Structure-activity relationships of middle-size cyclic peptides, KRAS inhibitors derived from an mRNA display.

Cyclic peptides are attracting attention as therapeutic agents due to their potential for oral absorption and easy access to tough intracellular targets. LUNA18, a clinical KRAS inhibitor, was transformed-without scaffold hopping-from the initial hit by using an mRNA display library that met our criteria for drug-likeness. In drug discovery using mRNA display libraries, hit compounds always possess a site linked to an mRNA tag. Here, we describe our examination of the Structure-Activity Relationship (SAR) using X-ray structures for chemical optimization near the site linked to the mRNA tag, equivalent to the C-terminus. Structural modifications near the C-terminus demonstrated a relatively wide range of tolerance for side chains. Furthermore, we show that a single atom modification is enough to change the pharmacokinetic (PK) profile. Since there are four positions where side chain modification is permissible in terms of activity, it is possible to flexibly adjust the pharmacokinetic profile by structurally optimizing the side chain. The side chain transformation findings demonstrated here may be generally applicable to hits obtained from mRNA display libraries.

Development of Orally Bioavailable Peptides Targeting an Intracellular Protein: From a Hit to a Clinical KRAS Inhibitor.

Cyclic peptides as a therapeutic modality are attracting a lot of attention due to their potential for oral absorption and accessibility to intracellular tough targets. Here, starting with a drug-like hit discovered using an mRNA display library, we describe a chemical optimization that led to the orally available clinical compound known as LUNA18, an 11-mer cyclic peptide inhibitor for the intracellular tough target RAS. The key findings are as follows: (i) two peptide side chains were identified that each increase RAS affinity over 10-fold; (ii) physico-chemical properties (PCP) including Clog P can be adjusted by side-chain modification to increase membrane permeability; (iii) restriction of cyclic peptide conformation works effectively to adjust PCP and improve bio-activity; (iv) cellular efficacy was observed in peptides with a permeability of around 0.4 × 10-6 cm/s or more in a Caco-2 permeability assay; and (v) while keeping the cyclic peptide's main-chain conformation, we found one example where the RAS protein structure was changed dramatically through induced-fit to our peptide side chain. This study demonstrates how the chemical optimization of bio-active peptides can be achieved without scaffold hopping, much like the processes for small molecule drug discovery that are guided by Lipinski's rule of five. Our approach provides a versatile new strategy for generating peptide drugs starting from drug-like hits.

Discovery of CH7057288 as an Orally Bioavailable, Selective, and Potent pan-TRK Inhibitor.

Kinase fusions involving tropomyosin receptor kinases (TRKs) have been proven to act as strong oncogenic drivers and are therefore recognized as attractive therapeutic targets. We screened an in-house kinase-focused library and identified a promising hit compound with a unique tetracyclic scaffold. Compound 1 showed high TRK selectivity but moderate cell growth inhibitory activity as well as a potential risk of inducing CYP3A4. In this report, chemical modification intended to improve TRK inhibition and avoid CYP3A4 induction enabled us to identify an orally bioavailable, selective, and potent TRK inhibitor 7.

Estimating the clinical risk of hypertension from VEGF signal inhibitors by a non-clinical approach using telemetered rats.

Anti-angiogenic drugs that target Vascular Endothelial Growth Factor (VEGF) signaling pathways caused hypertension as an adverse effect in clinical studies. Since the hypertension may limit the benefit provided for patients, the demand for non-clinical research that predicts the clinical risk of the hypertension has risen greatly. To clarify whether non-clinical research using rats can appropriately estimate the clinical risk of hypertension caused by VEGF signal inhibitors, we investigated the hemodynamic effects and pharmacokinetics (PK) of the VEGF signal inhibitors cediranib (0.1, 3, and 10 mg/kg), sunitinib (5, 10, and 40 mg/kg), and sorafenib (0.1, 1, and 5 mg/kg) in telemetered rats and examined the correlation between the non-clinical and the clinical hypertensive effect. The VEGF signal inhibitors significantly elevated blood pressure (BP) in rats within a few days of the initiation of dosing, and levels recovered after dosing ended. The trend of the hypertension was similar to that in clinical studies. We found that the AUC at which BP significantly increased by approximately 10 mmHg in rats was comparable to the clinical AUC at which moderate to severe hypertension occurred. These results represent correlations between the non-clinical and the clinical hypertensive effect of VEGF signal inhibitors, suggesting that non-clinical research using telemetered rats would be an effective approach to predict the clinical risk of hypertension caused by VEGF signal inhibitors.

Angiogenesis inhibitors identified by cell-based high-throughput screening: synthesis, structure-activity relationships and biological evaluation of 3-[(E)-styryl]benzamides that specifically inhibit endothelial cell proliferation.

Proliferation of endothelial cells is critical for angiogenesis. We report orally available, in vivo active antiangiogenic agents which specifically inhibit endothelial cell proliferation. After identifying human umbilical vein endothelial cell (HUVEC) proliferation inhibitors from a cell-based high-throughput screening (HTS), we eliminated those compounds which showed cytotoxicity against HCT116 and vascular endothelial growth factor receptor 2 (VEGFR-2) inhibitory activity. Evaluations in human Calu-6 xenograft model delivered lead compound 1. Following extensive lead optimization and alteration of the scaffold we discovered 32f and 32g, which both inhibited the proliferation and tube formation of HUVEC without showing inhibitory activity against any of 25 kinases or cytotoxicity against either normal fibroblasts or 40 cancer cell lines. Upon oral administration, 32f and 32g had good pharmacokinetic profiles and potent antitumor activity and decreased microvessel density (MVD) in Calu-6 xenograft model. Combination therapy with a VEGFR inhibitor enhanced the in vivo efficacy. These results suggest that 32f and 32g may have potential for use in cancer treatment.

The selective class I PI3K inhibitor CH5132799 targets human cancers harboring oncogenic PIK3CA mutations.

PURPOSE: The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in cell proliferation and survival in human cancer. PIK3CA mutations, which are found in many cancer patients, activate the PI3K pathway, resulting in cancer development and progression. We previously identified CH5132799 as a novel PI3K inhibitor. Thus, this study aimed to clarify the biochemical and antitumor activity of CH5132799 and elucidate the correlation between CH5132799 response and genetic alterations in the PI3K pathway. EXPERIMENTAL DESIGN: Kinase inhibitory activity was profiled in cell-free assays. A large panel of human breast, ovarian, prostate, and endometrial cancer cell lines, as well as xenograft models, were used to evaluate the antitumor activity of CH5132799, followed by analysis for genetic alterations. Effects on Akt phosphorylation induced by mTORC1 inhibition were tested with CH5132799 and compared with mTORC1 and PI3K/mTOR inhibitors. RESULTS: CH5132799 selectively inhibited class I PI3Ks and PI3Kα mutants in in vitro kinase assays. Tumors harboring PIK3CA mutations were significantly sensitive to CH5132799 in vitro and were remarkably regressed by CH5132799 in in vivo mouse xenograft models. In combination with trastuzumab, tumors disappeared in the trastuzumab-insensitive breast cancer model with the PIK3CA mutation. Moreover, CH5132799 did not reverse a negative feedback loop of PI3K/Akt/mTOR signaling and induced regression against tumors regrown after long-term mTORC1 inhibitor treatment. CONCLUSIONS: CH5132799 is a selective class I PI3K inhibitor with potent antitumor activity against tumors harboring the PIK3CA mutations. Prediction of CH5132799 response on the basis of PIK3CA mutations could enable patient stratification in clinical settings.

Discovery and biological activity of a novel class I PI3K inhibitor, CH5132799.

Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase and a promising therapeutic target for cancer. Using structure-based drug design (SBDD), we have identified novel PI3K inhibitors with a dihydropyrrolopyrimidine skeleton. Metabolic stability of the first lead series was drastically improved by replacing phenol with aminopyrimidine moiety. CH5132799, a novel class I PI3K inhibitor, exhibited a strong inhibitory activity especially against PI3Kα (IC(50)=0.014 µM). In human tumor cell lines with PI3K pathway activation, CH5132799 showed potent antiproliferative activity. CH5132799 is orally available and showed significant antitumor activity in PI3K pathway-activated human cancer xenograft models in mice.

Prediction of drug-induced QT interval prolongation in telemetered common marmosets.

Drug-induced QT interval prolongation is a critical issue in development of new chemical entities, so the pharmaceutical industry needs to evaluate risk as early as possible. Common marmosets have been in the limelight in early-stage development due to their small size, which requires only a small amount of test drug. The purpose of this study was to determine the utility of telemetered common marmosets for predicting drug-induced QT interval prolongation. Telemetry transmitters were implanted in common marmosets (male and female), and QT and RR intervals were measured. The QT interval was corrected for the RR interval by applying Bazett's and Fridericia's correction formulas and individual rate correction. Individual correction showed the least slope for the linear regression of corrected QT (QTc) intervals against RR intervals, indicating that it dissociated changes in heart rate most effectively. With the individual correction method, the QT-prolonging drugs (astemizole, dl-sotalol) showed QTc interval prolongations and the non-QT-prolonging drugs (dl-propranolol, nifedipine) did not show QTc interval prolongations. The plasma concentrations of astemizole and dl-sotalol associated with QTc interval prolongations in common marmosets were similar to those in humans, suggesting that the sensitivity of common marmosets would be appropriate for evaluating risk of drug-induced QT interval prolongation. In conclusion, telemetry studies in common marmosets are useful for predicting clinical QT prolonging potential of drugs in early stage development and require only a small amount of test drug.