Tubulin protein/microtubule

以剂量和时间依赖性方式，VERU-111（2.5-80 nM；24-48 小时）抑制 Panc-1、AsPC-1 和 HPAF-II 细胞的发育（24 小时：IC50 为 25、35 、 和 35 nM，分别；48 小时：IC50 分别为 11.8、15.5 和 25 nM）[4]。 VERU-111（5-20 nM；24 小时）以剂量依赖性方式将 Panc-1 和 AsPC-1 细胞阻滞在 G2/M 期 [4]。 VERU-111（5-20 nM；24 小时）表现出对 caspase 3 和 9 前体的剂量依赖性抑制以及 caspase-3 和 9 的激活，促进 Bax 和 Bad 的产生，并抑制 Bcl-2 AsPC 的表达Panc-1细胞中的-1和Bcl-xl蛋白[4]。

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化合物II/沙比沙布林和IAT对癌症细胞（包括多药耐药细胞）表现出潜在的细胞毒性使用SRB测定评估化合物II/ 化合物II/沙比扎布林和IAT与微管蛋白上的秋水仙碱结合位点结合，抑制微管蛋白聚合，并诱导癌症细胞凋亡开发了竞争性质谱结合测定法来研究小分子抑制剂与微管蛋白的相互作用，并应用于检测我们的先导化合物SMART-H，该化合物选择性结合微管蛋白上秋水仙碱位点。化合物II/Sabizabulin和IAT化合物与秋水仙素有效竞争微管蛋白结合（图1a），效力与鬼臼毒素相似。阴性对照长春花碱没有抑制秋水仙素与微管蛋白的结合，成功证明了这种竞争性质谱结合测定的特异性。将猪脑微管蛋白（纯度>97%）与II或IAT（5μM）一起孵育，以测试它们对微管蛋白聚合的影响（图1b）。化合物II和IAT在15分钟时分别抑制微管蛋白聚合47%和40%。以5μM的秋水仙素作为阳性对照，抑制微管蛋白聚合32%。这些数据表明，II和IAT都比秋水仙碱更好地抑制微管蛋白聚合，并表明这些化合物与SMART化合物结合在同一位点。将PC-3和PC-3/TxR细胞暴露于0.8-600 nmol/L的化合物II、IAT或多西他赛24小时。通过DNA-组蛋白复合物形成测量，化合物II/Sabizabulin和IAT在24小时内诱导PC-3（图1c）和PC-3/TxR（图1d）细胞凋亡的能力相同。尽管多西他赛能有效诱导PC-3细胞的细胞凋亡，但由于P-糖蛋白的过表达，PC-3/TxR细胞的凋亡明显较弱[7]。

将媒介物治疗组与 VERU-111（50 μg/小鼠；瘤内注射；每周 3 次，持续三周）进行比较，结果表明肿瘤生长显着减少。给予 VERU-111 的小鼠没有表现出任何明显的毒性，尽管它们的体重不断增加 [4]。

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化合物II/SabizabulinIAT抑制紫杉醇抗性前列腺（PC-3/TxR）异种移植生长亲本PC-3和紫杉醇抗性的前列腺癌症PC-3（PC-3/TxR）细胞接种到裸鼠中，并使肿瘤体积达到约150～300mm3。多烯紫杉醇（10或20mg/kg）是一种被批准用于晚期前列腺癌症临床的抗癌药物，用于比较。PC-3/TxR肿瘤异种移植物生长迅速，研究结束时肿瘤体积达到1500-2500mm3。尽管静脉注射多西他赛（10和20mg/kg）在两种模型中都显示出体内抗癌活性（图2a，b），但当静脉注射10mg/kg时，肿瘤生长抑制（TGI）效果从PC-3肿瘤的84%TGI降低到PC-3/TxR肿瘤的14%TGI（表V）。在较高剂量（20mg/kg）下，多西他赛引起PC-3肿瘤的部分消退（>100%TGI），但在PC-3/TxR肿瘤中仅引起56%的TGI。与PC-3肿瘤相比，多西他赛在PC-3/TxR肿瘤中的疗效降低，表明疗效受到P-糖蛋白介导的耐药性的损害。这些结果与我们的体外细胞毒性和凋亡数据一致。与多西他赛在PC-3/TxR肿瘤中缺乏疗效相反，化合物II/Sabizabulin（6.7mg/kg）口服给药，显示TGI超过100%，对体重没有影响（图2b和表V）。此外，在用化合物II治疗的4只携带PC-3/TxR肿瘤的裸鼠中，有2只在第19天没有肿瘤。进一步利用PC-3/TxR异种移植物模型来评估使用不同给药方案给药时化合物II和IAT的疗效。II的最大耐受剂量（体重减轻>20%）为10mg/kg，每天口服一次，持续4天；或每天两次（b.i.d.）给药5天时为3.3mg/kg（数据未显示）。如图2c所示，在第一周的前四天，每天两次服用3.3 mg/kg的II/Sabizabulin，然后在第2周和第4周将时间表改为每天一次。在第4-19天获得偏回归。TGI为97%，七只小鼠中的一只在第26天没有肿瘤。较高剂量（10mg/kg）和较低给药频率（q2d）的化合物II（图2d）在第13-29天引发了部分肿瘤消退，表明替代给药方案成功抑制了PC-3/TxR异种移植物的生长。化合物IAT以10或30mg/kg b.i.d.的剂量口服给裸小鼠，在第1至4周之间每周五次（图2c）。接受10mg/kg IAT的小鼠的TGI值为59%，而在接受更高剂量（30mg/kg）IAT治疗的动物中，从第19天到研究终止（第26天）观察到部分回归（>100%TGI）。载体组的一些小鼠在终点体重较低，部分原因是肌肉萎缩和/或癌症恶病质。相反，用化合物II（3.3mg/kg）或IAT（30mg/kg）治疗的小鼠体重增加（表V），表明这些优化剂量的II或IAT具有良好的耐受性[7]。

体外微管蛋白聚合测定[5,6]
根据Wang等人描述的方法，将猪脑微管蛋白（纯度>97%）与普通微管蛋白缓冲液（80 mM PIPES、2.0 mM MgCl2、0.5 mM EGTA和1 mM GTP）混合，在4°C下达到3 mg/mL的最终浓度。在96孔板中混合微管蛋白溶液和测试化合物后，立即在37°C的SYNERGY 4微孔板读取器中孵育微管蛋白聚合测定，并在340nm下每30秒监测65分钟。以紫杉醇作为微管蛋白聚合的阳性对照，秋水仙碱和ABI-274作为微管蛋白解聚的阳性对照进行重复实验。

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用于亲和性测定的SPR[5,6]
在配备有葡聚糖SPR传感器芯片（Reichert Polycarboxylate Hydrogel chip P/N 13206067）的Reicher4SPR系统中使用SPR技术分析与微管蛋白的结合亲和力。然后，将50μg/mL微管蛋白固定在传感器芯片表面，以获得12μRIU。芯片上的四个流动池中的一个作为阴性对照。在传感器芯片表面上注射不同浓度的4v或秋水仙碱进行缔合分析，然后进行离解分析。实验数据在25°C下使用运行缓冲液PBST（8 mM Na2HPO4、136 mM NaCl、2 mM KH2PO4、2.6 mM KCl和0.05%（v/v）Tween 20，pH 7.4）获得。平衡离解常数（KD）是用TraceDrawer软件通过稳态拟合模式计算的。

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竞争性质谱结合分析[7]
如前所述，进行了竞争性质谱结合研究。秋水仙素（1.2μM）与猪脑微管蛋白（1.0 mg/mL）在37°C的孵育缓冲液[80 mM哌嗪-N，N′-双（2-乙磺酸）（PIPES），2.0 mM氯化镁（MgCl2），0.5 mM乙二醇四乙酸（EGTA），pH 6.9]中孵育1小时。使用不同浓度（0.2-200μM）的鬼臼毒素（阳性对照）、化合物II/Sabizabulin、化合物IAT和长春花碱（阴性对照）与秋水仙素与微管蛋白的结合竞争。孵育1小时后，使用Li等人的超滤法（微浓缩器）获得滤液。作者的个人副本分子截留尺寸为30 kDa。在没有任何竞争对手的情况下，目标化合物抑制每种配体结合的能力表示为对照结合的百分比。每个实验进行三次。

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体外微管聚合试验[7]
将猪脑微管蛋白（0.4 mg）与5μM的目标化合物或载体[二甲基亚砜（DMSO）]混合，并在100μL缓冲液[80 mM PIPES、2.0 mM MgCl2、0.5 mM EGTA、pH 6.9和1 mM鸟苷-5′-三磷酸（GTP）]中孵育。每分钟监测一次340 nm波长的吸光度，持续15分钟。分光光度计保持在37°C进行微管蛋白聚合。

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代谢稳定性[7]
代谢稳定性研究是通过在37°C的振荡水浴中，将感兴趣的化合物（0.5μM）在总反应体积为1 mL的反应缓冲液中孵育进行的，该缓冲液中含有1 mg/mL微粒体蛋白[0.2 M磷酸盐缓冲溶液（pH 7.4）、1.3 mM烟酰胺腺嘌呤二核苷酸磷酸盐（NADP+）、3.3 mM葡萄糖-6-磷酸和0.4 U/mL葡萄糖-6-磷酸脱氢酶]。混合的人肝微粒体用于检查代谢稳定性。NADPH再生系统（溶液A和B）购自BD Biosciences（马萨诸塞州贝德福德）。反应溶液中DMSO的总浓度约为0.5%（v/v）。在5、10、20、30、60和90分钟时，从用于测定代谢稳定性的反应混合物中取样等分试样（100μL）。加入含有200 nM内标（23）的乙腈（150μL）以淬灭反应并沉淀蛋白质。然后在室温下以4000g离心样品15分钟，并通过液相色谱-串联质谱（LC-MS/MS）直接分析上清液。

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CYP酶特异性测定[7]
进行了五次CYP酶抑制试验，并通过LC-MS/MS进行分析。简而言之，对于CYP2D6、CYP2C9、CYP1A2和CYP2C19，将0.1 mg/mL微粒体蛋白与其特定底物在37°C的0.1 M磷酸钾缓冲液（pH 7.4）中孵育，而0.05 mg/mL微粒体蛋白用于CYP3A4试验。底物睾酮（50μM）、右美沙芬（7μM），（S）-苯妥英钠（80μM）；双氯芬酸（7μM）和非那西丁（100μM）分别用于CYP 3A4、2D6、2C19、2C9、1A2抑制试验。在这些测定中检测了0.04-50μM范围内的化合物II/Sabizabulin或IAT。用于CYP 3A4、2D6、2 C19、2 C9和1A2抑制试验的阳性对照分别为酮康唑（0.0009-1μM）、奎宁定（0.0009–1μM）、噻氯匹啶（0.019–20μM），磺胺苯唑（0.019-20μM，和呋喃茶碱（0.04–50μM）。

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水溶性[7]
化合物II/Sabizabulin和IAT的溶解度通过多屏幕溶解度滤板结合LC-MS/MS测定。简而言之，将198μL磷酸盐缓冲盐水（PBS）缓冲液（pH 7.4）装入96孔板中，分配2μL 10mM试验化合物（在DMSO中），在室温（N0 3）下轻轻摇动（200-300rpm）混合1.5小时。将平板在800g下离心10分钟，滤液用于通过LC-MS/MS方法测定其浓度和试验化合物的溶解度。

细胞增殖测定 [4]
细胞类型： Panc-1、AsPC-1、HPAF-II 细胞
测试浓度： 2.5、5、10、 20、40、80 nM
孵育时间：24、48 小时
实验结果：抑制 PanCa 细胞的生长和剂量- 和时间相关的方式。治疗 24 小时后，VERU-111 在 Panc-1、AsPC-1 和 HPAF-II 中的 IC50 分别为 25、35 和 35 nM，48 小时后)) 处理后它们分别为 11.8、15.5 和 25 nM。

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细胞凋亡分析 [4]
细胞类型： Panc-1、AsPC-1 细胞
测试浓度： 5、10、20 nM
孵育持续时间：24小时
实验结果：Panc-1和AsPC-1细胞在G2/M期方法中停滞。

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蛋白质印迹分析[4]
细胞类型： AsPC-1 和 Panc-1 细胞
测试浓度： 5、10、 20 nM
孵育持续时间：24 小时
实验结果：Caspase 3 和 9 前体以及 Caspase-3 的剂量依赖性抑制和 9 9 在激活的 AsPC-1 和 Panc-1 细胞中。诱导 Bax 和 Bad 的表达并抑制 Bcl-2 和 Bcl-xl 蛋白的表达。

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前列腺癌和胶质瘤细胞系的细胞培养和细胞毒性测定[7]
前列腺癌症细胞系（LNCaP，PC-3，DU145，PPC-1）和神经胶质瘤细胞系（U87MG）来源于ATCC。由于前列腺癌症的患病率和多西他赛治疗该疾病的常用性，选择前列腺癌症细胞进行这些研究。神经胶质瘤细胞系（U87MG）用于检测化合物在另一种类型的癌症（即神经胶质瘤）中的活性，并与脑-血屏障研究相协调。紫杉醇耐药性PC-3（PC-3/TxR；一种过度分泌P-糖蛋白的前列腺癌症细胞系）是密歇根大学安娜堡分校病理学系Evan T.Keller博士的礼物。PC-3/TxR被用作MDR模型。所有细胞系均经ATCC测试和认证，并立即扩增和冷冻，以便每2~3个月从同一批细胞的冷冻瓶中重新启动。细胞培养用品购自Cellgro Mediatech。前列腺癌症细胞系维持在补充有10%胎牛血清（FBS）的RPMI 1640培养基中，而神经胶质瘤癌症细胞系保持在含有2mML-谷氨酰胺和10%FBS的Eagle's MEM培养基中。如前所述，通过磺基罗丹明B（SRB）测定法在细胞系中测试感兴趣的化合物、紫杉醇和多烯紫杉醇的抗增殖活性。

Animal/Disease Models: Sixweeks old female athymic nude mice (carrying AsPC-1 cells)
Doses: 50 μg/animal
Route of Administration: intratumoral injection; 3 times a week for 3 weeks
Experimental Results: Effectively inhibited tumor growth.

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Pharmacokinetic Study [7]
Male ICR mice (N03 or 4 per group) 6–8 weeks of age were used to examine the pharmacokinetics (PK) of compound II/Sabizabulin or IAT. Both chemicals were formulated in DMSO/Polyethylene glycol 300 (PEG300), 1/9, v/v, and Tween80/DMSO/ H2O, 2/2/6, v/v/v for intravenous bolus (i.v., 10 mg/kg) and oral (p.o., 20 mg/kg) administration, respectively. Dosing volume for i.v. was 50 μL via tail vein, while the volume Orally Active Tubulin Antagonists Author's personal copy for p.o. was 200 μL through oral gavage. For i.v. administration, blood samples were collected at 2, 5, 15, and 30 min, 1, 2, 4, 8, 16, and 24 h after administration. For p.o. administration, blood samples were collected at 0.5, 1, 1.5, 2, 3, 4, 8, 16, and 24 h after administration. Plasma samples were prepared by centrifuging the blood samples at 8,000g for 5 min. All plasma samples were stored immediately at −80°C until analyzed. Female Sprague–Dawley rats (N03 or 4 per group) were used. Rat thoracic jugular vein catheters were purchased from Braintree Scientific Inc. (Braintree,MA). All animals were fed prior to dosing. Dosing volumes for intravenous bolus (i.v.) and oral (p.o.) solutions were 2 and 4 mL/kg, respectively. Compound II/Sabizabulin or IAT was administered i.v. into the thoracic jugular vein at a dose of 5 mg/kg (in DMSO/PEG300, 1/9, v/v). The dose in rats was chosen to be one-half of the dose in mice based onbody surface area. Catheters were flushed with 1 mL of heparinized saline after i.v. bolus. An equal volume of heparinized saline was injected to replace the removed blood, and blood samples (250 μL) were collected via the jugular vein catheter at 10, 20, 30 min, and 1, 2, 4, 8, 12, 24 h. Compounds II/Sabizabulin and IAT were also given (p.o.) by oral gavage at 10mg/kg (in Tween80/DMSO/H2O, 2/2/6, v/v/v) to evaluate their oral bioavailability. All blood samples (250 μL) after oral administration were collected via the jugular vein catheter at 30, 60, 90 min, 120 min, 150 min, 180 min, 210 min, 240 min, and 8, 12, 24 h. Heparinized syringes and vials were prepared prior to blood collection. Female beagle dogs weighing about 10 kg were used in this study. The dogs (N04) were given a single intravenous dose of compound II/Sabizabulin or IAT (2 mg/kg, in DMSO/PFG300, 1/9, v/v), in a dosing volume of 0.2 mL/kg. Blood was drawn at 10, 20, 30 min, and 1, 2, 4, 8, 12, 24, 48, 96 h. For p.o. administration (N04), the dogs (N04) were given a single oral dose of compound IAT (5 mg/kg, in Tween80/ DMSO/H2O, 2/2/6, v/v/v) in a dosing volume of 1 mL/ kg. We selected an oral dose level (5 mg/kg) in dogs that was one-fourth of the dose in mice and slightly higher than would be needed to correct for differences in body surface area (i.e., one sixth of the dose in mice) due to in vitro studies with liver microsomes from these species indicating less metabolic stability of the compounds in dogs (data not shown). Blood was drawn at 20, 40, 60, 80, 100, 120, 150, 180, 210 min and 4, 8, 12, 24, 48, 96 h. A protein precipitation method was used for sample preparation. An aliquot (200 μL) of acetonitrile (ACN) containing the internal standard was added to 100 μL of plasma and then was thoroughly vortexed for 15 s. After centrifugation, the supernatant was analyzed by LC-MS/MS. The pharmacokinetic parameters were determined using noncompartmental analysis.

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Brain Penetration Study [7]
Plasma and brain tissue were collected after a single dose oral administration (20 mg/kg) of compounds II/Sabizabulin and IAT, and single intraperitoneal administration (10 mg/kg) of docetaxel from nude mice. All three chemicals were formulated in Tween80/DMSO/H2O, 2/2/6, v/v/v. At the indicated time points (1 h and 4 h) after dosing, blood and brain tissue was collected from nude mice. Plasma was prepared as previously described and stored at −80°C until analyzed. Brain tissue samples were individually ground to a powder with a Bessman tissue pulverizer. The pulverizer was pre-cooled for 1 min in liquid nitrogen. Approximately 50 mg of tissue was placed on the pulverizer, and the whole apparatus was cooled in liquid nitrogen for 1 min and then the tissue was ground to a fine powder. The powder was immediately transferred to a sample vial, vortexed with 4 volumes of water, and then 10 volumes of acetonitrile containing the internal standard were added for extraction. After centrifugation, the supernatant was analyzed by LC-MS/MS to determine their brain and plasma concentrations.

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PC-3 and Paclitaxel-Resistant PC-3 (PC-3/TxR) Tumor Xenograft Studies [7]
PC-3 or PC-3/TxR cells (108 per mL) were prepared in growth media containing 10% FBS and mixed with high concentration, phenol red-free Matrigel at 1:1 ratio. Tumors were established by injecting 100 μL of the mixture (5×106 cells per animal) subcutaneously (s.c.) into the flank of 6–8-week-old male athymic nude mice. The length and width of tumors were measured and the tumor volume (mm3) was calculated by the formula, π/6 × L × W2, where length (L) and width (W) were determined in mm. When the tumor volumes reached about 150–300 mm3, the animals were treated with an intravenous formulation [Tween80/ethanol/saline (7.5/ 7.5/85)] or oral formulation [Tween80/DMSO/H2O (2/ 2/6)]. Docetaxel (10 or 20 mg/kg) was intravenously dosed on day 1 and day 9 in both PC-3 and PC-3/TxR xenograft models while compound II/Sabizabulin (6.7 mg/kg) was dosed orally (qd, five times a week) in PC-3/TxR xenograft model. In another PC-3/TxR xenograft study, compound II/Sabizabulin (3.3 mg/kg) was dosed twice a day (b.i.d.) for the first four days in the first week, and then the schedule was changed to once daily, five days a week during week 2–4 due to toxicity. While compound IAT (10 and 30 mg/kg) was orally dosed b.i.d. on mice, five times a week for four weeks, a higher dose of compound II (10 mg/kg) was also examined in PC-3/TxR xenografts, with every other day (q2d) treatments.

Compounds II/Sabizabulin and IAT Exhibited Favorable Drug-Like Properties [7]
The drug-like properties of II and IAT, such as metabolic stability, permeability, aqueous solubility, and drug-drug interactions, were examined (Table II). Compound II/Sabizabulin exhibited greater metabolic stability and aqueous solubility than IAT. Both compounds exhibited more than adequate permeability values, suggesting that they would be amenable to oral administration. In addition, both compounds showed high IC50 values (in the micromolar range) during CYP enzyme inhibition assays, suggesting that they will not cause CYP-mediated drug-drug interactions.

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Pharmacokinetic Studies in Mice, Rats and Dogs [7]
The pharmacokinetic parameters of compounds II/Sabizabulin and IAT after single intravenous or oral doses in ICR mice, Sprague–Dawley rats, and Beagle dogs are summarized in Table III. Compound II exhibited low clearance in mice and rats, suggesting that it exhibited prolonged metabolic stability and minimal first-pass hepatic metabolism in these species. In addition, II had a moderate volume of distribution in mice and rats, suggesting that it is widely distributed in tissues. Surprisingly, the total clearance of compound II in dogs was high. Two abundant metabolites in dog plasma, a hydroxylated metabolite and an unknown metabolite with +34 m/z than the parent (data not shown), were observed, which were consistent with those found in dog liver microsomes. In addition, abundant metabolites were observed when compound II was incubated with dog liver microsomes, but not in mouse, rat or human liver microsomes (data not shown). Nevertheless, compound II/Sabizabulin showed acceptable oral bioavailability of 21%, 36%, and 50% in rats, mice, and dogs, respectively. Compound IAT had low systemic clearance in rats and moderate clearance inmice and dogs. Similar to II, compound IAT exhibited moderate volume of distribution in these species. Compound IAT had comparable oral bioavailability among the three species (24%~36%).

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Brain Penetration of Compounds II/Sabizabulin and IAT in Nude Mice The ratios of whole brain to plasma concentrations of compound II and IAT were determined and compared to docetaxel in the nude mice (Table IV). Compound IAT exhibited a greater brain penetration than compound II and docetaxel. Compound II achieved slightly greater brain/plasma concentration ratios than docetaxel at both 1 and 4 h, while the IAT concentrations in brain reached 14–19% of plasma concentrations at 1 h and 4 h, respectively, showing a 3.2-fold higher brain/plasma ratio at both 1 h and 4 h compared to docetaxel.

[1]. Structure-Guided Design, Synthesis, and Biological Evaluation of (2-(1H-Indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl) Methanone (ABI-231) Analogues Targeting the Colchicine Binding Site in Tubulin. J Med Chem . 2019 Jul 25;62(14):6734-6750.

[2]. Discovery of novel 2-aryl-4-benzoyl-imidazole (ABI-III) analogues targeting tubulin polymerization as antiproliferative agents. J Med Chem . 2012 Aug 23;55(16):7285-9.

[3]. ABI-231: A novel small molecule suppresses tumor growth and metastatic phenotypes of cervical cancer cells via targeting Human papilloma virus (HPV) E6 and E7. Cancer Research 78(13 Supplement):679-679.

[4]. Therapeutic efficacy of a novel βIII/βIV-tubulin inhibitor (VERU-111) in pancreatic cancer. J Exp Clin Cancer Res. 2019 Jan 23;38(1):29.

[5]. Structure-Activity Relationship Study of Novel 6-Aryl-2-benzoyl-pyridines as Tubulin Polymerization Inhibitors with Potent Antiproliferative Properties. J Med Chem. 2020 Jan 23;63(2):827-846.

[6]. Discovery of novel 2-aryl-4-benzoyl-imidazole (ABI-III) analogues targeting tubulin polymerization as antiproliferative agents. J Med Chem . 2012 Aug 23;55(16):7285-9.

[6]. Orally bioavailable tubulin antagonists for paclitaxel-refractory cancer. Pharm Res. 2012 Nov;29(11):3053-63.

Sabizabulin is an orally bioavailable, small molecule tubulin inhibitor, with potential antineoplastic, antiviral and anti-inflammatory activities. Upon oral administration, sabizabulin binds to the colchicine-binding site of alpha- and beta-tubulin subunits of microtubules and crosslinks the microtubules, thereby inhibiting microtubule polymerization in tumor blood vessel endothelial cells and tumor cells. This blocks the formation of the mitotic spindle and leads to cell cycle arrest at the G2/M phase. As a result, this agent disrupts the tumor vasculature, tumor blood flow, deprives tumor cells of nutrients, and induces apoptosis. In addition, as microtubules plays an important role in intracellular transport, the inhibition of its polymerization may disrupt the transport of the androgen receptor (AR) into the cell nucleus, as well as virus trafficking around the cell. This may decrease viral replication and assembly. Inhibition of tubulin polymerization may also inhibit the release of pro-inflammatory cytokines and disrupt inflammatory cell activities. Sabizabulin is not a substrate of P-glycoprotein (Pgp), an efflux pump that when overexpressed, may confer resistance to taxane agents.

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VERU-111, an investigational drug intended for various therapeutic uses, was under investigation in clinical trials NCT04842747, NCT03752099, NCT04388826, NCT04844749, NCT05008510, and NCT05079360. These trials aimed to evaluate its efficacy, safety, and tolerability in conditions such as SARS-CoV-2 infection, metastatic castration-resistant prostate cancer (mCRPC), respiratory distress syndrome in adults, and metastatic triple-negative breast cancer.
SABIZABULIN is a small molecule drug with a maximum clinical trial phase of III (across all indications) and has 5 investigational indications.

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Mechanism of Action
Veru-111 is a selective tubulin inhibitor currently being tested for the treatment of pancreatic cancer. Veru-111 represses alpha- and beta-tublin subunits through enhanced expression of miR-200C. In both melanoma and prostate cancer cell lines, it has displayed strong antiproliferative activity. It also prevents microtubule polymerization and causes cell cycle arrest in the G2/M phase, which suggests anti-tumor properties.

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Background: The management of pancreatic cancer (PanCa) is exceptionally difficult due to poor response to available therapeutic modalities. Tubulins play a major role in cell dynamics, thus are important molecular targets for cancer therapy. Among various tubulins, βIII and βIV-tubulin isoforms have been primarily implicated in PanCa progression, metastasis and chemo-resistance. However, specific inhibitors of these isoforms that have potent anti-cancer activity with low toxicity are not readily available. Methods: We determined anti-cancer molecular mechanisms and therapeutic efficacy of a novel small molecule inhibitor (VERU-111) using in vitro (MTS, wound healing, Boyden chamber and real-time xCELLigence assays) and in vivo (xenograft studies) models of PanCa. The effects of VERU-111 treatment on the expression of β-tubulin isoforms, apoptosis, cancer markers and microRNAs were determined by Western blot, immunohistochemistry (IHC), confocal microscopy, qRT-PCR and in situ hybridization (ISH) analyses. Results: We have identified a novel small molecule inhibitor (VERU-111), which preferentially represses clinically important, βIII and βIV tubulin isoforms via restoring the expression of miR-200c. As a result, VERU-111 efficiently inhibited tumorigenic and metastatic characteristics of PanCa cells. VERU-111 arrested the cell cycle in the G2/M phase and induced apoptosis in PanCa cell lines via modulation of cell cycle regulatory (Cdc2, Cdc25c, and Cyclin B1) and apoptosis - associated (Bax, Bad, Bcl-2, and Bcl-xl) proteins. VERU-111 treatment also inhibited tumor growth (P < 0.01) in a PanCa xenograft mouse model. Conclusions: This study has identified an inhibitor of βIII/βIV tubulins, which appears to have excellent potential as monotherapy or in combination with conventional therapeutic regimens for PanCa treatment.[4]

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We recently reported the crystal structure of tubulin in complex with a colchicine binding site inhibitor (CBSI), ABI-231, having 2-aryl-4-benzoyl-imidazole (ABI). Based on this and additional crystal structures, here we report the structure-activity relationship study of a novel series of pyridine analogues of ABI-231, with compound 4v being the most potent one (average IC50 ∼ 1.8 nM) against a panel of cancer cell lines. We determined the crystal structures of another potent CBSI ABI-274 and 4v in complex with tubulin and confirmed their direct binding at the colchicine site. 4v inhibited tubulin polymerization, strongly suppressed A375 melanoma tumor growth, induced tumor necrosis, disrupted tumor angiogenesis, and led to tumor cell apoptosis in vivo. Collectively, these studies suggest that 4v represents a promising new generation of tubulin inhibitors. [5]

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Novel ABI-III compounds were designed and synthesized based on our previously reported ABI-I and ABI-II analogues. ABI-III compounds are highly potent against a panel of melanoma and prostate cancer cell lines, with the best compound having an average IC(50) value of 3.8 nM. They are not substrate of Pgp and thus may effectively overcome Pgp-mediated multidrug resistance. ABI-III analogues maintain their mechanisms of action by inhibition of tubulin polymerization.[6]

C21H19N3O4

377.393265008926

377.14

C, 66.83; H, 5.07; N, 11.13; O, 16.96

1332881-26-1

2635953-17-0 (HCl);1332881-26-1;

53379371

Light yellow to yellow solid powder

3.4

89.2Ų

2

5

6

28

534

0

O(C)C1C(=C(C=C(C=1)C(C1=CN=C(C2=CNC3C=CC=CC2=3)N1)=O)OC)OC

WQGVHOVEXMOLOK-UHFFFAOYSA-N

InChI=1S/C21H19N3O4/c1-26-17-8-12(9-18(27-2)20(17)28-3)19(25)16-11-23-21(24-16)14-10-22-15-7-5-4-6-13(14)15/h4-11,22H,1-3H3,(H,23,24)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮和光照。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2 mg/mL (5.30 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.0 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2 mg/mL (5.30 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 20.0mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
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