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.

rat LD50 oral >2 gm/kg Iyakuhin Kenkyu. Study of Medical Supplies., 6(386), 1975
rat LD50 intraperitoneal 286 mg/kg Drugs in Japan, 6(365), 1982
rat LD50 subcutaneous >2 gm/kg Iyakuhin Kenkyu. Study of Medical Supplies., 6(386), 1975
rat LD50 intravenous 50 mg/kg Archives Internationales de Pharmacodynamie et de Therapie., 119(245), 1959 [PMID:13628284]
mouse LD50 oral 1293 mg/kg Drugs in Japan, 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.

Syrosingopine is a yohimban alkaloid.

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We show direct evidence that syrosingopine is a dual inhibitor of the lactate transporters MCT1 and MCT4. Furthermore, we show that dual inhibition of MCT1 and MCT4 accounts for the synthetic lethality of syrosingopine in combination with metformin in human cancer cells. The MCTs are important for cancer cell growth and survival (Doherty and Cleveland, 2013), and accordingly, a lot of effort has been invested in developing lactate transporter inhibitors as potential anti-cancer agents. Several MCT1-specific inhibitors have been developed (Guile et al., 2006), with one (AZD3965) in phase I clinical trials for advanced cancer. The disadvantage of MCT1-specific inhibition is that it is ineffective when MCT4 is expressed. This is a particularly severe limitation, as MCT4 expression is induced by hypoxia in the majority of tumors. There are no reports of an effective small molecule inhibitor for MCT4. Pouysségur and colleagues have alluded to an MCT4-specific inhibitor, AZ93 (Marchiq and Pouysségur, 2016). However, similar to MCT1-specific inhibitors, this compound is ineffective in cells expressing both MCT1 and MCT4 due to the functional redundancy of the transporters. Acriflavine was recently reported to disrupt the MCT4-CD147 interaction (but not MCT1-CD147), but this had no effect on lactate secretion (Voss et al., 2017). We see no evidence that syrosingopine disrupts the interaction between MCT1 or MCT4 and CD147; instead, syrosingopine appears to interact directly with the transporters (Figure 4). Diclofenac was shown to prevent lactate uptake by MCT4 in a Xenopus oocyte lactate transport assay (Sasaki et al., 2016); however, its inhibitory effect on lactate uptake in human Caco-2 cells is unclear, as MCT isoform expression was not characterized. We show direct biochemical evidence that syrosingopine inhibits lactate transport by MCT1 and MCT4. In addition, synthetic lethality between syrosingopine and metformin in a cell panel comprising various combinations of MCT1-4 isoform expression suggests that syrosingopine is also able to inhibit MCT2 but is inactive against MCT3. How does syrosingopine in combination with metformin elicit synthetic lethality? Under physiological conditions, the reduction of pyruvate to lactate by LDH is favored and serves to regenerate NAD+ consumed upstream in the ATP-producing steps of the glycolytic pathway (Figure 6A). Lactate accumulation upon MCT inhibition leads to high intracellular lactate concentrations that can result in end-product inhibition of LDH and, consequently, loss of NAD+ regenerating capacity. The simultaneous inhibition by metformin of mitochondrial complex I, the other main source of NAD+ regeneration, results in a decrease in the NAD+/NADH ratio, loss of glycolytic ATP production, and cell death. The observation that ATP levels can be partly restored by exogenous NAD+ or the NAD precursor NMN suggests that the cause of synthetic lethality is NAD+ depletion. This rescue requires supra-physiological concentrations of NAD+ and NMN, which may be due to the poor permeability of these compounds in HL60 cells (Billington et al., 2008). We note that NAD+ and NMN are unable to prevent cell death after 48 hr of syrosingopine-metformin treatment (data not shown). The lack of NAD+ to fuel glycolysis is a plausible reason for syrosingopine-metformin synthetic lethality. This is reminiscent of the situation in DNA-damaged cells where activated PARP1 consumes excessive amounts of NAD+ (Ying et al., 2005), leading to cell death. Vitamin K2 partially rescues ATP production in syrosingopine-metformin-treated cells. Exogenous vitamin K2 gives a temporary boost in NAD+ levels that transiently supports glycolysis despite syrosingopine-metformin treatment. The rescue is short-lived due to the depletion of exogenous vitamin K2 and the absence of NAD+/NADH-regenerating mechanisms, which unfortunately precludes the opportunity of studying an effect on cell proliferation. Nevertheless, it provides supporting evidence for the proposed mechanism of synthetic lethality. Syrosingopine, as a dual MCT1 and MCT4 inhibitor, may have additional anti-tumor benefits in vivo. Most of the lactate secreted by cancer cells accumulates in the extracellular space, creating a tumor microenvironment that promotes cell invasion and metastasis (Kato et al., 2013). Extracellular acidification by lactate also has an immunosuppressive effect on tumor-infiltrating immune cells (Brand et al., 2016, Fischer et al., 2007). Functional differentiation in some tumors into highly glycolytic hypoxic cores surrounded by well-vascularized outer regions results in metabolic symbiosis where lactate generated as a waste product in the hypoxic core is utilized as a fuel by normoxic cancer cells at the tumor periphery. Tumor cells can also utilize lactate originating from surrounding stromal cells (the reverse Warburg effect) or directly take up lactate from the circulation (Faubert et al., 2017, Pavlides et al., 2009). Thus, in all these scenarios, syrosingopine-mediated trapping of lactate in tumor cells can provide an additional bonus beyond the effect of the drug combination on glycolysis. There is great interest in re-positioning metformin as an anti-cancer drug, and numerous clinical trials have been initiated to assess its anti-cancer activity. Concluded trials have reported mixed results, showing either no or weak clinical efficacy (Kordes et al., 2015, Tsilidis et al., 2014). There is considerable debate on the effective metformin concentration required for anti-neoplastic activity (Chandel et al., 2016, Dowling et al., 2016). The metformin concentration used in pre-clinical models demonstrating anti-cancer activity (mM range) is an order of magnitude greater than the serum metformin concentration attainable with routine anti-diabetic dosing (μM range), suggesting that this may be partly the reason behind the mixed results from the clinical trials. In this light, the ability of syrosingopine to elicit synthetic lethality with metformin in cancer cells and to substantially lower the effective concentration of metformin required in cellular models (Figure S5F) may be of potential clinical benefit. As lactate transport inhibition alone is at best cytostatic, this suggests that the rational combination of an MCT1 and MCT4 inhibitor with metformin may prove a viable anti-cancer strategy for both drug classes.[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.)