使用碳酸酐酶抑製劑治療消化性潰瘍的歷史和基本原理。紀念伊万·普斯卡斯 (Ioan Puşcaş) (1932–2015)
Review Article
The history and rationale of using carbonic anhydrase inhibitors in the treatment of peptic ulcers. In memoriam Ioan Puşcaş (1932–2015)
吉爾吉·M·布扎斯 匈牙利布達佩斯 Ferencváros 健康中心胃腸病科和
克勞迪烏·T·蘇普蘭 Neurofarba Department，Sezione di Scienzie Farmaceutiche，Polo Scientifico，Università degli Studi di Firenze，塞斯托·佛羅倫薩，佛羅倫薩，意大利
Review Article
抽象的
碳酸酐酶 (CA、EC 4.2.1.1) 抑製劑 (CAI) 在 20 世紀 70 年代開始用於治療消化性潰瘍，二十多年來，由 Ioan Puşcaş 領導的一個小組將其用於此目的，假設抑制胃粘膜CA同種型，鹽酸分泌減少。儘管乙酰唑胺和其他磺胺類CAI 確實能有效治療潰瘍，但在胃以外的其他器官中抑制CA 同工酶會導致許多嚴重的副作用，當組胺H2 受體拮抗劑和質子泵抑製劑出現時，這種治療方法就顯得過時了。幾十年後，即 2002 年，人們發現幽門螺桿菌是導致胃潰瘍和癌症的細菌病原體，編碼兩種 CA，一種屬於這些酶的 α 類，另一種屬於這些酶的 β 類。這些酶對於細菌的生命週期及其在胃高酸性環境中的適應至關重要。用乙酰唑胺（一種低納摩爾幽門螺桿菌CAI）等磺胺類藥物抑制這兩種細菌 CA 對病原體是致命的，這解釋了為什麼這些化合物在臨床上作為抗潰瘍藥物是有效的。因此， Ioan Puşcaş提倡的方法治療這種疾病是一個很好的方法，儘管其背後的原理是錯誤的。在這篇綜述中，我們對磺酰胺類 CAI 作為抗潰瘍藥物的歷史進行了概述，以紀念處於這一研究趨勢第一線的科學家。
Abstract
Carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) started to be used in the treatment of peptic ulcers in the 1970s, and for more than two decades, a group led by Ioan Puşcaş used them for this purpose, assuming that by inhibiting the gastric mucosa CA isoforms, hydrochloric acid secretion is decreased. Although acetazolamide and other sulfonamide CAIs are indeed effective in healing ulcers, the inhibition of CA isoforms in other organs than the stomach led to a number of serious side effects which made this treatment obsolete when the histamine H2 receptor antagonists and the proton pump inhibitors became available. Decades later, in 2002, it has been discovered that Helicobacter pylori, the bacterial pathogen responsible for gastric ulcers and cancers, encodes for two CAs, one belonging to the α-class and the other one to the β-class of these enzymes. These enzymes are crucial for the life cycle of the bacterium and its acclimation within the highly acidic environment of the stomach. Inhibition of the two bacterial CAs with sulfonamides such as acetazolamide, a low-nanomolar H. pylori CAI, is lethal for the pathogen, which explains why these compounds were clinically efficient as anti-ulcer drugs. Thus, the approach promoted by Ioan Puşcaş for treating this disease was a good one although the rationale behind it was wrong. In this review, we present a historical overview of the sulfonamide CAIs as anti-ulcer agents, in memoriam of the scientist who was in the first line of this research trend.
介紹
碳酸酐酶（CA）是一種普遍存在的金屬酶，催化二氧化碳可逆地轉化為碳酸氫鹽，在細菌代謝、呼吸、光合作用、腎酸化、胃酸和胰腺碳酸氫鹽分泌以及骨代謝等方面具有多種生物學功能。最重要的。本文在回顧了CA研究的歷史後，總結了羅馬尼亞研究員Ioan Puşcaş對數十年CA故事的貢獻，重點關注胃CA與消化性潰瘍疾病的關係，隨後介紹了細菌CA的主要成就。
Introduction
Carbonic anhydrase (CA) is a ubiquitous metalloenzyme catalysing the reversible conversion of carbon dioxide to bicarbonate and has various biological functions in bacterial metabolism, respiration, photosynthesis, renal acidification, gastric acid and pancreatic bicarbonate secretion as well as bone metabolism, to mention only the most important ones. After a historical overview of CA research, this article will summarize the contribution of Romanian researcher Ioan Puşcaş to the multi-decade CA story, focusing on the relationship between gastric CAs and peptic ulcer disease, followed by a presentation of main achievements of bacterial CAs.
CA的歷史
20 世紀初的生理學家和化學家對了解二氧化碳 (CO 2 ) 在血液中的運輸方式很感興趣引文9 ,引文10 . 實驗的時間表呈現在表格1。許多現代生理學的重要人物都參與其中，這項研究在 1933 年達到頂峰，當時諾曼·厄克特·梅爾德魯姆 (Norman Urquhart Meldrum)和弗朗西斯·約翰·沃斯利·勞頓(Francis John Worsley Roughton)首次從山羊、公牛、兔子、鯨魚和人的紅細胞中製備了這種酶，從而將其作用與血紅蛋白的作用區分開來。引文11、引文13 . CA 一詞是由他們的同事Philip Eggleton創造的，但文章中沒有提到他的名字引文1 . 1961 年，酶命名法制定後，CA 被列入酶委員會 4 級 (EC 4.2.1.1)。直到 1976 年，同工酶 I 和 II 才為人所知，但從那時起，總共有 18 種同工酶被描述，它們屬於不同的遺傳家族（α-、β- 和 γ-CA）。詳細描述了它們的功能差異，並通過倫琴衍射闡明了結構。
History of CA
Physiologists and chemists from the early decades of the 20th century were interested to find out how carbon dioxide (CO2) is transported in bloodCitation9,Citation10. The timeline of the experiments is presented in Table 1. Many important personalities of modern physiology were involved and the research culminated in 1933, when Norman Urquhart Meldrum and Francis John Worsley Roughton first prepared the enzyme from erythrocytes of goats, oxen, rabbits, whales and men, thus differentiating its effect from that of hemoglobinCitation11,Citation13. The term CA was coined by their co-worker, Philip Eggleton, but his name is not mentioned in the articlesCitation1. In 1961, after development of the enzyme nomenclature, CA was listed in Enyzme Commission Class 4 (EC 4.2.1.1). Until 1976, isoenzymes I and II were only known, but since then, a total of 18 isoenzymes have been described which belong to distinct genetic families (alpha-, beta- and gamma-CAs). Their functional differences have been described in detail and the structure was elucidated by Roentgen diffraction.
CA酸分泌理論
幾十年來，胃分泌的鹽酸的形成一直是胃生理學中一個有趣的話題。1934年，紐約西奈山醫院的富蘭克林·霍蘭德（ Franklin Hollander ，1899-1966）提出了兩部分理論。因此，胃液包含壁細胞分泌的酸性溶液和非壁細胞產生的鹼性溶液引文14 . 逆擴散理論屬於Torsten Teorell（1905-1992）（卡羅林斯卡學院，斯德哥爾摩）。氫離子主要由壁細胞分泌，並通過粘膜與血漿中的 NaCl 交換引文15 . 1932年，觀察到所謂的鹼潮（即刺激HCl分泌時血液碳酸氫鹽水平增加），據此，達文波特推測胃CA的存在，隨後於1939年得到證實。1940年，作者提出了酶通過催化CO 2水合併進一步解離為H +和HCO 3 ，是氫離子的主要來源引文12 . 並且，他還首次證明了硫氰酸鹽對CA和胃酸分泌的抑製作用。引文16 . 他的理論沒有解釋使 H +離子逆著胃腔和壁細胞之間巨大的濃度梯度移動的驅動力因此，1946年，作者本人撤回了他的理論引文1 .
The CA theory of acid secretion
The formation of hydrochloric acid secretion by the stomach was an interesting topic in gastric physiology for decades. In 1934, Franklin Hollander (1899–1966) of Mount Sinai Hospital, New York, formulated the two components theory. Accordingly, gastric juice comprises a parietal cell-secreted acid solution and an alkaline one produced by non-parietal cellsCitation14. The retrodiffusion theory belongs to Torsten Teorell (1905–1992) (Karolinska Institute, Stockholm). Hydrogen ions are primarily secreted by the parietal cells and are exchanged with NaCl from plasma through the mucosaCitation15. In 1932, the so-called alkaline tide was observed (i.e. increase of blood bicarbonate level during stimulation of HCl secretion) and based on this, Davenport presumed the existence of gastric CA, which was subsequently demonstrated in 1939. In 1940, the author formulated the CA theory in which by catalysing the hydration of CO2 and further dissociation to H+ and HCO3, the enzyme represents the main source of hydrogen ionsCitation12. And, for the first time, he also demonstrated the inhibition of CA and that of gastric acid secretion by thiocyanateCitation16. His theory did not explain the driving force which moves H+ ions against a huge concentration gradient between the gastric lumen and parietal cells. Thus, in 1946, the author himself withdrew his theoryCitation1.
Ioan Puşcaş還設想了 CA 在壁細胞中的核心作用。在一系列原創實驗中，使用Wilbur-Anderson的電測和比色法（1948年）和Maren的簡化微量法（1960年），他表明胃酸分泌介質（組胺、五肽胃泌素、乙酰膽鹼和鈣離子）激活胃粘膜CA，具體取決於劑量引文17、引文18 . 然而，羅馬尼亞藥理學家Dumitru Dobrescu (1927)於 1966 年在大鼠身上描述了組胺對 CA 的影響引文19並於 1972 年被俄羅斯作者證實引文20 . 1984年，這一現象的有效性在分離的豚鼠泌酸細胞上得到了驗證。所有促分泌劑的活性均被新的 AB Hassle 產品 H149/94 抑制，表明 CA 和質子泵之間存在功能聯繫引文21 . 1999 年， Ioan Puşcaş等人。引文圖22顯示奧美拉唑抑制胃粘膜H + /K + -ATP-酶和CA。最後，在 2011 年，Supran 等人。引文圖23證明了亞納摩爾濃度的單鹵代組胺衍生物和二鹵代組胺衍生物對CA I和II的激活。Ioan Puşcaş還表明H 2組胺受體阻滯劑（西咪替丁和雷尼替丁）、毒蕈鹼拮抗劑哌崙西平、鋅離子和前列腺素可抑制胃粘膜CA。然而，這些現像在壁細胞生理學中的重要性從未被顯示出來。
Ioan Puşcaş also envisaged a central role for CA in the parietal cells. In a series of original experiments, using Wilbur–Anderson’s electrometric and colorimetric method (1948) and Maren’s simplified micromethod (1960), he showed that mediators of gastric acid secretion (histamine, pentagastrin, acetylcholine and calcium ions) activate gastric mucosa CA depending on the doseCitation17,Citation18. The effect of histamine on CA was, however, described in 1966 by Romanian pharmacologist Dumitru Dobrescu (1927) on ratsCitation19 and confirmed in 1972 by Russian authorsCitation20. In 1984, the validity of this phenomenon was verified on isolated guinea pig oxyntic cells. The activity of all the secretagogues was inhibited by the new AB Hassle product H149/94, suggesting a functional link between CA and the proton pumpCitation21. In 1999, Ioan Puşcaş et al.Citation22 showed that omeprazole inhibits both gastric mucosa H+/K+-ATP-ase and CA. Finally, in 2011, Supuran et al.Citation23 demonstrated the activation of CA I and II by mono- and dihalogenated histamine derivatives at subnanomolar concentrations. Ioan Puşcaş also showed that H2 histamine receptor blockers (cimetidine and ranitidine), muscarinic antagonist pirenzepine, Zn ions and prostaglandins inhibit gastric mucosa CA. However, the importance of these phenomena in the parietal cell physiology was never shown.
CA抑製劑治療胃和十二指腸潰瘍
發現乙酰唑胺後不久，Henry D. Janowitz（1922-2008）（紐約西奈山醫院）將其用於狗身上引文24和人類引文25 . 幾年後，研究表明需要更高劑量的乙酰唑胺利尿劑持續幾天才能確保有效抑制胃酸分泌引文26 . 1956 年在利物浦化學防禦實驗中心完成的工作表明，常用劑量的乙酰唑胺並不能消除 HCl 的分泌引文27，但是，1956 年， Rodolfo Cheli (1928-1997) 報導了對正常受試者和慢性胃炎患者的一些抑製作用引文28 .
Treatment of gastric and duodenal ulcers by CA inhibitors
Shortly after the discovery of acetazolamide, Henry D. Janowitz (1922–2008) (Mount Sinai Hospital, New York) administered it in dogsCitation24 and humansCitation25. Some years later, it was shown that higher diuretic doses of acetazolamide are needed for several days to ensure the efficient inhibition of acid secretionCitation26. Work done in 1956 at the Chemical Defence Experimental Establishment, Liverpool, showed that acetazolamide in usual doses did not abolish HCl secretionCitation27, but, in 1956, Rodolfo Cheli (1928–1997) reported some inhibitory effects in normal subjects and chronic gastritis patientsCitation28.
1960年，美國伊利諾伊州一家軍隊醫院將乙酰唑胺引入治療消化性潰瘍引文29 . 125例患者接受乙酰唑胺250mg每日一次，連續7～20天，隨著胃酸分泌減少，典型的潰瘍症狀迅速緩解。然而，沒有進行放射線檢查，該藥物產生了可逆性代謝性酸中毒。1963年，一位在加納工作的德國醫生引文30 名患者每天給 11 名患者服用 3 × 2 片 250 毫克乙酰唑胺，持續 9 天，還發現組胺刺激的胃酸分泌減少了 70-92%。與此同時，CA抑製劑也被用於其他幾種情況（表2）。
In 1960, acetazolamide was introduced to the therapy of peptic ulcers in a military hospital from Illinois, USACitation29. Acetazolamide was given in a dose of 250 mg q.i.d. to 125 patients for 7–20 days and along with a decrease of acid secretion, the typical ulcer symptoms were relieved promptly. However, no radiographic control was performed and the drug produced reversible metabolic acidosis. In 1963, a German physician working in GhanaCitation30 administered 3 × 2 tablets of 250 mg acetazolamide/day to 11 patients for 9 days and also noted a 70–92% decrease in histamine-stimulated acid secretion. In the same time, CA inhibitors were used in several other conditions (Table 2).
表 2. 乙酰唑胺 (AAZ) 的臨床應用時間表。
1968 年，Ioan Puşcaş開始在 Şimleu Silvaniei 醫院使用乙酰唑胺，並很快在大量患者身上積累了經驗。他的研究結果大部分發表在國內和國際醫學媒體上。在幾項連續研究中，他表明乙酰唑胺的劑量為 250-500 mg/tid，持續 10-21 天，胃和十二指腸潰瘍的治愈率很高，接近 100%。這些結果是當時任何其他治療方法都無法比擬的引文31、引文32 . 為了補償藥物利尿作用引起的電解損失，添加了鈉鹽、鉀鹽和鎂鹽的混合物——沒有任何抗酸作用——整個組合物以Ulcosilvanil（潰瘍 + 特蘭西瓦尼亞）的名稱銷售。這些高癒合百分比通過放射學記錄，隨後通過光纖或視頻內窺鏡檢查記錄。使用乙氧唑胺獲得了類似的結果引文33 . 當每月服用 10 天時，該藥物作為維持治療也能有效預防消化性潰瘍復發引文34 . 然而，在臨床藥理學快速發展的時代，當藥物的“安全性”受到質疑時，其高功效卻被廣泛的副作用（疲勞、感覺異常、腎絞痛、食慾不振、視力障礙、性慾下降和頭痛）所掩蓋。任何藥物都比它的“功效”更重要。儘管這些副作用從乙酰唑胺使用之初就已被認識到，但它們在已發表的研究中經常被掩蓋或僅部分報告。因此， Ulcosilvanil儘管在全國范圍內銷售，但在其母鎮 Şimleu Silvaniei 二十多年來一直是一種神奇藥物。更安全的組胺 H 2受體阻滯劑、質子泵抑製劑的出現以及幽門螺桿菌的發現嚴重削減了硫磺土的使用量，並且由於銷量下降，其生產在上世紀末之前就已停產。Ioan Puşcaş知道幽門螺桿菌的發現，但他從未認識到這種細菌在消化性潰瘍病中的重要性（見附錄和補充表）。
In 1968, Ioan Puşcaş began to use acetazolamide in Şimleu Silvaniei Hospital and he rapidly gained experience with a large number of patients. The results of his studies were largely published in the national and international medical press. In several consecutive studies, he showed that acetazolamide, given in dose of 250–500 mg/t.i.d. for 10–21 days, healed gastric and duodenal ulcer in high percentages, approaching 100%. These results were unparalleled with any other treatment in those daysCitation31,Citation32. To compensate the electrolytic losses stemming from the diuretic effect of the drug, a mixture of sodium, potassium and magnesium salts was added – without any antacid effect – and the whole composition was marketed as Ulcosilvanil (ulcer + Transylvania). These high-healing percentages were documented radiologically and later by fibre-optic or videoendoscopy. Similar results were obtained with ethoxzolamideCitation33. The drug was also efficient as maintenance treatment for preventing peptic ulcer relapses when administered for 10 days/monthCitation34. However, the high efficacy was overshadowed by a wide spectrum of side effects (fatigue, paraesthesias, renal colic, loss of appetite, vision disturbances, decreased libido and headache) in an era of rapid development of clinical pharmacology, when the “safety” of any drug was more important than its “efficacy”. Although these side-effects were recognized from the beginning of acetazolamide use, they were often masked or only partially reported in the published studies. Thus, Ulcosilvanil, although marketed countrywide, remained a miracle drug given in its parent town, Şimleu Silvaniei, for more than two decades. The advent of the safer histamine H2 receptor blockers, proton pump inhibitors and the discovery of Helicobacter pylori seriously curtailed the use of Ulcosilvanil and because of a drop in sales, its production was terminated before the end of the last century. Ioan Puşcaş was aware of the discovery of H. pylori, but he never recognized the importance of the bacterium in peptic ulcer disease (see Appendix and Supplementary Table).
幽門螺桿菌CA 及其抑制：用 CA 抑製劑治療消化性潰瘍的基本原理
Helicobacter pylori CAs and their inhibition: the rationale of treating peptic ulcers with CA inhibitors
與其他細菌一樣，幽門螺桿菌編碼兩種 CA，一種 α 類酶和一種 β 類酶，分別稱為 HpαCA 和 HpβCA引文35 ,引文36 . 經多個研究小組測定，這兩種酶具有顯著的 CO 2水合酶活性，這可能是由於它們對細菌至關重要的生理作用
As other bacteria, H. pylori encodes for two CAs, an α-class and a β-class enzyme, called HpαCA and HpβCA, respectivelyCitation35,Citation36. The two enzymes possess a significant CO2 hydrase activity, as determined by several groups, which is probably due to their crucial physiological roles for the bacterium
HpαCA 由 247 個氨基酸殘基組成，與其他 α 類細菌 CA 有 27-36% 的相似性，包括肺炎克雷伯菌、淋病奈瑟菌、糞腸球菌、魚腥藻 PCC7120和聚球藻 PCC7942引文37、引文38、引文45 . 令人驚訝的是，HpαCA 氨基酸序列與15 種人類CA（hCA）顯示出相對較高的相似性（23-36%），儘管這兩個物種之間存在很大的進化距離，這表明CA 具有基本的生物學功能，在地球生命的進化過程中一直被保存下來。大多數已知參與催化機製或形成氫鍵網絡的氨基酸殘基在hCA 和HpαCA 之間都是保守的，包括三個鋅配體的His 殘基（hCA I 編號系統中的位置94、96 和119 ） ) 以及幾個與底物、抑製劑和激活劑結合的重要殘基，例如 Tyr7、His64 和 Leu198引文45–48。
HpαCA is composed of 247 amino acid residues and shows 27–36% similarity with other α-class bacterial CAs, including Klebsiella pneumonia, Neisseria gonorrhoeae, Enterococcus faecalis, Anabaena PCC7120 and Synechococcus PCC7942Citation37,Citation38,Citation45. Surprisingly, the HpαCA amino acid sequence shows relatively high similarity (23–36%) with the 15 types of human CAs (hCA), although there is a large evolutionary distance between the two species, indicating thus that CAs possess a fundamental biological function which has been conserved during the evolution of life on earth. Most amino acid residues which are known to be involved in the catalytic mechanism or to form a network of hydrogen bonds are conserved between hCAs and HpαCA, including the three zinc-liganded His residues (at position 94, 96 and 119, hCA I numbering system) and several important residues for the binding of the substrates, inhibitors and activators, such as Tyr7, His64 and Leu198Citation45–48.
HpβCA 由 221 個氨基酸殘基組成，與其他 β 類細菌 CA 有 25-34% 的相似性，包括來自大腸桿菌的兩種酶，一種來自細長聚球藻、豬布魯氏菌和流感嗜血桿菌的酶引文35 ,引文36、引文49、引文50 . 據報導，大多數細菌 β-CA 由三個連續組件組成：(i) 包括兩個 α 螺旋（H1 和 H2）的 N 端臂，(ii) 包括三個 β- 的鋅結合核心。 (S1– S3) 和兩個α 螺旋（H3 和H4）和(iii) C 末端子結構域，包括兩個β 折疊(S4–S5) 和五個α 螺旋(H5–H9)引文51、引文52 . HpβCA 鋅結合核心的氨基酸序列在迄今為止測序的其他細菌 β-CA 中高度保守。這些殘基包括 Zn(II) 配位氨基酸 Cys42、Asp44、His98 和 Cys101（殘基編號基於大腸桿菌CynT2 編號系統）引文51、引文52 . α-CA 和 β-CA 之間的主要區別在於，β-CA 通常是低聚物，通常由 2 至 6 個分子量為 25-30 kDa 的單體組成引文51、引文52 .
HpβCA is composed of 221 amino acid residues and shows 25–34% similarity with other β-class bacterial CAs, including the two enzymes from Escherichia coli, the one from Synechococcus elongatus, Brucella suis and Haemophilus influenzaeCitation35,Citation36,Citation49,Citation50. It has been reported that most of the bacterial β-CAs are composed of three sequential components: (i) an N-terminal arm including two α-helixes (H1 and H2), (ii) a zinc-binding core including three β-sheets (S1–S3) and two α-helixes (H3 and H4) and (iii) a C-terminal subdomain including two β-sheets (S4–S5) and five α-helixes (H5–H9)Citation51,Citation52. The amino acid sequence of the zinc-binding core of HpβCA is highly conserved among other bacterial β-CAs sequenced to date. These residues include the Zn(II)-coordinating amino acids Cys42, Asp44, His98 and Cys101 (residue numbering are based on the E. coli CynT2 numbering system)Citation51,Citation52. The principal difference between α- and β-CAs consists in the fact that generally β-CAs are oligomers usually consisting of two to six monomers of molecular weight of 25–30 kDaCitation51,Citation52.
重組、純化的 HpαCA 和 HpβCA 對生理反應（CO 2水合）的催化活性與幾種人類來源的α-CA 的催化活性相比，例如hCA I–III（胞質同工酶）、hCA VA 和VB （線粒體同工酶）同種型）和 hCA XII 和 XIV（跨膜同工酶），顯示在表3。可以看出，HpαCA和HpβCA是催化效率高的CA，具有高酶活性（β類酶的活性是該細菌的α-CA的3.2倍）引文38-41。此外，該活性與 hCA I 的活性幾乎相同（以k cat / K m值表示），而細菌酶的K m值比 hCA I 更接近 hCA II。事實上，HpβCA 是一種中效CA，催化活性高於hCA III、hCA VA、hCA XII和hCA XIV等。只有 hCA VB，尤其是 hCA II（自然界中已知的最好的催化劑之一）表現出比 HpβCA 更好的活性引文38-41。此外，還可以觀察到所有這些酶的活性均受到卓越的CA 抑製劑 (CAI)、磺酰胺藥物乙酰唑胺 AAZ 的引文53-57，這可以解釋為什麼 Ulcosilvanil 顯示出一系列嚴重的副作用，這是由於除胃以外的其他器官中 CA 同工型的抑制，或者更好地說，是由於 hCA 而不是 Hpα/βCA 抑制
The catalytic activity of recombinant, purified HpαCA and HpβCA for the physiologic reaction (CO2 hydration), in comparison with that of several α-CAs of human origin, such as hCA I–III (cytosolic isozymes), hCA VA and VB (mitochondrial isoforms) and hCA XII and XIV (transmembrane isozymes), are shown in Table 3. It may be observed that HpαCA and HpβCA are catalytically efficient CAs, possessing a high enzymatic activity (the β-class enzyme is 3.2 times more active than the α-CA from this bacterium)Citation38–41. Furthermore, this activity is almost identical (as kcat/Km value) to that of hCA I, whereas the Km value of the bacterial enzyme is closer to that of hCA II than that of hCA I. In fact, HpβCA is a medium-efficiency CA, possessing a catalytic activity higher than that of hCA III, hCA VA, hCA XII and hCA XIV among others. Only hCA VB and especially hCA II, one of the best catalysts known in nature, show a better activity than HpβCACitation38–41. Also, it may be observed that the activity of all these enzymes is inhibited by the CA inhibitor (CAI) par excellence, the sulfonamide drug, acetazolamide AAZCitation53–57, which may explain why Ulcosilvanil showed a range of severe side effects, due to inhibition of CA isoforms in other organs than the stomach, or better to say, due to hCAs and not Hpα/βCA inhibition
奇里卡等人。引文37使用免疫陰性染色和 SDS 消化的冷凍斷裂免疫金標記的電子顯微鏡研究了 HpαCA 和 HpβCA 的亞細胞定位，然後表明 hpαCA 附著在外膜上，而 hpβCA 定位在細胞質中，位於細胞質側。內膜上，以及面向周質空間的外膜上引文
Chirica et al.Citation37 studied the subcellular localization of HpαCA and HpβCA by using electron microscopy with immunonegative staining and SDS-digested freeze-fracture immunogold labelling and then showed that hpαCA was attached to the outer membrane and that hpβCA was localized in the cytosol, on the cytosolic side of the inner membrane, and on the outer membrane facing the periplasmic space
幽門螺桿菌週質 HpαCA 和胞質 HpβCA 的生物學作用是什麼？斯塔勒等人。引文58報導HpαCA和HhpβCA缺失突變體以及雙突變體顯示脲酶活性顯著降低，表明脲酶和CA這兩種酶類型的代謝聯繫。他們還表明，與親本菌株、HpαCA 缺失突變體和雙突變體相比，只有 HpβCA 缺失突變體在 pH 6.0-6.25 培養 48 小時時表現出明顯的生長減少，這表明胞質 HpβCA 僅對當週質HpαCA 發揮功能時的耐酸性引文58 . 在 Marcus 等人的另一項研究中，引文59與野生型菌株相比，暴露於 pH 2.0 30 分鐘後，HpαCA 缺失突變體的存活率降低了約 3 log 10 。這些差異的發現可能是由於兩項研究中採用的培養條件不同造成的。在存在 CAI 乙酰唑酰胺 (AAZ) 的情況下，同時對抗 hpαCA ( K I : 21 nM) 和 hpβCA ( K I : 40 nM) (表3），另一種幽門螺桿菌菌株（ATCC 43504）的存活率也下降了∼3 log 10引文59 . 這些發現表明 hpαCA 和 HhpβCA 可能對細菌的生存至關重要。
What are the biological roles of periplasmic HpαCA and cytosolic HpβCA in H. pylori? Stähler et al.Citation58 reported that HpαCA and HhpβCA deletion mutants as well as the double mutant displayed a significant decrease in urease activity, indicating a metabolic link of these two enzyme types, urease and CAs. They also showed that only the HpβCA deletion mutant showed a clearly reduced growth at pH 6.0–6.25 for 48 h in culture, compared to the parental strain, the HpαCA deletion mutant and the double mutant, suggesting thus that the cytosolic HpβCA is only important for acid resistance when the periplasmic HpαCA is functionalCitation58. In another study by Marcus et al.,Citation59 the HpαCA deletion mutant showed an ∼3 log10 decrease in survival after 30 min of exposure to pH 2.0 when compared with the wild-type strain. These discrepant findings are probably due to the different culture conditions employed in the two studies. In the presence of the CAI acetazolamide (AAZ), acting against both hpαCA (KI: 21 nM) and hpβCA (KI: 40 nM) (Table 3), another H. pylori strain also (ATCC 43504) showed an ∼3 log10 decrease in surviveCitation59. These findings indicate that both hpαCA and HhpβCA might be essential for the bacterial survival.
馬庫斯等人。引文59證明HpαCA對於病原體的酸性適應和生存至關重要，並提出了脲酶和hpαCA在酸性環境中維持週質pH的作用模型引文58、引文59 . 馬庫斯等人。引文59採用了幽門螺桿菌的CA缺失突變體，並表明脲酶產生NH 3和hpαCA產生碳酸氫鹽在酸性條件下調節週質pH 6.1和內膜電位-101 mV中具有主要作用引文59 . 因此，將周質緩衝至與活力一致的pH不僅取決於從細胞質流出的氨，還取決於週質hpαCA將CO 2 （由脲酶產生）轉化為碳酸氫鹽（圖1）。事實上，碳酸/碳酸氫鹽對的 pKa 約為 6.1 非常適合此類任務，與 pKa 為 9.2 的銨/氨緩衝液不同，對於將周質緩衝到接近中性的 pH 值不太有用。與上面提到的周質反應類似的反應將發生在細胞質中，即細胞質hpβCA水合CO 2產生碳酸氫鹽，將細胞質pH緩沖在~8.0（圖1）引文59、引文60 . hpβCA催化反應的另一種產物，即質子，被脲酶產物NH 3中和。NH 3還可以中和進入的質子，這可能發生在胃中存在的高酸性條件下。因此，hpαCA和HpβCA與細胞質脲酶合作，分別在幽門螺桿菌的周質和細胞質中進行酸適應，這表明這些CA可能是開發抗幽門螺桿菌藥物的有吸引力的藥物靶點。
Marcus et al.Citation59 has proved that HpαCA is essential for the acid acclimation and survival of the pathogen and then proposed a model for the role of urease and hpαCA in maintenance of the periplasmic pH in acidic environmentCitation58,Citation59. Marcus et al.Citation59 employed a CA deletion mutant of H. pylori and showed that the generation of NH3 by urease and bicarbonate by hpαCA has a major role in the regulation of the periplasmic pH 6.1 and inner membrane potential −101 mV under acidic conditionsCitation59. Thus, buffering of the periplasm to a pH consistent with viability depends not only on the ammonia efflux from the cytoplasm but also on the conversion of CO2 (produced by urease) to bicarbonate by the periplasmic hpαCA (Figure 1). In fact, the pKa of the carbonic acid/bicarbonate couple of ∼6.1 is very appropriate for such a task, unlike the ammonium/ammonia buffer, which having a pKa of 9.2, is less useful for buffering the periplasm to pH values close to neutrality. The similar reactions to the one mentioned above for the periplasm will occur in the cytoplasm, i.e. hydration of CO2 by cytoplasmic hpβCA producing bicarbonate to buffer the cytoplasmic pH at ∼8.0 (Figure 1)Citation59,Citation60. Another product of the hpβCA-catalysed reaction, i.e. the protons, are neutralized by NH3, the product of urease. NH3, can also neutralize entering protons, which may occur in the highly acidic condition present in the stomach. Accordingly, in cooperation with cytoplasmic urease, hpαCA and HpβCA work for acid acclimation of H. pylori in its periplasm and cytoplasm, respectively, suggesting that these CAs may be attractive drug targets for developing anti-H. pylori agents.
圖 1. 幽門螺桿菌CA 和脲酶及其參與胃粘膜內病原體的酸適應引文46 .
Figure 1. Helicobacter pylori CAs and urease and their involvement in the acid acclimation of the pathogen within the gastric mucosaCitation46.
初步體外研究，使用有效且臨床使用的 CAI、AAZ（KI ： HpαCA為 21 nM，HpβCA 為 40 nM）Lindskog小組的41人37在 5 µM 濃度下未表現出對細菌生長的任何抑製作用。然而，在 1 mM AAZ 存在的情況下，幽門螺桿菌的酸存活率下降了 ∼3 log 1037 . 西森組41還發表的數據顯示，另一種 CAI，醋甲唑胺 (MZA)，在體外表現出對某些幽門螺桿菌菌株的生長抑製作用。MZA 的抑制活性比 AAZ 弱，但它是 HpαCA 和 HpβCA 的強抑製劑。有趣的是，100μg/ml的MZA完全抑制了菌株SS1的生長，但該濃度的藥物對另一種菌株的生長沒有抑製作用41 , 11 637。然而，在500 μg/ml濃度的MZA下，菌株11 637的生長也受到抑制。這些發現表明，CAI抑制幽門螺桿菌的生長，但每種菌株對藥物的敏感性差異很大。
Preliminary in vitro studies, with the potent and clinically used CAI, AAZ (KI: 21 nM for HpαCA and 40 nM for HpβCA)41 from Lindskog’s group37 did not show any inhibition of bacterial growth at the concentration of 5 µM. However, in the presence of 1 mM of AAZ, there was an ∼3 log10 decrease in acid survival of H. pylori37. Nishimori’s group41 also published data showing that another CAI, methazolamide (MZA), displayed growth inhibition of certain strains of H. pylori in vitro. MZA possesses weaker inhibitory activity than AAZ but it is however a strong inhibitor of both HpαCA and HpβCA. Interestingly, the growth of the bacterial strain SS1 was totally inhibited at 100 µg/ml of MZA, but there was no inhibitory effect of the drug at this concentration on the growth of another strain41, 11 637. However, at a concentration of 500 µg/ml of MZA, the growth of strain 11 637 was also inhibited. These findings indicate that CAIs suppress the growth of H. pylori, but the susceptibility to the drug greatly varies in each strain41.
結論
幽門螺桿菌中存在的兩種 CA與脲酶一起對該病原體胃的高酸性條件下的酸適應和生存發揮著核心作用。Ioan Puşcaş表示，幾種磺胺類 CAI 已被證明對體外幽門螺桿菌的生長具有抑製作用，並能抑制壁細胞的酸分泌32 . 這些發現強烈建議臨床使用 CAI 作為根除幽門螺桿菌治療的新藥。當然，這可能包括 CAI 與各種抗生素的聯合治療。幸運的是，迄今為止，在之前的臨床研究中，除了經常報導的肢體和口腔周圍出現輕度感覺異常之外，尚未觀察到 AAZ 的嚴重並發症。32、33、61 . 為了開發具有與以前使用的藥物不同的藥理機制的替代根除療法，應該保證使用一組 CAI 進行仔細設計的方案的臨床試驗。此外，最近的報告HpαCA 的 X 射線晶體結構的62與乙酰唑胺和醋甲唑胺複合可能導致開發出與人類酶親和力較低的細菌 CA 選擇性抑製劑，這可能導致與 AAZ 相比副作用減少的抗潰瘍藥物。總之，儘管用磺胺類 CAIs 治療消化性潰瘍的最初假設是錯誤的，但治療是有效的，這是由於幽門螺桿菌CA的抑製作用，而幽門螺桿菌 CA 參與了細菌的關鍵生理過程。發現對細菌而非人類酶具有選擇性的 CAI 可能會為這種疾病帶來有效的治療方法，這在某種程度上是Ioan Puşcaş和他的合作者首創的，但尚未進行徹底探索。
Conclusions
The two CAs present in H. pylori together with the urease play a central role for acid acclimation and survival in the highly acid condition of the stomach of this pathogen. Several sulfonamide CAIs have been shown to possess inhibitory effect on the growth of H. pylori in vitro, as well as inhibition of acid secretion from the parietal cells, as shown by Ioan Puşcaş32. These findings strongly recommend the clinical use of CAIs as novel agents for the eradication therapy of H. pylori. This may include, of course, combination therapy of a CAI with various antibiotics. Fortunately, no serious complication of AAZ were observed so far in previous clinical studies, apart for the appearance of mild paraesthesias in the limb and around the mouth, which were frequently reported32,33,61. For the development of alternative eradication therapies that possess different pharmacological mechanism from those of the previously used drugs, clinical trials with a carefully designed protocol using a panel of CAIs should be warranted. Furthermore, the recent report62 of the X-ray crystal structure of HpαCA complexed with acetazolamide and methazolamide might lead to the development of bacterial CA-selective inhibitors with less affinity for the human enzymes, which might lead to anti-ulcer agents with reduced side effects when compared with AAZ. In conclusion, although the starting hypothesis for treating peptic ulcers with sulfonamide CAIs was wrong, the treatment was efficacious and this was due to inhibition of H. pylori CAs, which are involved in crucial physiologic processes in the bacterium. Finding CAIs selective for the bacterial over the human enzymes may lead to efficient treatment approaches for this disease, which was in a way pioneered, but not thoroughly explored by Ioan Puşcaş and his collaborators.
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