The NLRP3 inhibitor Dapansutrile improves the therapeutic action of lonafarnib on progeroid mice

Abstract

The role of the inflammasomes in aging and progeroid syndromes remain understudied. Recently, MCC950, a NLRP3 inhibitor, was used in Zmpste24^-/- mice to ameliorate the phenotypes. However, the safety of MCC950 was questioned due to liver toxicity observed in humans. Nevertheless, inhibition of the inflammasomes would be a beneficial therapy for progeria. Here, we show that OLT1177 (dapansutrile), other NLRP3 inhibitor, improved cellular and animal phenotypes using progeroid fibroblasts and a Lmna^G609G/G609G mouse model. In both cases dapansutrile reduced progerin accumulation, NLRP3-inflammasome activation and secretory phenotype of senescence, extended the lifespan of progeroid animals, preserved bodyweight, and reduced kyphosis, inflammation, and senescence. Interestingly, dapansutrile further improved the effect of lonafarnib, the only FDA-approved drug for the progeria. The combination of both drugs reduced the inflammation and senescence, extended survival and ameliorated various progeroid defects both in vitro and in vivo, compared with treatment using lonafarnib alone. These findings and the safety of dapansutrile demonstrated in several clinical trials proposes it as a possible co-adjuvant treatment with lonafarnid in HGPS.

Keywords: Dapansutrile; Lonafarnib; NLRP3; progeria.

FIGURE 1

Dapansutrile reduces skin fibroblasts inflammasome activation, senescence and progeroid phenotypes. (a) Cell growth with Dapansutrile determined in healthy and HGPS fibroblasts from eight patients. (b) Effect of Dapansutrile on mRNA expression of NLRP1, NLRP3, ASC and Caspase 1 determined by qPCR experiments in skin fibroblasts, n = 3 (controls) and n = 8 (patients). (c, d) Western blot analysis showing representative blots of NLRP1, NLRP3, caspase 1, and β‐Actin levels in skin fibroblasts from patients with HGPS, n = 2 (controls) and n = 2 (patients). (e) Immunofluorescence (IF) visualization of γH2AX (green) and nuclei (blue) in skin fibroblasts from a representative patient and control. (f, g) Protein expression of p21 and p16 in skin fibroblasts from patients with HGPS, n = 4 (controls) and n = 4 (patients) (Two more patients and control in Figure S1). Data are shown as means ± SD. ***p < 0.001, **p < 0.005.

FIGURE 2

NLRP3 Inhibition by dapansutrile ameliorates Lmna^G609G/G609G mice progeroid phenotypes. (a) Body weights of the indicated groups at 8 and 12 weeks after treatments. (b) Kyphosis percentage of untreated (n = 6 males) and dapansutrile‐treated (n = 6 males) Lmna^G609G/G609G mice. Image show two representative animal after 4 months of treatment. (c) Kaplan–Meier graph showing a significant increase in the medium and maximum lifespan in treated mice compared with Lmna^G609G/G609G mice. N = 8 per group. (d) Western blot analysis showing representative blots of NLRP3, total and active caspase 1, active IL‐1β, progerin and β‐Actin levels in heart and liver tissues from wild‐type, Lmna^G609G/G609G and dapansutrile‐treated Lmna^G609G/G609G mice. n = 5 mice per group. Densitometry in Figure S5. (e) A heat map depicting the expression of 44 mouse cytokines in the serum of WT, Lmna^G609G/G609G, and dapansutrile‐treated Lmna^G609G/G609G mice. (f) Western blot analysis showing representative blots of p53, p21 and β‐Actin levels in heart and liver from wild‐type, Lmna^G609G/G609G and dapansutrile‐treated Lmna^G609G/G609G mice. Densitometry in Figure S6. n = 5 mice per group. ***p < 0.001, **p < 0.005.

FIGURE 3

Dapansutrile works as a co‐treatment of lonafarnib in Lmna^G609G/G609G mice. (a–d) Effect of lonafarnib, dapansutrile, and lonafarnib combined with dapansutrile on mRNA levels of NLRP1, NLRP3, ASC and Caspase 1 determined by qPCR experiments in skin fibroblasts, n = 3 (controls) and three patients per group. (e) Heat map depicting expression of 48 human cytokines in the medium of skin fibroblasts with lonafarnib, dapansutrile and lonafarnib combined with dapansutrile, n = 3 (controls) and n = 3 (patients) per group. (f, g) Effect of culturing macrophages in conditioned medium (CM). CM was collected from control, HGPS, HGPS+lonafarnib, HGPS+dapansutrile and HGPS+lonafarnib combined with dapansutrile fibroblasts. Protein levels of NLRP1, NLRP3, and caspase 1 are shown in (f, Densitometry in Figure S9), and levels of IL1β and IL‐6 release in (g). (h) Kaplan–Meier graph showing a significant increase in the medium and maximum lifespan in untreated versus treated Lmna^G609G/G609G mice with the different treatments indicated. N = 8 per group. (i) Body weights of the indicated groups at 8 and 12 weeks after the indicated treatment. (j) Representative image of kyphosis of untreated and treated Lmna^G609G/G609G mice with lonafarnib and lonafarnib+dapansutrile. Data are shown as means ± SD. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.005.