NRAS is the second most frequently mutated gene in melanoma. Previous reports have demonstrated the sensitivity of cancer cell lines carrying KRAS mutations to apoptosis initiated by inhibition of protein kinase C delta (PKCδ). Here, we report that PKCδ inhibition is cytotoxic in melanomas with primary NRAS mutations. Novel small-molecule inhibitors of PKCδ were designed as chimeric hybrids of two naturally-occurring PKCδ inhibitors, staurosporine and rottlerin. The specific hypothesis interrogated and validated is that combining two domains of two naturally-occurring PKCδ inhibitors into a chimeric or hybrid structure retains biochemical and biological activity, and improves PKCδ isozyme selectivity. We have devised a potentially general synthetic protocol to make these chimeric species using Molander trifluorborate coupling chemistry. Inhibition of PKCδ, by siRNA or small molecule inhibitors, suppressed the growth of multiple melanoma cell lines carrying NRAS mutations, mediated via caspase-dependent apoptosis. Following PKCδ inhibition, the stress-responsive JNK pathway was activated, leading to the activation of H2AX. Consistent with recent reports on the apoptotic role of phospho-H2AX, knockdown of H2AX prior to PKCδ inhibition mitigated the induction of caspase-dependent apoptosis. Furthermore, PKCδ inhibition effectively induced cytotoxicity in BRAF-mutant melanoma cell lines that had evolved resistance to a BRAF inhibitor, suggesting the potential clinical application of targeting PKCδ in patients who have relapsed following treatment with BRAF inhibitors. Taken together, the present work demonstrates that inhibition of PKCδ by novel small molecule inhibitors causes caspase-dependent apoptosis mediated via the JNK-H2AX pathway in melanomas with NRAS mutations or BRAF inhibitor-resistance.
The concept of targeting cancer therapeutics towards specific mutations or abnormalities in tumor cells which are not found in normal tissues has the potential advantages of high selectivity for the tumor and correspondingly low secondary toxicities. Many human malignancies display activating mutations in the Ras family of signal-transducing genes or over-activity of p21Ras-signaling pathways. Carcinoid and other neuroendocrine tumors similarly have been demonstrated to have activation of Ras signaling directly by mutations in Ras, indirectly by loss of Ras-regulatory proteins, or via constitutive activation of upstream or downstream effector pathways of Ras, such as growth factor receptors or PI3-Kinase and Raf/MAP kinases. We previously reported that aberrant activation of Ras signaling sensitizes cells to apoptosis when the activity of the PKCδ isozyme is suppressed, and that PKCδ suppression is not toxic to cells with normal levels of p21Ras signaling. We demonstrate here that inhibition of PKCδ by a number of independent means, including genetic mechanisms (shRNA) or small molecule inhibitors, is able to efficiently and selectively repress the growth of human neuroendocrine cell lines derived from bronchopulmonary, foregut or hindgut tumors. PKCδ inhibition in these tumors also efficiently induced apoptosis. Exposure to small-molecule inhibitors of PKCδ over a period of 24 hr is sufficient to significantly suppress cell growth and clonogenic capacity of these tumor cell lines. Neuroendocrine tumors are typically refractory to conventional therapeutic approaches. This Ras-targeted therapeutic approach, mediated through PKCδ suppression, which selectively takes advantage of the very oncogenic mutations which contribute to the malignancy of the tumor, may hold potential as a novel therapeutic modality.
A synthesis of racemic oleocanthal has been accomplished in eleven steps from 1,3 propanediol by a key tandem intramolecular Michael cyclization-Horner Wadsworth Emmons olefination.The secoiridoids are a class of plant-derived monoterpenes containing the substituted pyran core of secologanin (1). This class of natural products arises in biological systems from the oxidative cleavage of the loganin skeleton (2) by the cytochrome P450 enzyme secologanin synthase. 1 Secologanin then undergoes various modifications to produce a wide array of structures displaying diverse biological activity including analgesic 2 , anti-inflammatory 3 , antiarthritic4, anti-allergenic5, antibacterial6, and antiviral 7 activities. Coupling of the simple secoiridoids with tryptamine gives rise to a large class (>250 examples) of indole and oxindole alkaloids including geissoschizine, strychnine, reserpene, ajmaline, and the Vinca alkaloids with highly varied carbon frameworks and biological profiles.We reasoned that, given the structural similarities of the secologanin-derived natural products, synthetic access to the more complex secoiridoid and secologanin tryptamine alkaloids could be obtained through a single strategically functionalized intermediate. As a test case for our strategy, our attention focused on the secoiridoid oleocanthal (3) both for its relative structural simplicity which retains the compact arrangement of functionality of the secoiridoids as well as its demonstrated potency as an inhibitor of the COX-1 and COX-2 enzymes 3,8 , making it an ideal entry point into the synthesis of this class of natural products.Important to our retrosynthetic analysis was proceeding through an intermediate with functionalities that could be independently manipulated allowing for future adaptation of this synthesis to produce a diverse selection of natural product targets. Lactone 5 appeared ideal for this purpose as it contains the requisite carbon backbone as well as properly situated synthetic handles with orthogonal reactivities.We envisioned the dialdehyde moiety of 3 arising from the reduction and oxidation of lactone 5 whose carbon framework could be assembled through a key tandem Michael cyclization and Horner-Wadsworth-Emmons (HWE) olefination 9 of phosphonoacetic ester 6. This allows for the simultaneous assembly of the desired lactone core, the addition of the two-carbon olefin sidechain, and the introduction of a stereogenic center. Studies to control the absolute configuration of this new stereogenic center through the introduction of chiral auxiliaries are currently underway. Assembly of lactone 5 began with the synthesis of aldehyde 7 from 1,3-propanediol utilizing the method reported by Schaus and coworkers. 10 Reaction of 7 with commercially available trimethyl acetophosphonate under Masamune-Roush conditions produced the unsaturated ester 8 as a single isomer and in good yield (Scheme 2). Deprotection and subsequent esterification gave the desired substrate 6, which upon treatment with Cs 2 CO 3 and acetaldeh...
The syntheses of D,L-geissoschizol, D,L-corynantheidol, D,L-dihydrocorynantheol, D,L-protoemetinol, and D,L-3-epi-protoemetinol have been accomplished from a single synthetic intermediate.
Terpenes U 0200Synthesis of (±)-Oleocanthal via a Tandem Intramolecular Michael Cyclization-HWE Olefination. -A short and scalable total synthesis of racemic (V) is reported based on the transformation of phosphorane (II) to pyran (IV) as the key step. -(ENGLISH, B. J.; WILLIAMS*, R. M.; Tetrahedron Lett. 50 (2009) 23, 2713-2715; Dep. Chem., Colo. State Univ., Fort Collins, CO 80523, USA; Eng.) -Mais 38-181
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