic productcarboxylic acid. Error bars represent 95% CI. Blue denotes significant hydrolysis. Left: Comparison of measured absorbance after incubation without (control) and with Mtb-PncA. POA and PePOA are included as controls to verify the formation and detection of the complex. Right: Normalized percentage increase of absorbance of the sample after incubation with Mtb-PncA.
To further experimentally support our findings, we attempted to test the activity of Mtb-PncA preincubated with the corresponding 5-alkylpyrazinecarbonitriles (1:100). Our rationale was that if the compounds could correctly enter the active site of Mtb-PncA, we should observe lower activity of the enzyme due to irreversible, covalent inhibition as previously described for unsubstituted pyrazine-2-carbonitrile. No inhibition of enzyme activity was detected (). This means that the 5-alkylcarbonitriles, and by extension also the 5-alkylcarboxamides discussed above, were probably unable to correctly interact within the active site of Mtb-PncA.
[4] Inhibition of pyrazinamidase (PncA) with pyrazine-2-carbonitriles. Error bars represent 95% CI. Wwater; Eenzyme (PncA); PCNpyrazine-2-carbonitrile. Grayed columnswith enzyme; white columnscontrols w/o enzyme. Bluefunctional enzyme, significant hydrolysis. Redenzyme inhibited.
Reasons for insufficient hydrolysis of 5-substituted PZA derivatives by Mtb-PncA were rationalized also computationally by analyzing the available crystallographic structure of Mtb-PncA (PDB ID: 3PL1), to which we modeled the product of the enzymatic hydrolysis, POA, from the complex of a related nicotinamidase from Acinetobacter baumannii (PDB ID: 2WTA). We found that the 5-substitution of the pyrazine core is incompatible with the small size and shape of the binding cavity of Mtb-PncA as exemplified in. The pyrazine ring is buried deep inside the Mtb-PncA active site pocket, and the H5 hydrogen is facing toward the wall of the protein (specifically toward Trp68) and is thus unable to accommodate any larger substituent.
[5] Active site of mycobacterial pyrazinamidase Mtb-PncA, PDB ID: 3PL1 (pyrazinoic acid as the hydrolytic product was modeled based on PDB ID: 2WTA).
Final Considerations on the Mechanism of Action
The molecular mechanism of action of the active compounds remains to be determined. In the group of 5-alkylpyrazine-2-carboxamides (series A), we proved that the compounds are not hydrolyzed by Mtb-PncA. We cannot exclude hydrolysis by other less specific means. Still, if we take it as a premise that the carboxamides act in their nonhydrolyzed form, then the mechanism of action could be similar to 5-chloropyrazine-2-carboxamide (5-Cl-PZA), that is, inhibition of the fatty acid synthesis by interfering with Fatty Acid Synthase I (FAS I, UniProt P95029) complex., Unfortunately, the interaction of 5-Cl-PZA with FAS I was never described at the molecular level.
The activity in the 5-alkylpyrazinoic acids was observed only in the highest (heptyl) homologue 8. In this case, the long alkyl chain might enhance penetration through the mycobacterial cell wall, increasing the effectiveness of the nonspecific mechanism of action based on proton shuttling and acidification of the cytoplasm, consistent with the theory of the nonspecific mechanism of action of PZA.
In the series of binuclear 5-alkyl-N-phenylpyrazine-2-carboxamides (series C), we did not confirm InhA as the target. There are several publications on antimycobacterial N-phenylpyrazine-2-carboxamides,,,,,, but none of them proved a specific target, except for the work of Bouz et al., in which the specifically substituted derivatives bearing the 4-aminosalicylic acid fragment inhibited the folate pathway of mycobacteria. However, this is not applicable to our compounds in series C, which had activity with various substituents on the benzene ring.
Conclusions
In this work, we have prepared 37 simple derivatives of pyrazinamide with 5-alkyl (series A and C) or 5-alkanamido (series B and D) substitution on the pyrazine ring. The series contained both derivatives with unsubstituted carboxamidic groups (series A and B), N-phenyl-substituted carboxamide (series C and D), and free carboxylic moiety (5-alkylpyrazinoic acids). Generally, elongating the 5-alkyl chain increased the in vitro antimycobacterial activity, whereas the 5-alkanamido-substituted compounds were inactive. In series C (N-phenylpyrazine-2-carboxamides), different simple substituents were tolerated on the benzene ring (both electron-donating and electron-withdrawing, both lipophilic and hydrophilic), and the length of the alkyl chain was the main determinant of the antimycobacterial activity. Compounds 4 (HpPZA) and 24 (Hx-3-CF3-anilide), selected as active representatives of their structural types, retained their antimycobacterial activity against MDR clinical isolates of M. tuberculosis.
Selected 5-alkyl-N-phenylpyrazine-2-carboxamides (series C) were tested for the inhibition of mycobacterial enoyl-ACP reductase (InhA). Despite their structural similarity to known inhibitors of the diphenyl ether type (triclosan and its alkyl derivatives) and the positive results of molecular docking to InhA, the title compounds showed no significant activity. As the last step, we tested whether the 5-alkylpyrazine-2-carboxamides (series A) could be the substrates of mycobacterial pyrazinamidase Mtb-PncA. We found that our compounds are not hydrolyzed by Mtb-PncA and concluded that the 5-alkyl substituent is incompatible with the small catalytic site of Mtb-PncA. This was also confirmed by the inability to inhibit Mtb-PncA with 5-alkylpyrazine-2-carbonitriles, while unsubstituted pyrazine-carbonitrile is a known covalent inhibitor of Mtb-PncA. Although 5-substitution was incompatible with proper binding to Mtb-PncA, analysis of the Mtb-PncA:POA complex suggested that 6-substitution might be tolerated in the case of minor conformational adjustments in the catalytic site.
To sum up, this study brought several promising 5-alkyl derivatives of the first-line anti-TB drug pyrazinamide. To our knowledge, for the first time, we addressed the issue of whether simple pyrazinamide derivatives can be substrates of Mtb-PncA, which is the enzyme responsible for the activation of pyrazinamide.
Materials and Methods
General Information
All chemicals, reagents, and solvents were of reagent or higher grade purity and were purchased from Merck (Darmstadt, Germany) unless stated otherwise. Solvents for flash chromatography (hexane and ethyl acetate) were purchased from Penta (Prague, Czech Republic). The progress of reactions was checked using Merck Silica 60 F254 TLC plates (Merck) with UV detection at 254 nm wavelength. Microwave-assisted reactions were performed in a CEM Discover microwave reactor with a focused field (CEM Corporation, Matthews, NC, USA) connected to an Explorer 24 autosampler (CEM Corporation). The instrument was running under CEM’s Synergy software to set and monitor the conditions of reactions. An internal infrared sensor monitored the bulk temperature of the reaction mixture. All obtained products were purified with a preparative flash chromatograph CombiFlash Rf (Teledyne Isco Inc., Lincoln, NE, USA). Gradient elution was performed by using a mixture of hexane and ethyl acetate as the mobile phase. Silica gel (0.040–0.063 mm, Merck) was used as the stationary phase.
NMR spectra were recorded using a Varian VNMR S500 (Varian, Palo Alto, CA, USA) at 500 MHz for 1H and 125 MHz for 13C. Chemical shifts were reported in ppm and were referred indirectly to tetramethylsilane via the signal of the solvent (2.49 for 1H and 39.7 for 13C in DMSO-d 6; 7.26 for 1H and 77.0 for 13C in CDCl3). Infrared spectra were recorded with a spectrometer FT-IR Nicolet 6700 (Thermo Scientific, Waltham, MA, USA) using attenuated total reflectance (ATR) methodology on a germanium crystal. Elemental analysis was measured with a Vario MICRO cube Element Analyzer (Elementar Analysensysteme, Hanau, Germany). All values regarding elemental analyses are given as percentages. Melting points were determined in open capillaries on a Stuart SMP30 melting point apparatus (Bibby Scientific Limited, Staffordshire, UK) and are uncorrected. Yields are expressed as percentages of theoretical yields and refer to the isolated products (chromatographically pure) after all purification steps.
The theoretical lipophilicity parameter log P was calculated with ChemDraw 22.2.0 (64-bit version, PerkinElmer Informatics, USA). Molecular modeling was done in MOE 2022.09 (Chemical Computing Group, Montreal, Canada) under the AMBER10:EHT force field.
Chemistry
Full characterization and purity checks of the synthesized compounds can be found in Supporting Information, Section 2.
5-Alkylpyrazine-2-carbonitriles (BuCN, PeCN, HxCN, HpCN)
A 250 mL beaker was charged with a solution of pyrazine-2-carbonitrile (5.0 g, 0.048 mol) in water (150 mL), heated to 80 °C, and silver nitrate (0.82 g, 0.005 mol, 0.1 equiv), and the corresponding carboxylic acid (0.048 mol, 1 equiv) were added. Ammonium peroxydisulfate (12.1 g, 0.053 mol, 1.1 equiv) in water (40 mL) was then added dropwise while stirring, and the temperature was maintained at 75–80 °C for 1 h. After cooling, the mixture was extracted with EtOAc (3 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate; the solvents were evaporated under reduced pressure, and residue was subjected to flash chromatography on silica, using gradient elution with 0–25% EtOAc in hexane.
5-Alkylpyrazine-2-carboxamides (1–4)
In a 250 mL beaker, a mixture of concentrated hydrogen peroxide (30% v/v water solution, 10 equiv, 20 mL) and distilled water (140 mL) was stirred at 50 °C, and its pH was adjusted to pH = 9 using a 10% (w/w) aqueous solution of sodium hydroxide. The corresponding 5-alkylpyrazine-2-carbonitrile (0.02 mol) was added dropwise. The mixture was heated to 55 °C and stirred for 2 h with continuous pH control, maintaining pH = 9 by intermittently adding several drops of 10% NaOH solution. During the reaction, the carboxamide product usually precipitated as a white solid. After the reaction, the mixture was cooled in an ice bath and the precipitate was filtered. The crude product was washed with a small amount of hexane to get rid of the unreacted carbonitrile (liquid), dried, and submitted to flash chromatography on silica, using gradient elution with 0–50% EtOAc in hexane.
5-Alkylpyrazine-2-carboxylic Acids (5–8)
In a 250 mL beaker, 10 mmol of the corresponding 5-alkylpyrazine-2-carboxamide (1–4) was mixed with 80 mL of 10% (m/m) aqueous solution of sodium hydroxide. The mixture was stirred and heated to 50 °C for 1 h. After the reaction, the still-warm reaction mixture was acidified to pH = 3 by diluted hydrochloric acid (10% v/v). The final acids precipitated as solids (6–8) were filtered and washed with cold water to yield products of sufficient purity. The butyl-substituted acid (5) formed as a viscous, water-immiscible liquid, which was extracted using EtOAc. The combined organic layers were dried over anhydrous sodium sulfate and evaporated to yield the final product 5 as a viscous liquid, which solidified after prolonged standing. All acids 5–8 were low-melting solids (mp <70 °C).
5-Alkanamidopyrazine-2-carboxamides (9–13 and 33–37)
The 5-amino intermediates were prepared in bulk following the previously reported procedures, that is 5-aminopyrazine-2-carboxamide (5-NH2-PZA, III) and 5-amino-N-phenyl-pyrazine-2-carboxamide (5-NH2-POA-anilide, IV). Final 5-alkanamidopyrazine-2-carboxamides (9–13: R = H, 33–37: R = Ph) were prepared by the acylation of the corresponding 5-aminopyrazine-2-carboxamide III or IV with aliphatic acyl chlorides as follows. The corresponding 5-aminopyrazine-2-carboxamide III or IV (1.5 mmol) was placed in a round-bottom flask (50 mL), dissolved in 30 mL of dichloromethane (DCM), and 237 mg of pyridine (3 mmol, 2 equiv) was added. The flask was covered with parafilm and placed in an ice bath. In a separate flask, the corresponding alkanoyl chloride (1.8 mmol, 1.2 equiv) was diluted with 10 mL of DCM. This solution was added dropwise into the main flask stirring in the ice bath, covered with parafilm, and stirred for 2 h. Then the flask was removed from the ice bath and stirred for an additional 2 h at room temperature. After the reaction, the reaction mixture was adsorbed on silica under reduced pressure and subjected to flash chromatography on silica, using gradient elution with 0–50% EtOAc in hexane.
Anilides of 5-Alkylpyrazine-2-carboxylic Acids (14–32)
The corresponding 5-alkylpyrazine-2-carboxylic acid (1 mmol) was mixed with 1,1′-carbonyldiimidazole (CDI) in a round-bottom flask (305 mg, 1.9 mmol, 1.9 equiv) in dimethyl sulfoxide (DMSO, 1 mL). The flask with the reactants was covered with foil and left to stir for 15–20 min. During the reaction course, CO2 bubbles were released, and once the effervescence subsided, 1 mmol (1 equiv) of the corresponding substituted aniline was added. The mixture was then stirred for 12 h at room temperature with a TLC check (mobile phase: hexane + EtOAc 2:1 (v/v)). The contents of the flask were diluted with distilled water (10 mL), and the product precipitated as a solid. The product was then extracted into EtOAc (3 × 20 mL), and the combined organic phases were dried over anhydrous sodium sulfate, adsorbed on silica gel under reduced pressure, and subjected to flash chromatography (silica, gradient elution 0–20% EtOAc in hexane).
In Silico Studies
In silico studies were performed in Molecular Operating Environment (MOE) 2022.09 (Chemical Computing Group Inc., Montreal, QC, Canada) under the Amber10:EHT force field. The standard docking protocol as implemented in the software was used. For the full description of the methodology used, see Supporting Information, Section 1.6.
Biology
In Vitro Antimycobacterial Activity
The microdilution broth method based on the Microplate Alamar Blue Assay (MABA) was used. The initial antimycobacterial assay was performed with fast-growing Mycolicibacterium smegmatis DSM 43465 (ATCC 607) and Mycolicibacterium Aurum DSM 43999 (ATCC 23366), obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Subsequent assays were performed with the reference strains Mycobacterium avium subsp. avium Chester CNCTC My 80/72 (ATCC 15769), Mycobacterium kansasii Hauduroy CNCTC My 235/80 (ATCC 12478), and Mycobacterium tuberculosis H37Rv CNCTC My 331/88 (ATCC 27294), obtained from the Czech National Collection of Type Cultures (CNCTC), National Institute of Public Health (Prague, Czech Republic). The Middlebrook 7H9 broth with a declared pH = 6.6 (Merck), enriched with 0.4% glycerol (Merck) and 10% OADC growth supplement (Himedia, Mumbai, India), was used for cultivation. Tested compounds were dissolved in DMSO and diluted with broth (the final concentration of DMSO did not exceed 2.5% (v/v) and did not affect the growth of mycobacteria). Standards used for activity determination were isoniazid (INH), rifampicin (RIF), and ciprofloxacin (CIP) (Merck). Bacterial viability was assessed after the addition of Alamar Blue (resazurin sodium salt 0.01%), with growth determined by visual inspection of the blue-to-pink color change. The activity was expressed as minimum inhibitory concentration (MIC) in μg/mL. Multidrug-resistant isolates of M. tuberculosis (MDR Mtb) were obtained from the Department of Clinical Microbiology, University Hospital Hradec Králové, Hradec Králové, Czech Republic, and tested by the same procedure. For a full description of the methodology used, see Supporting Information, Section 1.1.
In Vitro Antibacterial Activity
Antibacterial activity evaluation was performed by using a microdilution broth method. Antibacterial evaluation was performed against five reference bacterial strains from the Czech Collection of Microorganisms (CCM, Brno, Czech Republic) (Staphylococcus aureus subsp. aureus CCM 4223 (ATCC 29213), methicillin-resistant Staphylococcus aureus subsp. aureus CCM 4750 (ATCC 43300), Enterococcus faecalis CCM 4224 (ATCC 29212), Escherichia coli CCM 3954 (ATCC 25922), Pseudomonas aeruginosa CCM 3955 (ATCC 27853)), and three clinical isolate strains kindly provided from the Department of Clinical Microbiology, University Hospital and Faculty of Medicine in Hradec Králové, Charles University, Czech Republic (Staphylococcus epidermidis lab. id. 112-2016, Klebsiella pneumoniae lab. id. 64-2016, Serratia marcescens lab. id. 62-2016). Antibacterial activity of the tested compounds was expressed as minimum inhibitory concentration (MIC in μM) after 24 and 48 h of static incubation in a dark and humidified atmosphere at 35 ± 2 °C. Visual inspection was used for MIC endpoint evaluation. The internal quality standards of gentamicin and ciprofloxacin (both from Merck) were involved in the assays. For a full description of the methodology used, see Supporting Information, Section 1.2.
In Vitro Antifungal Activity
Antifungal activity evaluation was performed using a microdilution broth method. Eight fungal strains (four yeasts and four molds) were used for antifungal activity screening, namely: Candida albicans CCM 8320 (ATCC 24433), Candida krusei CCM 8271 (ATCC 6258), Candida parapsilosis CCM 8260 (ATCC 22019), Candida tropicalis CCM 8264 (ATCC 750), Aspergillus fumigatus ATCC 204305, Aspergillus flavus CCM 8363, Lichtheimia corymbifera CCM 8077, and Trichophyton interdigitale CCM 8377 (ATCC 9533). Tested strains were purchased from the Czech Collection of Microorganisms (CCM, Brno, Czech Republic) or the American Type Culture Collection (ATCC, Manassas, VA, USA). Visual inspection was used for MIC endpoint evaluation. The internal quality standards amphotericin B (Merck) and voriconazole (Toronto Research Chemicals, CA) were involved in the assays. For a full description of the methodology used, see Supporting Information, Section 1.3.
In Vitro Cytotoxicity
The human hepatocellular liver carcinoma cell line HepG2 purchased from Health Protection Agency Culture Collections (ECACC, Salisbury, UK) was cultured in EMEM (Minimum Essential Medium Eagle) (Sigma-Aldrich via Merck, Darmstadt, Germany) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 1% l-glutamine solution (Sigma-Aldrich), and nonessential amino acid solution (Sigma-Aldrich) in a humidified atmosphere containing 5% CO2 at 37 °C. The cytotoxicity of the tested compounds was investigated spectrophotometrically at 490 nm (TECAN, Infinite M200, Austria) using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (CellTiter 96, PROMEGA, Fitchburg, WI, USA). A standard toxicological parameter IC50 was calculated with nonlinear regression from a semilogarithmic plot of incubation concentration versus the percentage of absorbance relative to untreated controls using GraphPad Prism 10.1 software (GraphPad Software, San Diego, CA, USA). For a full description of the methodology used, see Supporting Information, Section 1.4.
Investigation of the Mechanism of Action
Preparation of Pyrazinamidase from M. tuberculosis (Mtb-PncA)
The coding sequence of M. tuberculosis pyrazinamidase (Mtb-PncA, Uniprot ID: I6XD65), gene pncA (Rv2043c), GenBank accession NC_000962.3, coordinates 2 288 681–2 289 241 (− strand), was amplified from genomic DNA isolated from M. tuberculosis H37Rv by polymerase chain reaction (PCR). The reaction was performed by using Q5 DNA polymerase (New England Biolabs, Ipswich, MA, USA) with the forward primer 5′-gcgaacagattggtggtggaATGCGGGCGTTGATCATCGTC-3′ and the reverse primer 5′-ttgttagcagaagcttattaGGAGCTGCAAACCAACTCGAC-3′, where the capital letters represent the annealing sequence, while the lowercase letters represent the adaptor sequence complementary to the plasmid. An additional GGA (underlined lowercase in the forward primer) corresponding to the glycine residue was introduced in front of the start codon to facilitate the proteolytic removal of the SUMO tag during protein purification. After separation via agarose gel electrophoresis and gel extraction, the purified Mtb-PncA PCR product was cloned into a linearized pETRUK vector, encoding an N-terminal charge-modified SUMO fusion tag, by In Fusion (Takara, Kusatsu, Japan. The sequence was validated by Sanger sequencing (LGC Genomics, Germany).
The recombinant construct was transformed into Escherichia coli Rosetta 2 (DE3) pLysS and overexpressed by using ZYP-5052 autoinduction media at 18 °C. The bacterial cells were harvested by centrifugation for 10 min at 4 °C at 6000 × g. After removing the medium, the cell pellets were frozen and stored at −80 °C. For cell lysis, the frozen pellets were resuspended in lysis buffer containing 20 mM Hepes-NaOH with pH = 7, NaCl 200 mM, and β-mercaptoethanol (β-ME) 5 mM supplemented with 5 mM MgCl2 and cold-active cryonase (Takara, Kusatsu, Japan). The resuspended cell pellet was lysed on ice by repeating three times a 5 min sonication step at an amplitude of 70%, waiting 30 min between each cycle. The lysate was clarified by centrifugation at 18,000 × g for 45 min at 4 °C. The supernatant was applied onto a 5 mL SP HP column (Cytiva, Marlborough, USA) pre-equilibrated with cation exchange buffer A (20 mM Hepes-NaOH with pH = 7, NaCl 200 mM, β-ME 5 mM). The protein was eluted using a linear gradient of cation exchange buffer B (20 mM Hepes-NaOH pH 7, NaCl 1000 mM, β-ME 5 mM) over 30 column volumes. After examination by SDS-PAGE, the fractions corresponding to the tagged proteins were combined. To remove the SUMO tag, SUMO hydrolase was added (1:1000 mass ratio) to the protein, and the cleavage reaction was then transferred to dialysis tubing and dialyzed overnight at 4 °C in 1 L of buffer (20 mM Bis-tris with pH = 6, 30 mM NaCl, 5 mM β-ME, 10% (v/v) glycerol). The salt and small molecular weight impurities were further removed by an additional dialysis step in fresh dialysis buffer for another 4 h. Then the protein was loaded onto a 5 mL SP HP column (Cytiva, Marlborough, USA) to remove the SUMO tag and SUMO hydrolase, and the flow-through, containing the Mtb-PncA, was applied directly onto a Q HP column (Cytiva, Marlborough, USA) and eluted using a linear gradient of dialysis buffer containing an additional 1000 mM NaCl. Pooled fractions were further purified by size exclusion chromatography in a column equilibrated with 10 mM Tris-HCl pH 7, 100 mM NaCl, and 5 mM β-ME. Fractions containing the protein were then concentrated to 20 mg/mL. Aliquots were then flash frozen in liquid nitrogen and stored at −80 °C.
Enzymatic Hydrolysis with Mycobacterial Pyrazinamidase (Mtb-PncA)
Enzymatic hydrolysis by mycobacterial pyrazinamidase (Mtb-PncA) was tested as previously described with minor adjustments. The method is based on the formation of colored Fe2+ complexes of pyrazine-2-carboxylic acid as the product of enzymatic hydrolysis. In a 96-well microtiter plate, 10 μM Mtb-PncA in 50 mM PBS with pH = 6.5 was mixed with 2 mM of tested compounds dissolved in DMSO (or DMSO as a control). The total volume in the well was 100 μL, and the final concentration of DMSO during incubation was 2%. The plate was incubated at room temperature for 15 min followed by the addition of 10 μL of freshly prepared 20% (NH4)2Fe(SO4)2 (Merck) solution in Milli-Q water. The plate was manually shaken and immediately measured. Pyrazinamide (PZA) was used as a positive control. The results were additionally confirmed visually by observing a dark orange-brown coloration.
For the carbonitrile inhibition assay, 50 μM Mtb-PncA in 50 mM PBS with pH = 6.5 was preincubated on ice with 5 mM of the respective pyrazine-2-carbonitrile in Milli-Q water for 2–3 min. The molar ratio of Mtb-PncA to carbonitrile was 1:100. In the 96-well microtiter plate, 20 μL of the enzyme-carbonitrile preincubated solution was mixed with 2 μL of 100 mM PZA in Milli-Q water (the substrate) and diluted with 50 mM PBS with pH = 6.5 to a final volume of 100 μL and incubated for 15 min at laboratory temperature. The activity was determined spectrophotometrically with visual confirmation. Pyrazine-2-carbonitrile was used as a positive control (known as an inhibitor of Mtb-PncA).
All enzymatic assays were measured on a Spark Multimode Microplate Reader (Tecan Austria GmbH, Grödig, Austria) spectrophotometer at 458 nm (maximum absorbance of the POA–Fe2+ complex). Experiments were run in quadruplicates, and the mean and 95% confidence intervals (CI) were reported. All data processing was done using GraphPad Prism 10.1 (GraphPad Software, LLC).
Supplementary Material
Acknowledgements
The authors extend their gratitude to Dr. Katarina Grabrijan for the help with the InhA enzyme inhibition assays and Dr. Izidor Sosič for providing the InhA enzyme. This work was supported by the project National Institute of Virology and Bacteriology (Programme EXCELES, ID Project No. LX22NPO5103)Funded by the European UnionNext Generation EU. M.J. acknowledges the support from the research project 2200/04/2024-2026 as part of the “Competition for 2024-2026 Postdoctoral Job Positions at the University of Hradec Králové,” at the Faculty of Science, University of Hradec Králové.