Discovery of Sulanemadlin (ALRN-6924), the First Cell-Permeating, Stabilized α-Helical Peptide in Clinical Development.

We report the discovery of sulanemadlin (ALRN-6924), the first cell-permeating, stabilized α-helical peptide to enter clinical trials. ALRN-6924 is a "stapled peptide" that mimics the N-terminal domain of the p53 tumor suppressor protein. It binds with high affinity to both MDM2 and MDMX (also known as MDM4), the endogenous inhibitors of p53, to activate p53 signaling in cells having a non-mutant, or wild-type TP53 genotype (TP53-WT). Iterative structure-activity optimization endowed ALRN-6924 with favorable cell permeability, solubility, and pharmacokinetic and safety profiles. Intracellular proteolysis of ALRN-6924 forms a long-acting active metabolite with potent MDM2 and MDMX binding affinity and slow dissociation kinetics. At high doses, ALRN-6924 exhibits on-mechanism anticancer activity in TP53-WT tumor models. At lower doses, ALRN-6924 transiently arrests the cell cycle in healthy tissues to protect them from chemotherapy without protecting the TP53-mutant cancer cells. These results support the continued clinical evaluation of ALRN-6924 as an anticancer and chemoprotection agent.

Stapled α-helical peptide drug development: a potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy.

Stapled α-helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. Here, we report a potent and selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo. Specifically, ATSP-7041 binds both MDM2 and MDMX with nanomolar affinities, shows submicromolar cellular activities in cancer cell lines in the presence of serum, and demonstrates highly specific, on-target mechanism of action. A high resolution (1.7-Å) X-ray crystal structure reveals its molecular interactions with the target protein MDMX, including multiple contacts with key amino acids as well as a role for the hydrocarbon staple itself in target engagement. Most importantly, ATSP-7041 demonstrates robust p53-dependent tumor growth suppression in MDM2/MDMX-overexpressing xenograft cancer models, with a high correlation to on-target pharmacodynamic activity, and possesses favorable pharmacokinetic and tissue distribution properties. Overall, ATSP-7041 demonstrates in vitro and in vivo proof-of-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein-protein interaction and may offer a viable modality for cancer therapy.

Role of ghrelin in regulating rabbit ovarian function and the response to LH and IGF-I.

The aim of these in vivo and in vitro studies was to examine the role of ghrelin in the control of plasma hormone concentrations, the proliferation, apoptosis and secretory activity of ovarian granulosa cells and the response of these cells to hormonal treatments. Female rabbits were injected with ghrelin (10 microg/animal/day for one week before ovulation induced by 25IU PMSG and 0.25IU LHRH). On the day of ovulation, blood samples were collected and analyzed for concentrations of progesterone (P(4)), testosterone (T), estradiol (E(2)), estrone-sulphate (ES), insulin-like growth factor I (IGF-I) and leptin (L) by RIA. Some control and ghrelin-treated animals were killed in the periovulatory period, their ovaries were weighed and granulosa cells were isolated and cultured for 2d. Cell proliferation (expression of PCNA) and apoptosis (expression of TdT) were evaluated by immunocytochemistry and TUNEL respectively. Secretion of P(4), T, E(2), IGF-I, and prostaglandin F (PGF) by granulosa cells cultured with and without LH or IGF-I (1, 10 or 100 ng/ml medium) was assessed by RIA. The remaining control and treated animals were kept until parturition, while the number, viability and body weight of pups were recorded. Ghrelin treatment increased rabbit plasma T and decreased ES concentrations but did not influence P(4), E(2), IGF-I or L. Granulosa cells from ghrelin-treated animals showed higher expression of PCNA and lower expression of TdT, than those from control animals. They also secreted less P(4), T, E(2), IGF-I and PGF than granulosa cells from untreated animals. Treatment of cultured granulosa cells with ghrelin (1, 10 or 100 ng/ml medium) either increased (at 1 ng/ml) or decreased (at 10 ng/ml) P(4) secretion, increased (at 100 ng/ml) or decreased (at 10 ng/ml) IGF-I secretion, decreased T (at 1 and 10 ng/ml) and OT (at 1 ng/ml) secretion, and increased (at 100 ng/ml) PGF secretion. LH treatment of cells from control animals stimulated P(4) (at 1 and 10 ng/ml), E(2), and IGF-I (both at 10 and 100 ng/ml), but not T secretion. IGF-I stimulated P(4) (all concentrations) and PGF (at 100 ng/ml) but suppressed T (all concentrations) and E(2) (at 1 and 10 ng/ml) secretion. Pre-treatment of animals with ghrelin stimulated, suppressed or even reversed subsequent LH and IGF-I effects on hormone secretion by cultured granulosa cells. Ghrelin injections did not affect ovarian weight or the number and body mass of pups born, although pup mortality was significantly lower in ghrelin-treated than in control mothers. These observations suggest that ghrelin is involved in the control of ovarian cell proliferation, apoptosis and secretion of hormones, as well as in the response of these cells to physiological stimulators such as LH and IGF-I.

Effects of chronic food restriction and treatments with leptin or ghrelin on different reproductive parameters of male rats.

The existence of a close relationship between energy status and reproductive function is well-documented, especially in females, but its underlying mechanisms remain to be fully unfolded. This study aimed to examine the effects of restriction of daily calorie intake, as well as chronic treatments with the metabolic hormones leptin and ghrelin, on the secretion of different reproductive hormones, namely pituitary gonadotropins and prolactin, as well as testosterone, in male rats. Restriction (50%) in daily food intake for 20 days significantly reduced body weight as well as plasma PRL and T levels, without affecting basal LH and FSH concentrations and testicular weight. Chronic administration of leptin to rats fed ad libitum increased plasma PRL levels and decreased circulating T, while it did not alter other hormonal parameters under analysis. In contrast, in rats subjected to 50% calorie restriction, leptin administration increased plasma T levels and reduced testis weight. Conversely, ghrelin failed to induce major hormonal changes but tended to increase testicular weight in fed animals, while repeated ghrelin injections in food-restricted males dramatically decreased plasma LH and T concentrations and reduced testis weight. In sum, we document herein the isolated and combined effects of metabolic stress (50% food restriction) and leptin or ghrelin treatments on several reproductive hormones in adult male rats. Overall, our results further stress the impact and complex way of action of different metabolic cues, such as energy status and key hormones, in reproductive function also in the male.

Effect of ghrelin on activities of some lysosomal hydrolases in rabbits.

OBJECTIVES: Observing the changes of activity of some lysosomal enzymes in blood serum of female rabbits subjected to injection of 10 microg of ghrelin/kg of body weight. METHODS: In the blood serum the activity of cathepsins D and L, alanine aminopeptidase, acid phosphatase, lysosomal lipase and lysosomal esterase was determined. RESULTS: As a result of ghrelin injection the activity of all the enzymes examined in blood serum increased markedly. CONCLUSION: Changes of lysosomal enzymes activities in the blood serum caused by the effects of ghrelin should be regarded as the response of the lysosomal system.