MCT1/MCT4 (lactate transporters)

In HeLa cells, silosepine (10 μM; 1, 3, 4 hours) starts to accumulate intracellular acidity after 1 hour and peaks at 4 hours [1]. Silosepine (10 μM; 1, 2) inhibits MCT4 and MCT1 in MCT1-KO and MCT4-KO HAP1 cells, respectively, to prevent oscillatory disruption [1]. In MCT1-KO cells, the combination of dimethosine (10-100 μM) and syrosingopine (10-100 μM) inhibits HAP1 relay missynthesis, rendering it deadly [1].

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Syrosingopine Causes Intracellular Lactate Accumulation and Acidification. Syrosingopine and F3-syro Inhibit the Lactate Transporters MCT1 and MCT4. Lactate Efflux by MCT1 and MCT4 Is Inhibited by Syrosingopine and F3-syro. Lactate Import and Export Are Affected Differently by Syrosingopine and F3-syro. Syrosingopine and F3-syro Bind MCT1 and MCT4 In Vitro. Inhibition of Lactate Transport Is Required for Synthetic Lethality between Syrosingopine and Metformin [1]

The antihypertensive effect of syloserpine (5 mg/kg; subcutaneous injection; once) is equivalent to that observed when ganglionic nerve blockage via dormant peripheral norepinephrine reserves [2].

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In conscious spontaneously hypertensive rats (SHR), 2, 3, 6, 9, 12, and 16 months of age, the blockade of autonomic ganglia (with chlorisondamine) or postjunctional alpha 1-adrenergic receptors (with prazosin) or the depletion of peripheral norepinephrine stores (with syrosingopine), in contrast to the blockade of alpha 2-adrenergic receptors (with yohimbine, rauwolscine), produced a sustained decrease in the directly measured mean tail artery blood pressure. In 3- to 9-month-old SHR, the fall in blood pressure after prazosin pretreatment was significantly smaller than that after chlorisondamine or syrosingopine pretreatment. In ganglion-blocked SHR, prazosin decreased blood pressure only when this parameter had been elevated by an intra-arterial infusion of epinephrine or norepinephrine. In contrast, under the same experimental conditions, yohimbine or rauwolscine administration failed to modify the pressor effects of either phenylephrine or epinephrine but partially reduced those of norepinephrine and, unlike prazosin, strongly antagonized those of B-HT 920. In either intact or ganglion-blocked SHR, a 30-minute intra-arterial infusion of diltiazem at 100.0, but not 25.0, micrograms/kg/min significantly decreased baseline mean tail artery blood pressure. In ganglion-blocked SHR, the smaller dose of diltiazem antagonized by 40 and 80% the pressor effects of norepinephrine and B-HT 920, respectively, but failed to change the vasoconstrictor responses of phenylephrine, epinephrine, or vasopressin, which were, however, reduced by the higher dose of diltiazem. These results indicate that, in conscious adult SHR, norepinephrine released by peripheral sympathetic nervous terminals and humorally borne epinephrine stimulate almost exclusively post-junctional alpha 1-adrenergic receptors. The latter findings may account for the lack of blood pressure-lowering effects of the studied calcium antagonists at doses that effectively antagonize alpha 2-adrenergic receptor-mediated vasoconstriction in conscious SHR [2].

Cell viability assay [1]
Cell Types: HeLa, MCT1-KO, MCT4-KO HAP1, HAP1 MCT1-KO
Tested Concentrations: 10 µM
Incubation Duration: 1, 2, 3, 4 hrs (hours)
Experimental Results: Causes intracellular lactic acid accumulation and acidification1] . . Slows lactate efflux by inhibiting MCT4 and MCT1. Induces synthetic lethality by inhibiting lactate transport when used in combination with metformin.

Animal/Disease Models: Spontaneously hypertensive rats (SHR) (2 to 17 months old) [2].
Doses: 5 mg/kg
Route of Administration: subcutaneous injection; once (16 hrs (hrs (hours)) before blood pressure study).
Experimental Results: Exhibits antihypertensive activity by depleting peripheral norepinephrine stores.

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Mouse experiments [1]
Mice were injected intra-peritoneally with syrosingopine (7.5mg/kg body weight) 16 hours and 1 hour before sacrifice. Mice were euthanized with CO2 and blood taken from the body cavity for lactate measurement. Serum lactate levels were measured using an Arkray Lactate Pro 2 lactate test meter with corresponding test strips. Intracellular lactate was measured in liver tumor nodules. Nodules were excised (3 per mouse) and ground to a fine powder in liquid nitrogen. Pulverized tumor material was resuspended in 20 μL water and freeze-thawed 3 times (dry-ice/37° water bath) to release cell contents. Lactate was measured with the lactate test meter. Protein concentration was measured by BCA to normalize the lactate measurements between the nodules.

Oral LD50 in rats >2 gm/kg Iyakuhin Kenkyu. Medical Products Research. , 6(386), 1975
Intraperitoneal LD50 in rats 286 mg/kg Japanese Pharmaceuticals, 6(365), 1982
Subcutaneous LD50 in rats >2 gm/kg Iyakuhin Kenkyu. Medical Products Research. , 6(386), 1975
Intravenous LD50 in rats 50 mg/kg Archives Internationales de Pharmacodynamie et de Therapie., 119(245), 1959 [PMID:13628284]
Oral LD50 in mice 1293 mg/kg Japanese Pharmaceuticals, 6(365), 1982

[1]. Dual Inhibition of the Lactate Transporters MCT1 and MCT4 Is Synthetic Lethal with Metformin due to NAD+ Depletion in Cancer Cells. Cell Rep. 2018 Dec 11;25(11):3047-3058.e4.

[2]. Role of the sympathetic nervous system in blood pressure maintenance and in the antihypertensive effects of calcium antagonists in spontaneously hypertensive rats. Hypertension. 1988 Apr;11(4):360-70.

Sirosingopine is a yohimbine alkaloid. We provide direct evidence that sirosingopine is a dual inhibitor of lactate transporters MCT1 and MCT4. Furthermore, we demonstrate that this dual inhibition of MCT1 and MCT4 is the reason why sirosingopine, in combination with metformin, produces a synthetic lethal effect in human cancer cells. MCTs are crucial for cancer cell growth and survival (Doherty and Cleveland, 2013), therefore, considerable effort has been devoted to developing lactate transporter inhibitors as potential anticancer drugs. Several MCT1-specific inhibitors have been developed (Guile et al., 2006), one of which (AZD3965) is undergoing a phase I clinical trial in advanced cancer. A drawback of MCT1-specific inhibition is that it is ineffective when MCT4 is expressed. This is a particularly serious limitation because MCT4 expression is hypoxia-induced in most tumors. There are currently no reports of effective small-molecule inhibitors of MCT4. Pouysségur et al. previously mentioned an MCT4-specific inhibitor, AZ93 (Marchiq and Pouysségur, 2016). However, similar to MCT1-specific inhibitors, this compound was ineffective against cells simultaneously expressing MCT1 and MCT4 due to functional redundancy in the MCT1 and MCT4 transporters. Recently, it has been reported that acridine fibrin disrupts the MCT4-CD147 interaction (but not the MCT1-CD147 interaction) without affecting lactate secretion (Voss et al., 2017). We found no evidence that syrosingopine disrupts the interaction between MCT1 or MCT4 and CD147; instead, syrosingopine appears to interact directly with these transporters (Figure 4). In Xenopus oocyte lactate transport experiments, diclofenac was shown to inhibit MCT4 lactate uptake (Sasaki et al., 2016); however, its inhibitory effect on lactate uptake in human Caco-2 cells remains unclear due to the lack of characterization of MCT isoform expression. We provide direct biochemical evidence that cerosingopine can inhibit lactate transport by both MCT1 and MCT4. Furthermore, in cell lines containing different combinations of MCT1-4 isoform expression, the synthetic lethality of cerosingopine combined with metformin indicates that cerosingopine also inhibits MCT2, but has no activity against MCT3. How does the combination of cerosingopine and metformin produce synthetic lethality? Under physiological conditions, the reduction of pyruvate to lactate by LDH is promoted, thereby regenerating NAD+ consumed in the upstream ATP generation step of the glycolysis pathway (Figure 6A). MCT inhibition leads to lactate accumulation, resulting in increased intracellular lactate concentration, ultimately inhibiting LDH activity and causing loss of NAD+ regeneration capacity. Metformin simultaneously inhibits mitochondrial complex I (another major source of NAD+ regeneration), leading to a decrease in the NAD+/NADH ratio, reduced ATP production from glycolysis, and ultimately cell death. Exogenous NAD+ or the NAD precursor NMN can partially restore ATP levels, suggesting that the synthetic lethality is caused by NAD+ depletion. This restoration requires supraphysiological concentrations of NAD+ and NMN, possibly due to the poor permeability of these compounds in HL60 cells (Billington et al., 2008). Notably, after 48 hours of combined syrosingopine-metformin treatment, neither NAD+ nor NMN could prevent cell death (data not shown). Insufficient NAD+ to drive glycolysis may be a plausible reason for syrosingopine-metformin synthetic lethality. This is similar to the situation in DNA-damaged cells, where activated PARP1 consumes excess NAD+ (Ying et al., 2005), ultimately leading to cell death. Vitamin K2 can partially rescue ATP production in syrosingopine-metformin-treated cells. Exogenous vitamin K2 can temporarily increase NAD+ levels, thus transiently supporting glycolysis in the presence of syrosingopine-metformin. However, this rescue effect is transient due to the depletion of exogenous vitamin K2 and the loss of the NAD+/NADH regeneration mechanism, making it impossible to investigate its impact on cell proliferation. Nevertheless, it still provides supporting evidence for the proposed synthetic lethal mechanism. Syrosingopine, as a dual inhibitor of MCT1 and MCT4, may have additional antitumor effects in vivo. A large portion of the lactate secreted by cancer cells accumulates in the extracellular space, forming a tumor microenvironment that promotes cell invasion and metastasis (Kato et al., 2013). Lactic acidification also has an immunosuppressive effect on tumor-infiltrating immune cells (Brand et al., 2016; Fischer et al., 2007). Some tumors functionally differentiate into a hypoxic core of high glycolysis surrounded by a highly vascularized peripheral region, thus forming a metabolic symbiosis: the metabolic waste product lactate produced by the hypoxic core is used as fuel by normally oxygenated cancer cells in the tumor periphery. Tumor cells can also utilize lactate from surrounding stromal cells (reverse Wahlberg effect) or directly absorb lactate from the bloodstream (Faubert et al., 2017; Pavlides et al., 2009). Therefore, in all these cases, syrosingopine-mediated lactate retention in tumor cells provides additional benefits beyond the effects of drug combinations on glycolysis. There has been considerable interest in repositioning metformin as an anticancer drug, and several clinical trials have been initiated to evaluate its anticancer activity. Completed trials have reported mixed results, showing poor or no clinical efficacy (Kordes et al., 2015; Tsilidis et al., 2014). There is still considerable controversy regarding the effective concentration required for metformin to exert its antitumor activity (Chandel et al., 2016; Dowling et al., 2016). The concentrations of metformin that have demonstrated anticancer activity in preclinical models (mM level) are an order of magnitude higher than the serum metformin concentrations (μM level) achievable at conventional antidiabetic doses, suggesting that this may be part of the reason for the inconsistent results in clinical trials. Given this, the ability of syrosingopine to produce a synthetic lethal effect with metformin in cancer cells and to significantly reduce the effective concentration of metformin required in cell models (Figure S5F) may have potential clinical application value. Since inhibiting lactate transport alone can only have a cellular inhibitory effect at best, this suggests that the rational combination of MCT1 and MCT4 inhibitors with metformin may be an effective anticancer strategy against both classes of drugs. [1]

C35H42N2O11

666.71478

666.279

C, 63.05; H, 6.35; N, 4.20; O, 26.40

84-36-6

6769

White to yellow solid powder

1.35g/cm3

795ºC at 760 mmHg

175ºC

434.6ºC

1.615

4.635

1

12

13

48

1130

6

CCOC(=O)OC1=C(C=C(C=C1OC)C(=O)O[C@@H]2C[C@@H]3CN4CCC5=C([C@H]4C[C@@H]3[C@@H]([C@H]2OC)C(=O)OC)NC6=C5C=CC(=C6)OC)OC

ZCDNRPPFBQDQHR-SSYATKPKSA-N

InChI=1S/C35H42N2O11/c1-7-46-35(40)48-31-26(42-3)12-18(13-27(31)43-4)33(38)47-28-14-19-17-37-11-10-22-21-9-8-20(41-2)15-24(21)36-30(22)25(37)16-23(19)29(32(28)44-5)34(39)45-6/h8-9,12-13,15,19,23,25,28-29,32,36H,7,10-11,14,16-17H2,1-6H3/t19-,23+,25-,28-,29+,32+/m1/s1

3beta,20alpha-Yohimban-16beta-carboxylic acid, 18beta-hydroxy-11,17alpha-dimethoxy-, methyl ester, 4-hydroxy-3,5-dimethoxybenzoate (ester) ethyl carbonate (ester) (8CI)

HF41T; SU3118; HF 41T; syrosingopine; 84-36-6; Syringopine; Isotense; Londomin; Neoreserpan; Siringina; Menatensina; SU 3118; Syrosingopine; HF-41T; SU-3118

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~62.5 mg/mL (~93.74 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.12 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.12 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)