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.

An inhibitor of KDM5 demethylases reduces survival of drug-tolerant cancer cells.

The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.

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.

IKKbeta inhibition protects against bone and cartilage destruction in a rat model of rheumatoid arthritis.

OBJECTIVE: The IKK complex regulates NF-kappaB activation, an important pathway implicated in the rheumatoid arthritis (RA) disease process. This study was undertaken to assess the efficacy of N-(6-chloro-7-methoxy-9H-beta-carbolin-8-yl)-2-methylnicotinamide (ML120B), a potent and selective small molecule inhibitor of IKKbeta. METHODS: Polyarthritis was induced in rats by injection of Freund's complete adjuvant into the hind footpad. ML120B was administered orally twice daily, either prophylactically or therapeutically. Paw volumes and body weights were measured every 2-3 days throughout the study. We assessed bone erosions by several methods: histologic evaluation, quantitative micro-computed tomography (micro-CT) imaging analysis, and measurement of type I collagen fragments in the serum. Quantitative polymerase chain reaction was used to evaluate expression of messenger RNA for genes related to inflammation and to bone and cartilage integrity. RESULTS: Oral administration of ML120B inhibited paw swelling in a dose-dependent manner (median effective dosage 12 mg/kg twice daily) and offered significant protection against arthritis-induced weight loss as well as cartilage and bone erosion. We were able to directly demonstrate that NF-kappaB activity in arthritic joints was reduced after ML120B administration. Also, we observed that down-regulation of the NF-kappaB pathway via IKKbeta inhibition dampened the chronic inflammatory process associated with rat adjuvant-induced arthritis. CONCLUSION: The results of the present study suggest that IKKbeta inhibition is an effective therapeutic approach to treat both the inflammation and the bone/cartilage destruction observed in RA. Methods for the determination of serum markers for bone and cartilage destruction, as well as micro-CT analysis, may aid in predicting and evaluating the therapeutic efficacy of IKKbeta inhibition therapy in humans.

A selective small molecule IkappaB Kinase beta inhibitor blocks nuclear factor kappaB-mediated inflammatory responses in human fibroblast-like synoviocytes, chondrocytes, and mast cells.

IkappaB kinase (IKK) beta is essential for inflammatory cytokine-induced activation of nuclear factor kappaB (NF-kappaB). NF-kappaB plays a pivotal role in the function of major cell types that contribute to the pathophysiological process of rheumatoid arthritis (RA). Here, we report the mechanism and the effect of the IKKbeta inhibitor N-(6-chloro-7-methoxy-9H-beta-carbolin-8-yl)-2-methylnicotinamide (ML120B), a beta-carboline derivative, on NF-kappaB signaling and gene activation in RA-relevant cell systems. ML120B is a potent, selective, reversible, and ATP-competitive inhibitor of IKKbeta with an IC50 of 60 nM when evaluated in an IkappaBalpha kinase complex assay. ML120B does not inhibit other IKK isoforms or a panel of other kinases. ML120B concentration-dependently inhibits tumor necrosis factor alpha (TNFalpha)-stimulated NF-kappaB signaling via inhibition of IkappaBalpha phosphorylation, degradation, and NF-kappaB translocation into the nucleus. For the first time, we have demonstrated that in human fibroblast-like synoviocytes, TNFalpha- or interleukin (IL)-1beta-induced monocyte chemoattractant protein-1 regulated on activation, normal T cell expressed and secreted and production is IKKbeta-dependent. In addition, for the first time, we have demonstrated that lipopolysaccharide- or peptidoglycan-induced cytokine production in human cord blood-derived mast cells is IKKbeta-dependent. In addition, in human chondrocytes, ML120B inhibited IL-1beta-induced matrix metalloproteinase production with an IC50 of approximately 1 microM. ML120B also blocked IL-1beta-induced prostaglandin E2 production. In summary, ML120B blocked numerous NF-kappaB-regulated cell responses that are involved in inflammation and destructive processes in the RA joint. Our findings support the evaluation of IKKbeta inhibitors as anti-inflammatory agents for the treatment of RA.

Persistent HIF-1alpha activation in gut ischemia/reperfusion injury: potential role of bacteria and lipopolysaccharide.

In both animal models of hemorrhagic shock and clinical settings, shock-induced gut ischemia has been implicated in the development of the systemic inflammatory response syndrome and distant organ injury, yet the factors transducing these events remain to be fully determined. Because hypoxia-inducible factor (HIF-1), a transcription factor composed of oxygen-labile HIF-1alpha and constitutive HIF-1beta subunits, regulates the physiologic/pathophysiologic response to hypoxia and ischemia, we examined the HIF-1 response in two rat models of gut ischemia-reperfusion. We found that ileal nuclear HIF-1alpha protein levels were induced in rats subjected to trauma (laparotomy) plus hemorrhagic shock for 90 min relative to their trauma sham-shock and naïve counterparts and that this trauma hemorrhagic shock-induced mucosal HIF-1alpha protein response persisted after 1 h and 3 h of reperfusion. Likewise, in a model of isolated gut ischemia-reperfusion injury, where the superior mesenteric artery was occluded for 45 min, nuclear HIF-1alpha were induced in the gut mucosa relative to their sham counterparts and persisted after 1 h and 3 h or reperfusion. Similar to the in vivo response, in vitro hypoxia induced HIF-alpha expression in three different enterocyte cell lines (rat IEC-6 and human Caco-2 and HT-29 cell lines). However, in contrast to the in vivo response, HIF-1 expression rapidly disappeared on subsequent reoxygenation. Because in vivo enterocytes are exposed to bacteria, we tested whether the in vitro HIF-1alpha response would persist on reoxygenation if the enterocytes were cocultured with bacteria. P. aeruginosa, an enteric bacterium, markedly induced enterocyte HIF-1alpha protein levels under normoxic conditions. Furthermore, the addition of P. aeruginosa during either the hypoxic or reoxygenation phase prevented the degradation of HIF-1alpha protein levels. Moreover, the observation that lipopolysaccharide induced HIF-1alpha expression in a time-dependent manner in IEC-6 cells indicated that the induction of HIF-1 by exposure to P. aeruginosa is not dependent on bacterial viability. In conclusion, these results suggest that HIF-1alpha activation is an early reperfusion-independent event in models of gut ischemia-reperfusion and that this HIF-1alpha response is potentiated by the presence of P. aeruginosa or lipopolysaccharide.