The Kirsten rat sarcoma viral oncoprotein homolog (KRAS) is currently a primary focus of oncologists and translational scientists, driven by exciting results with KRAS-targeted therapies for ...non-small cell lung cancer (NSCLC) patients. While KRAS mutations continue to drive high cancer diagnosis and death, researchers have developed unique strategies to target KRAS variations. Having been investigated over the past 40 years and considered “undruggable” due to the lack of pharmacological binding pockets, recent breakthroughs and accelerated FDA approval of the first covalent inhibitors targeting KRAS G12C , have largely sparked further drug development. Small molecule development has targeted the previously identified primary location alterations such as G12, G13, Q61, and expanded to address the emerging secondary mutations and acquired resistance. Of interest, the non-covalent KRAS G12D targeting inhibitor MRTX-1133 has shown promising results in humanized pancreatic cancer mouse models and is seemingly making its way from bench to bedside. While this manuscript was under review a novel class of first covalent inhibitors specific for G12D was published, These so-called malolactones can crosslink both GDP and GTP bound forms of G12D. Inhibition of the latter state suppressed downstream signaling and cancer cell proliferation in vitro and in mouse xenografts. Moreover, a non-covalent pan-KRAS inhibitor, BI-2865, reduced tumor proliferation in cell lines and mouse models. Finally, the next generation of KRAS mutant-specific and pan-RAS tri-complex inhibitors have revolutionized RAS drug discovery. This review will give a structural biology perspective on the current generation of KRAS inhibitors through the lens of emerging secondary mutations and acquired resistance.
Previous chemical investigation of the Irish deep-sea soft coral Duva florida led to the identification of tuaimenal A (10), a new merosesquiterpene containing a highly substituted chromene core and ...modest cytotoxicity against cervical cancer. Further MS/MS and NMR-guided investigation of this octocoral has resulted in the isolation and characterization of seven additional tuaimenal analogs, B–H (1–7), as well as two known A-ring aromatized steroids (8, 9), and additional tuaimenal A (10). Tuaimenals B, F, and G (1, 5, 6), bearing an oxygen at the C5 position, as well as monocyclic tuaimenal H (7), show increased cervical cancer inhibition profiles in comparison to that of 10. Tuaimenal G further displayed potent, selective cytotoxicity with an EC50 value of 0.04 μM against the C33A cell line compared to the CaSki cell line (EC50 20 μM). These data reveal the anticancer properties of tuaimenal analogs and suggest unique antiproliferation mechanisms across these secondary metabolites.
Abstract
The aim of this study is to address the challenge of the expression of recombinant atypical protein kinase C (aPKCs) in a bacterial system and understand the binding interactions of novel ...anti-cancer inhibitors of aPKCs. The Protein kinase Cs (PKCs) expression at a high rate is a key indicator of cancer cell proliferation and survival. In particular, the over-expressions of aPKCs were found to be directly associated with various cancer cells in benign and malignant stages. Several cancer growth blockers (inhibitors) are available for targeted therapy. However, these inhibitors had to pass through rigorous in silico screening, in vitro, and in vivo assays before FDA approval. During the characterization studies of the protein, insights into binding interactions between aPKCs and drug molecules are crucial. For successive studies such as for biophysical characterization, the recombinant bioactive full-length PKC- ι (an aPKC) expression in the bacterial system is still a challenge. In addition, the binding interaction studies of aPKCs with inhibitors in physiological conditions providing structural details are limited to some X-ray crystallography structures and computational studies. Here we addressed these two challenges and report a protocol detailing the bioactive full-length and catalytic domain of PKC- ι expression and purification from the bacterial system. The further characterization of the recombinant protein by the determination of melting temperature, functional activity, and circular dichroism reported here provides important information on the characteristics of the protein in its native state. Moreover, we show the chemical interactions of five novel anti-cancer inhibitors ICA-1S (Nucleosidic homolog) 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide), ICA-1T (Phosphorylated nucleosidic homolog of ICA-1S, 4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl methyl dihydrogen phosphate), ACPD (2-acetyl-1,3-cyclopentanedione), DNDA (3,4-diaminonaphthalene-2,7-disulfonic acid) and ζ(ZETA)-Stat (8-hydroxy-1,3,6- naphthalenetrisulfonic acid) to locate their regions of binding on the catalytic domain of the aPKCs (PKC- ι and PKC- ζ). We have utilized the chemical shift changes obtained from various multi-dimensional Nuclear Magnetic Resonance (NMR) experiments. Moreover, we have applied the isothermal calorimetry method to understand the biophysical properties of the aPKCs in presence of these inhibitors. The observed biophysical properties of the protein and chemical shift perturbations in the amide backbone of the catalytic domain upon binding shed light on the molecular recognition process, binding characteristics, and atomic-level structural details of the aPKCs.
Citation Format: Radwan Ebna Noor, Tracess B. Smalley, Mildred Acevedo-Duncan. A biophysical investigation into the binding interactions of novel anti-cancer inhibitors of atypical protein kinase C (aPKCs). abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3844.
Research has demonstrated that the atypical protein kinase C-zeta (PKC-ζ) is a component of many dysregulated pathways in breast and ovarian cancer, including cellular proliferation, survival, and ...cell cycle upregulation. Breast and ovarian cancers affect women every day and are second and fifth leading cause of cancer death. Women who seek treatments are commonly met with invasive surgeries or chemotherapy. Protein kinase C (PKC) is a family of serine and threonine phosphorylating kinases that have been shown to modulate and transduce signaling cascades that play roles in the development and survival of cancers. Atypical PKC (aPKC), have been heavily suggested to participate in phosphoinositide 3-kinase (PI3K) misregulated cancers (such as breast and ovarian) and inhibition of these pathway branches have demonstrated decreases in malignant behaviors. In our studies, we investigated (1) PKC-ζ protein in breast tissue to evaluate its potential as a biomarker for breast cancer invasion and (2) the effects of the atypical PKC-ζ inhibitor ζ-Stat on ovarian cell lines (TOV21G and ES-2) to determine the outcomes on proliferation and cellular invasion. In breast cancer tissues, we showed that an overexpression of PKC-ζ protein can be indicative of carcinogenesis. We also evaluated the invasive behavior of MDA-MB-231 breast cancer cells upon the knockdown of PKC-ζ with a Transwell invasion assay and an immunofluorescent probe for filamentous actin (F-actin) organization. In ovarian cancer, we utilized clear cell ovarian carcinoma cells (CCOC) because this subtype of ovarian cancer represents 5% of incidence, presents unique pathological features, has poor prognosis and has a high reoccurrence rate after treatment. Overall, our results showed that PKC-ζ is linked to proliferation and invasion in both breast and ovarian cancer. These results illustrate that PKC-ζ plays a role in invasion pathways through the Ras-related C3 botulinum toxin substrate 1 (Rac1) and Ras homolog gene family member A (RhoA). Additionally, our results showed that TOV21G tumor growth in athymic female mice was decreased when treated with the PKC-ζ inhibitor, ζ-Stat. These data suggests that PKC-ζ is a biomarker and a novel target in the carcinogenesis of breast and CCOC and its inhibition by way of ζ-Stat decreased the rate of proliferation, tumor growth and expression of invasive protein pathways.
