Structure based discovery of clomifene as a potent inhibitor of cancer-associated mutant IDH1

Abstract

Isocitrate dehydrogenase (IDH) plays an indispensable role in the tricarboxylic acid cycle, and IDH mutations are present in nearly 75% of glioma and 20% of acute myeloid leukemia. One IDH1R132H inhibitor (clomifene citrate) was found by virtual screening method, which can selectively suppress mutant enzyme activities in vitro and in vivo with a dose-dependent manner. The molecular docking indicated that clomifene occupied the allosteric site of the mutant IDH1. Enzymatic kinetics also demonstrated that clomifene inhibited mutant enzyme in a non-competitive manner. Moreover, knockdown of mutant IDH1 in HT1080 cells decreased the sensitivity to clomifene. In vivo studies indicated that clomifene significantly suppressed the tumor growth of HT1080-bearing CB-17/Icr-scid mice with oral administration of 100 mg/kg and 50 mg/kg per day. In short, our findings highlight clomifene may have clinical potential in tumor therapies as a safe and effective inhibitor of mutant IDH1.

Keywords: IDH1; cancer; clomifene; drug repurposing; virtual ligand screening.

Figure 1. Clomifene specifically binds with IDH1R132H and selectively inhibits mutant IDH1 activity in vitro

(a) Chemical structure of clomifene and AGI-5198. (b) Detailed view of clomifene binding in the ligand binding pocket. (c) Overlay of one monomer of the IDH1 R132H-clomifene binary complex. (d, e) Measurements of affinity of clomifene and AGI5198 with IDH1R132H by MST in standard treated capillaries, the resulting binding curve was shown. From the binding curve, the Kd values of 18.45 ± 1.61 μM (Clomifene) and 2.90 ± 0.14 μM (AGI5198) were calculated. (f) IC₅₀ of clomifene against wild-type and mutant IDH1 and IDH1 (g, h) Clomifene inhibits IDH1R132H noncompetitively with respect to NADPH and α-KG, respectively.

Figure 2. Clomifene suppresses IDH1 activity in celluro

(a) Inhibitory activities of IDH1R132H enzyme lead to decreased production of D-2HG in IDH1R132H mutant HT1080 cells after treatment with clomifene for 48 h, and D-2HG was measured by LC/MS method. (b) HT1080 cells were treated for 24 h in the different concentrations of clomifene and then processed for FACS by using Annexin V/propidium iodide staining. (c) Treatment of clomifene significantly reduced methylation levels of histone lysine residues in HT1080 cells. All data are the average of results from triplicate experiments.

Figure 3. Knocking down IDH1R132C attenuates the inhibitory effect of HT1080 cell growth by clomifene

(a) Expression level of IDH1 in HT1080 cells is decreased by knockdown of IDH1. HT1080 cells were transiently transfected with shMOCK or shIDH1 and cell lysates were analyzed by western blot. (b) Clomifene has less effect on cell growth of shIDH1 transfected cells than that of shMOCK cells. (c) Mutant IDH1-mediated H3K9me3 was substantially decreased dose dependently with clomifene treatment.

Figure 4. Effect of Clomifene(CMF) on fibrosarcoma cancer growth in an HT1080 xenograft mouse model

(a) The average tumor volume of vehicle-treated control mice (n = 6) and clomifene treated mice (n = 6, 50 or 100 mg/kg per day via gavage) plotted over 21 days after tumor cell injection. (b) Clomifene has no effect on mouse body weight. (c, d) After 2 weeks’ treatment, differences in tumor size and weight are shown. The asterisk * indicates a significant increased tumor size (P < 0.05) in the vehicle-treated group compared with the clomifene-treated group as determined by one-way analysis of variance.

Figure 5. Clomifene (CMF) suppresses IDH1 activity in vivo

(a) Immunohistochemical analysis for the H3K9me3 antibodies was analyzed using mouse tumor samples. The magnification of representative photographs for the immunohistochemistry staining is 400×. (b, c) Clomifene inhibits D-2HG production in serum and tumor tissues.