complex I of oxidative phosphorylation (OXPHOS)
Mitochondrial electron transport chain complex I (no specific IC50, Ki, or EC50 values provided) [2]
Oxidative phosphorylation complex I (inhibited ATP production and oxygen consumption at single-digit nM concentrations in the presence of malate/glutamate; inhibited the conversion of NADH to NAD+ at low nM concentrations in an immunoprecipitated complex I assay) [1]

IACS-010759 hydrochloride (10, 30, 100 nM; for 4 or 5 days) lowers the viability of primary AML and promotes apoptosis [1]. IACS-010759 hydrochloride (0.001, 0.01, 0.1, 1, 10, 100, 1000 nM; 72 h) substantially suppresses OCR and galactose-dependent H460 cell survival with practically the same IC50 value of 1.4 nM[1]. IACS-010759 hydrochloride has similar action in mouse (average IC50=5.6 nM), rat (IC50=12.2 nM) and cynomolgus monkey (IC50=8.7 nM) cell lines [1].

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Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in brain cancer and acute myeloid leukemia (AML) cell models reliant on oxidative phosphorylation (OXPHOS). The inhibitory effects are likely attributed to energy depletion and reduced aspartate production, which further impairs nucleotide biosynthesis [2]
In isolated mitochondria or permeabilized cells, IACS-010759 inhibited ATP production and oxygen consumption at single-digit nM concentrations when malate/glutamate was present, but showed no inhibitory effect in the presence of succinate. Additionally, in an immunoprecipitated complex I assay, the compound inhibited the conversion of NADH to NAD+ at low nM concentrations [1]

In mice with NB-1 (PGD null) subcutaneous xenografts, IACS-010759 hydrochloride (5, 10, 25 mg/kg/day; oral; for 21 days) at dosages of 5 or 10 mg/kg caused tumor reduction, negligible weight loss, and intolerance to the 25 mg/kg dose [1]. Less frequent dosing regimens (Q2D or Q3D) may somewhat enhance survival, but IACS-010759 hydrochloride (10?mg/kg; oral; QD (daily) or QD×5 (5 d on/2 d off); for 35 d) prolonged median survival from 28 days to over 60 days [1]. With a high volume of distribution and low plasma clearance, IACS-010759 hydrochloride (0.3 mg/kg for intravenous; 1 mg/kg for oral) has a prolonged terminal half-life (>24 hours) [1].

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In in vivo models of brain cancer and AML, IACS-010759 potently inhibited tumor growth at well-tolerated doses [2]
In an orthotopic AML cell line xenograft model, daily oral administration of IACS-010759 at doses ranging from 10 mg/kg to 25 mg/kg resulted in a >50-day extension of median survival. In diffuse large B-cell lymphoma (DLBCL) and glioblastoma multiforme (GBM) xenograft models, the same dosing regimen led to robust tumor regression [1]

Isolated mouse complex I assay[1]
Complex I was isolated from mouse heart mitochondria using an adaptation of the method of Sharpley and colleagues. The concentration of IACS-010759 in Fig. 1f was 60 nM. The NADH:decylubiquinone assay is described in Sharpley et al, and the APAD+ and H2O2 assays are described in Birrell et al.

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An immunoprecipitated complex I assay was performed to assess the inhibitory effect of IACS-010759 on complex I activity. The key reaction monitored in this assay was the conversion of NADH to NAD+. Different concentrations of the compound were tested, and it was found that IACS-010759 could inhibit this conversion reaction at low nM concentrations [1]

Generation of clonal cell lines resistant to IACS-010759[1]
H292 cells (1 × 106 cells/plate) were seeded in 15-cm dishes in galactose growth medium and treated with 1 nM IACS-010759 (IC65) for 3 weeks, followed by exposure to 8 nM IACS-010759 (IC95) until resistant clones emerged. Twenty-six resistant clones were isolated from four independent experiments and were seeded at 5 × 103 cells/well in 96-well plates in 100 µl galactose growth medium. After cells became fully attached, IACS-010759 or rotenone was added to a final concentration of 370 nM to 18 pM for 3 d. Plates were scanned in the IncuCyte live-cell analysis system before analysis via Hoechst and PI. Subsequently, both Hoechst and PI using an Operetta high-content imaging system. RNA-seq was conducted on the parental line and 12 resistant clones, uncovering a single nonsynonymous, heteroplasmic (35–50%), recurrent mutation in the mitochondrial-encoded gene MT-ND1 in 9 of the 12 resistant clones that conferred the L55F (T3469C) amino acid change. Paired-end reads were initially aligned to transcript sequences of complex I genes with Bowtie 2 (ref.53), and the aligned fragments were probabilistically assigned to transcripts using eXpress54. Variants from the reference genome were called using the ‘mpileup’ command in SAMtools. MutPred55 analysis of the L55F variant classifies the alteration as potentially pathogenic (MutPred score = 0.8); this alteration is found at a very low frequency in mtDNA sequences in Genebank (1:30,589 based on full-length mitochondrial genomes deposited in Genebank before 28 October 2015), suggesting it is unlikely to be a polymorphism. The mutation was confirmed in four of the resistant clones by cloning the MT-ND1 gene sequence and analyzing purified plasmid DNA via Sanger sequencing using the following primers: Forward: 5′-GTAAAACGACGGCCAGT-3′ and Reverse: 5′-AACAGCTATGACCATG-3′.

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Brain cancer and AML cell lines that are reliant on OXPHOS were selected as the in vitro cell models. These cells were treated with IACS-010759, and subsequent assessments were conducted to measure cell proliferation and apoptosis. The results showed that the compound significantly inhibited cell proliferation and induced obvious apoptosis in these cell models [2]
Isolated mitochondria or permeabilized cells were used for the experiment. The cells or mitochondria were exposed to IACS-010759 under two different conditions: one with malate/glutamate present and the other with succinate present. After treatment, the levels of ATP production and oxygen consumption were detected to evaluate the impact of the compound on mitochondrial function and OXPHOS activity [1]

IACS-010759 exhibits good oral bioavailability and demonstrates excellent physicochemical properties in preclinical animal models [1]

IACS-010759 was well tolerated at the doses used in in vivo studies [2]

[1]. Protopopova M. IACS-10759: A novel OXPHOS inhibitor which selectively kill tumors with metabolic vulnerabilities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4380. doi:10.1158/1538-7445.AM2015-4380.

[2]. An inhibitor of oxidative phosphorylation exploits cancer vulnerability. Nat Med. 2018 Jul;24(7):1036-1046.

Tumor cells typically rely on glycolysis and oxidative phosphorylation (OXPHOS) to provide energy and macromolecular building blocks for sustained growth. Genetic or epigenetic inactivation of either of these redundant pathways represents a metabolic vulnerability that should be susceptible to inhibitors of the other pathway. Through extensive medicinal chemistry studies, IACS-10759 has been identified as a potent inhibitor of oxidative phosphorylation complex I. In isolated mitochondria or permeabilized cells, IACS-10759 inhibits ATP production or oxygen consumption at nanomolar concentrations in the presence of malate/glutamate, but has no effect in the presence of succinate. More directly, in immunoprecipitation complex I assays, IACS-10759 inhibits the conversion of NADH to NAD+ at low nanomolar concentrations. The specific complex I subunit inhibited by IACS-10759 has been identified using genetic and pharmacological methods, and the mechanism of its inhibition of complex I is currently under investigation. Importantly, IACS-10759 exhibits good oral bioavailability, demonstrates excellent physicochemical properties in preclinical animal models, and achieves significant in vivo efficacy at daily oral doses of 10-25 mg/kg. Specifically, in orthotopic AML cell line xenograft models, median survival was extended by more than 50 days; in DLBCL and GBM xenograft models, tumor regression was also significant. Based on these results and its drug-like properties, IACS-10759 has entered the IND application preparation stage, with plans to conduct its first human trial in the third quarter of 2015.

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IACS-010759 is a clinical-grade small molecule inhibitor targeting mitochondrial electron transport chain complex I. Metabolic reprogramming is an emerging hallmark of tumor biology. Although therapeutic targeting of glycolysis has been extensive, drug development targeting mitochondrial oxidative phosphorylation (OXPHOS) remains underdeveloped, partly due to insufficient understanding of the tumor environment crucial to OXPHOS. This compound takes advantage of the metabolic vulnerability of OXPHOS-dependent tumors, making it a potential therapeutic for cancers such as brain cancer and acute myeloid leukemia (AML)[2]. IACS-010759 is a potent inhibitor of OXPHOS complex I discovered through extensive medicinal chemistry studies. The specific complex I subunit that this compound inhibits has been identified using genetic and pharmacological methods, and the mechanism of complex I inhibition is currently under investigation. Due to its good in vivo efficacy and drug-like properties, IACS-010759 has entered the Investigational New Drug (IND) application stage and is scheduled to begin its first human trial in the third quarter of 2015[1]. IACS-010759 is currently undergoing a Phase I clinical trial for relapsed/refractory acute myeloid leukemia (AML) and solid tumors[2].

C25H26CLF3N6O4S

599.024953365326

598.137

C, 50.13; H, 4.38; Cl, 5.92; F, 9.51; N, 14.03; O, 10.68; S, 5.35

1807523-99-4

IACS-010759; 1570496-34-2; 1570496-34-2 (HCl); 1807524-00-0 (besylate); 1807524-05-5; 1807524-01-1 (mesylate)

91864637

Typically exists as white to off-white solids at room temperature

1

12

7

40

909

0

LUSCFOVOISLXTM-UHFFFAOYSA-N

InChI=1S/C25H25F3N6O4S.ClH/c1-16-29-23(24-30-22(32-38-24)18-6-8-20(9-7-18)37-25(26,27)28)31-34(16)15-17-4-3-5-19(14-17)33-12-10-21(11-13-33)39(2,35)36;/h3-9,14,21H,10-13,15H2,1-2H3;1H

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.17 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 25.0 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.)