Advances in Molecular Mechanisms for Traditional Chinese Medicine Actions in Regulating Tumor Immune Responses.

Traditional Chinese medicine (TCM) has been developed for thousands of years with its various biological activities. The interest in TCM in tumor prevention and treatment is rising with its synergistic effect on tumor cells and tumor immunosuppressive microenvironment (TIM). Characteristic of TCM fits well within the whole system and multi-target cancer treatment. Herein we discuss the underlying mechanisms of TCM actions in TIM via regulating immunosuppressive cells, including restoring the antigen presentation function of dendritic cells, enhancing NK cells-mediated killing activity, restraining the functions of myeloid cell-derived suppressor cells, and inhibiting cancer-associated fibroblasts. TCM also regulates tumor progression through enhancing immune response, preventing immune escape and inducing cell death of tumor cells, which triggers immune response in nearby cells. In addition, we discuss TCM in clinical applications and the advantages and disadvantages of TCM in cancer prevention and treatment, as well as current therapeutic challenges and strategies. It might be helpful for understanding the therapeutic potential of TCM for cancer in clinic.

Discovery of novel glycogen synthase kinase-3α inhibitors: Structure-based virtual screening, preliminary SAR and biological evaluation for treatment of acute myeloid leukemia.

Glycogen synthase kinase 3α (GSK-3α) plays a constitutive role in various physiological processes and has been proved to be a therapeutic target for acute myeloid leukemia (AML). In this paper, by means of computer-aided drug design, we discovered a novel chemical series of GSK-3α inhibitors with an IC50 value of 0.033-2.804 µM. The preliminary structure-activity relationship was concluded and, notably, the most potent and isoform-selective compound G28_14 was identified with IC50 values of 33 nM and 218 nM against GSK-3α and -3ß, respectively, exhibiting a nearly ten-fold isoform-selectivity. Further cell viability assays and colony formation assays revealed that G28_14 suppressed cell survival by impairing cell proliferation by up to 90% in two AML cell lines. Moreover, surface marker expression analysis demonstrated that G28_14 induced terminal differentiation with a high level of CD11b, CD11c, and CD14. Western immunoblotting showed that G28_14 isoform-selectively inhibited the phosphorylation of GSK-3α in-cell without activating Wnt/ß-catenin signaling. In addition, to elucidate its structure-activity relationship, the binding mode of this chemical series was proposed using molecular docking and molecular dynamics simulations. Taken together, this chemical series is worth developing as differentiation therapies for the treatment of AML.

Synthesis of Site-Specific Radiolabeled Antibodies for Radioimmunotherapy via Genetic Code Expansion.

Radioimmunotherapy (RIT) delivers radioisotopes to antigen-expressing cells via monoantibodies for the imaging of lesions or medical therapy. The chelates are typically conjugated to the antibody through cysteine or lysine residues, resulting in heterogeneous chelate-to-antibody ratios and various conjugation sites. To overcome this heterogeneity, we have developed an approach for site-specific radiolabeling of antibodies by combination of genetic code expansion and click chemistry. As a proof-of-concept study, model systems including anti-CD20 antibody rituximab, positron-emitting isotope 64Cu, and a newly synthesized bifunctional linker (4-dibenzocyclooctynol-1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid, DIBO-DOTA) were used. The approach consists of three steps: (1) site-specific incorporation of an azido group-bearing amino acid (NEAK) via the genetic code expansion technique at the defined sites of the antibody as a "chemical handle"; (2) site-specific and quantitative conjugation of bifunctional linkers with the antibodies under a mild condition; and (3) radiolabeling of the chelate-modified antibodies with the appropriate isotope. We used heavy-chain A122NEAK rituximab as proof-of-concept and obtained a homogeneous radioconjugate with precisely two chelates per antibody, incorporated only at the chosen sites. The conjugation did not alter the binding and pharmacokinetics of the rituximab, as indicated by in vitro assays and in vivo PET imaging. We believe our research is a good supplement to the genetic code expansion technique for the development of novel radioimmunoconjugates.

Development of oleanane-type triterpenes as a new class of HCV entry inhibitors.

Development of hepatitis C virus (HCV) entry inhibitors represents an emerging approach that satisfies a tandem mechanism for use with other inhibitors in a multifaceted cocktail. By screening Chinese herbal extracts, oleanolic acid (OA) was found to display weak potency to inhibit HCV entry with an IC50 of 10 µM. Chemical exploration of this triterpene compound revealed its pharmacophore requirement for blocking HCV entry, rings A, B, and E, are conserved while ring D is tolerant of some modifications. Hydroxylation at C-16 significantly enhanced its potency for inhibiting HCV entry with IC50 at 1.4 µM. Further modification by conjugation of this new lead with a disaccharide at 28-COOH removed the undesired hemolytic effect and, more importantly, increased its potency by ~5-fold (54a, IC50 0.3 µM). Formation of a triterpene dimer via a linker bearing triazole (70) dramatically increased its potency with IC50 at ~10 nM. Mechanistically, such functional triterpenes interrupt the interaction between HCV envelope protein E2 and its receptor CD81 via binding to E2, thus blocking virus and host cell recognition. This study establishes the importance of triterpene natural products as new leads for the development of potential HCV entry inhibitors.

Synthesis of N-substituted ε-hexonolactams as pharmacological chaperones for the treatment of N370S mutant Gaucher disease.

A series of N-substituted ε-hexonolactams have been designed and prepared by a concise route with a tandem ring-expansion reaction as the key step. Some of the N-substituted ε-hexonolactams show better enhancements to N370S mutant ß-glucocerebrosidase activity than NB-DNJ and NN-DNJ. Both the experimental results and computational studies highlight the importance of the carbonyl group for stabilizing protein folds in the mutant enzyme. The structure-activity relationships are also discussed. These novel N-alkylated iminosugars are promising pharmacological chaperones for the treatment of N370S mutant Gaucher disease.

Synthesis and evaluation of novel neamine derivatives effectively targeting to RNA.

Three new derivatives of neamine, 3 (NE), 6 (NEA) and 9 (NEL), were synthesized by connecting arginine or lysine to 5-hydroxyl group of neamine using ethylenediamine as a linker. The binding affinities of these derivatives to A site of 16S RNA and TAR RNA indicate that the modification on 5-hydroxyl of neamine by amino acid can enhance the binding affinity of neamine. Compound 9 (NEL) shows some antibacterial activities. These results demonstrate that modification on 5-hydroxyl group of neamine may provide a promising way for the development of potential candidates effectively targeting to RNAs.

Z-ajoene induces apoptosis of HL-60 cells: involvement of Bcl-2 cleavage.

Garlic organosulfur components exhibit antitumor activity, but the molecular mechanisms underlying these effects have not been well characterized. We showed that Z-ajoene, a sulfur-rich compound purified from garlic, induced time- and dose-dependent apoptosis in HL-60 cells. This process implied the activation of caspase-3 and the cleavage of the antiapoptotic protein Bcl-2. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-[OMe]-fluoromethylketone inhibited Bcl-2 cleavage and apoptosis induced by Z-ajoene. This effect was partially prevented by treatment of HL-60 cells with the antioxidant N-acetylcysteine. Hence, the transmission of apoptotic signal induced by Z-ajoene involved a reactive oxygen species-dependent pathway leading to caspase-dependent Bcl-2 cleavage.

Antitumor activity of Z-ajoene, a natural compound purified from garlic: antimitotic and microtubule-interaction properties.

Ajoene, a garlic stable oil-soluble sulfur rich compound was generally isolated as a mixture of two isomers [(E, Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide]. It has been described essentially as a potent inhibitor of platelet aggregation in vitro and in vivo. The antiproliferative effects of ajoene and experiments using a single isomer had received little attention. The present study aims at defining the antitumor activities of cis-Z-ajoene in vitro and in vivo. Antiproliferative activity of Z-ajoene was demonstrated against a panel of human tumor cell lines with IC(50) values varying from 5.2 mM to 26.1 mM and at a lower extent in normal marsupial kidney cells (PtK2). Meanwhile, Z-ajoene arrested HL60 cells in G(2)/M phase of cell cycle in a dose and time-dependent way. In PtK2 cells, exposure to 20 microM Z-ajoene for 6 h induced a complete disassembly of the microtubule network, that was associated with an increased number of cells blocked in early mitotic stages. An IC(50) for microtubule disassembly of 1 microM was determined by a fully automated microplate-based multi-detection reader. In vitro, a reversible inhibition of the microtubule protein assembly was observed with an IC(50) of 25 microM Z-ajoene. In vivo, Z-ajoene inhibited tumor growth by 38% and 42% in mice grafted with sarcoma 180 and hepatocarcinoma 22, respectively. For the first time, Z-ajoene was shown to be a potent inhibitor of tumor cell growth both in vitro and in vivo. The microtubule cytoskeleton appeared to be one of the Z-ajoene targets, but the mechanisms by which Z-ajoene interacted with microtubule appeared different from those of other microtubule poisons such as those of the Vinca alkaloids family. The ability of Z-ajoene to preferentially suppress the growth of neoplastic cells could provide a new approach in tumor therapy.