Discovery of RMC-5552, a Selective Bi-Steric Inhibitor of mTORC1, for the Treatment of mTORC1-Activated Tumors.

Hyperactivation of mTOR kinase by mutations in the PI3K/mTOR pathway or by crosstalk with other mutant cancer drivers, such as RAS, is a feature of many tumors. Multiple allosteric inhibitors of mTORC1 and orthosteric dual inhibitors of mTORC1 and mTORC2 have been developed as anticancer drugs, but their clinical utility has been limited. To address these limitations, we have developed a novel class of "bi-steric inhibitors" that interact with both the orthosteric and the allosteric binding sites in order to deepen the inhibition of mTORC1 while also preserving selectivity for mTORC1 over mTORC2. In this report, we describe the discovery and preclinical profile of the development candidate RMC-5552 and the in vivo preclinical tool compound RMC-6272. We also present evidence that selective inhibition of mTORC1 in combination with covalent inhibition of KRASG12C shows increased antitumor activity in a preclinical model of KRASG12C mutant NSCLC that exhibits resistance to KRASG12C inhibitor monotherapy.

Selective inhibitors of mTORC1 activate 4EBP1 and suppress tumor growth.

The clinical benefits of pan-mTOR active-site inhibitors are limited by toxicity and relief of feedback inhibition of receptor expression. To address these limitations, we designed a series of compounds that selectively inhibit mTORC1 and not mTORC2. These 'bi-steric inhibitors' comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. Structural modification of these components modulated their affinities for their binding sites on mTOR and the selectivity of the bi-steric compound. mTORC1-selective compounds potently inhibited 4EBP1 phosphorylation and caused regressions of breast cancer xenografts. Inhibition of 4EBP1 phosphorylation was sufficient to block cancer cell growth and was necessary for maximal antitumor activity. At mTORC1-selective doses, these compounds do not alter glucose tolerance, nor do they relieve AKT-dependent feedback inhibition of HER3. Thus, in preclinical models, selective inhibitors of mTORC1 potently inhibit tumor growth while causing less toxicity and receptor reactivation as compared to pan-mTOR inhibitors.

Complexation of ytterbium to human transferrin and its uptake by K562 cells.

There is an increasing interest in the use of lanthanides in medicine. However, the mechanism of their accumulation in cells is not well understood. Lanthanide cations are similar to ferric ions with regard to transferrin binding, suggesting transferrin-receptor mediated transport is possible; however, this has not yet been confirmed. In order to clarify this mechanism, we investigated the binding of Yb3+ to apotransferrin by UV-Vis spectroscopy and stopped-flow spectrophotometry, and found that Yb3+ binds to apotransferrin at the specific iron sites in the presence of bicarbonate. The apparent binding constants of these sites showed that the affinity of Yb3+ is lower than that of Fe3+and binding of Yb3+ in the N-lobe is kinetically favored while the C-lobe is thermodynamically favored. The first Yb3+ bound to the C-lobe quantitatively with a Yb/apotransferrin molar ratio of < 1, whereas the binding to the other site is weaker and approaches completeness by a higher molar ratio only. As demonstrated by 1H NMR spectra, Yb3+ binding disturbed the conformation of apotransferrin in a manner similar to Fe3+. Flow cytometric studies on the uptake of fluorescein isothiocyanate labeled Yb3+-bound transferrin species by K562 cells showed that they bind to the cell receptors. Laser scanning confocal microscopic studies with fluorescein isothiocyanate labeled Yb3+-bound transferrin and propidium iodide labeled DNA and RNA in cells indicated that the Yb3+ entered the cells. The Yb3+-transferrin complex inhibited the uptake of the fluorescein labeled ferric-saturated transferrin (Fe2-transferrin) complex into K562 cells. The results demonstrate that the complex of Yb3+-transferrin complex was recognized by the transferrin receptor and that the transferrin-receptor-mediated mechanism is a possible pathway for Yb3+ accumulation in cells.

Syntheses and calcium-mobilizing evaluations of N1-glycosyl-substituted stable mimics of cyclic ADP-ribose.

Cyclic ADP-ribose (cADPR) is not only a potent endogenous calcium modulator but also a second messenger. However, studies on the mechanism of cADPR action were limited due to its instability and lack of available structural modifications in the N1-glyosyl unit of cADPR. In the present work, a series of N1-glycosyl mimics with different configurational glycosyls or an ether strand were designed and synthesized mimicking the furanose ring. S(N)2 substitutions were carried out between the protected inosine and glycosyl triflates to form the N1-glycosylinosine derivatives, accompanied with some O6-glycosyl-substituted as side products. The intramolecular cyclization was followed the strategy described by Matsuda et al. It was found that the 8-unsubstituted substrate could also be used to construct the intramolecular cyclic pyrophosphate. The activities of N1-glycosyl-substituted cADPR mimics were evaluated by induced Ca2+ release in rat brain microsomes and HeLa cells. It was found that the configuration of the N1-glycosyl moiety in cADPR is not a critical structural factor for retaining the activity of mobilizing Ca2+ release. More interestingly, the N1-acyclic analogue 6 exhibited strong activity by inducing Ca2+ release in both rat brain microsomes and HeLa cells. It constitutes a useful tool for further studies.

Chemical synthesis and calcium release activity of N(1)-ether strand substituted cADPR mimic.

8-Chloro cyclic inosine 5'-diphosphate ethoxymethyl ether 3 was synthesized by means of chemical method from protected inosine via phenylthio-type biphosphate substrate. The detection of Ca(2+) release activity shows that 3 is a potent agonist of cADPR and has activity in intact Hela cells.