Marinopyrrole derivative MP1 as a novel anti-cancer agent in group 3 MYC-amplified Medulloblastoma.

BACKGROUND: Medulloblastoma (MB) patients with MYC oncogene amplification or overexpression exhibit extremely poor prognoses and therapy resistance. However, MYC itself has been one of the most challenging targets for cancer treatment. Here, we identify a novel marinopyrrole natural derivative, MP1, that shows desirable anti-MYC and anti-cancer activities in MB. METHODS: In this study, using MYC-amplified (Group 3) and non-MYC amplified MB cell lines in vitro and in vivo, we evaluated anti-cancer efficacies and molecular mechanism(s) of MP1. RESULTS: MP1 significantly suppressed MB cell growth and sphere counts and induced G2 cell cycle arrest and apoptosis in a MYC-dependent manner. Mechanistically, MP1 strongly downregulated the expression of MYC protein. Our results with RNA-seq revealed that MP1 significantly modulated global gene expression and inhibited MYC-associated transcriptional targets including translation/mTOR targets. In addition, MP1 inhibited MYC-target metabolism, leading to declined energy levels. The combination of MP1 with an FDA-approved mTOR inhibitor temsirolimus synergistically inhibited MB cell growth/survival by downregulating the expression of MYC and mTOR signaling components. Our results further showed that as single agents, both MP1 and temsirolimus, were able to significantly inhibit tumor growth and MYC expression in subcutaneously or orthotopically MYC-amplified MB bearing mice. In combination, there were further anti-MB effects on the tumor growth and MYC expression in mice. CONCLUSION: These preclinical findings highlight the promise of marinopyrrole MP1 as a novel MYC inhibition approach for MYC-amplified MB.

Assessment of Tissue Distribution and Metabolism of MP1, a Novel Pyrrolomycin, in Mice Using a Validated LC-MS/MS Method.

MP1 is a novel marinopyrrole analogue with activity in MYCN amplified neuroblastoma cell lines. A rapid, selective, and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of MP1 in mouse plasma. Analyte separation was achieved using a Waters Acquity UPLC®BEH C18 column (1.7 µm, 100 × 2.1 mm). Mobile phase consisted of 0.1% acetic acid in water (10%) and methanol (90%) at a total flow rate of 0.25 mL/min. The mass spectrometer was operated at unit resolution in the multiple reaction monitoring (MRM) mode, using precursor ion > product ion transitions of 324.10 > 168.30 m/z for MP1 and 411.95 > 224.15 m/z for PL-3. The MS/MS response was linear over the concentration range from 0.2-500 ng/mL for MP1, correlation coefficient (r2) of 0.988. Precision (% RSD) and accuracy (% bias) were within the acceptable limits as per FDA guidelines. MP1 was stable under storage and laboratory handling conditions. The validated method was successfully applied to assess the solubility, in-vitro metabolism, plasma protein binding, and bio-distribution studies of MP1.

Effects of novel pyrrolomycin MP1 in MYCN amplified chemoresistant neuroblastoma cell lines alone and combined with temsirolimus.

BACKGROUND: The activity of MP1, a pyrrolomycin, was studied in MYCN amplified neuroblastoma (NB) alone and combined with temsirolimus (TEM). METHODS: Activity of MP1 was tested in MYCN amplified (BE-2c, IMR) and non amplified (SKN-AS) NB cells. The effect of MP1 on MYCN, MCL-1, cleaved PARP, LC3II/LC3I, bcl-2, BAX, and BRD-4 were determined by western blot and RNAseq. The effect of MP1 on metabolism, mitochondrial morphology, and cell cycle was determined. Toxicology and efficacy of MP1 plus TEM were evaluated. RESULTS: The IC50 of MP1 was 0.096 µM in BE-2c cells compared to 0.89 µM in IMR, and >50 µM in SKN-AS. The IC50 of MP1 plus TEM in BE-2c cells was 0.023 µM. MP1 inhibited metabolism leading to quiescence and produced a decline in cell cycle S-phase. Electron microscopy showed cristae loss and rounding up of mitochondria. Gene and protein expression for MYCN and MCL-1 declined while LCII and cleaved PARP increased. Protein expression of BAX, bcl-2, and BRD-4 were not significantly changed after MP1 treatment. The in-vivo concentrations of MP1 in blood and tumor were sufficient to produce the biologic effects seen in-vitro. MP1 plus TEM produced a complete response in 3 out of 5 tumor bearing mice. In a second mouse study, the combination of MP1 and TEM slowed tumor growth compared to control. CONCLUSIONS: MP1 has a potent inhibitory effect on the viability of MYCN amplified NB. Inhibition of metabolism by MP1 induced quiescence and autophagy with a favorable toxicology and drug distribution profile. When combined with TEM anti-tumor activity was potentiated in-vitro and in-vivo.

Kinase and Histone Deacetylase Hybrid Inhibitors for Cancer Therapy.

Histone deacetylases (HDACs), encompassing at least 18 members, are promising targets for anticancer drug discovery and development. To date, five histone deacetylase inhibitors (HDACis) have been approved for cancer treatment, and numerous others are undergoing clinical trials. It has been well validated that an agent that can simultaneously and effectively inhibit two or more targets may offer greater therapeutic benefits over single-acting agents in preventing resistance to treatment and in potentiating synergistic effects. A prime example of a bifunctional agent is the hybrid HDAC inhibitor. In this perspective, the authors review the majority of reported kinase/HDAC hybrid inhibitors.

Structure-Based Identification of Novel Ligands Targeting Multiple Sites within a Chemokine-G-Protein-Coupled-Receptor Interface.

CXCL12 is a human chemokine that recognizes the CXCR4 receptor and is involved in immune responses and metastatic cancer. Interactions between CXCL12 and CXCR4 are an important drug target but, like other elongated protein-protein interfaces, present challenges for small molecule ligand discovery due to the relatively shallow and featureless binding surfaces. Calculations using an NMR complex structure revealed a binding hot spot on CXCL12 that normally interacts with the I4/I6 residues from CXCR4. Virtual screening was performed against the NMR model, and subsequent testing has verified the specific binding of multiple docking hits to this site. Together with our previous results targeting two other binding pockets that recognize sulfotyrosine residues (sY12 and sY21) of CXCR4, including a new analog against the sY12 binding site reported herein, we demonstrate that protein-protein interfaces can often possess multiple sites for engineering specific small molecule ligands that provide lead compounds for subsequent optimization by fragment based approaches.

Drug Design Targeting the CXCR4/CXCR7/CXCL12 Pathway.

Under physiological conditions, CXCL12 modulates cell proliferation, survival, angiogenesis, and migration mainly through CXCR4. Interestingly, the newly discovered receptor CXCR7 for CXCL12 is highly expressed in many tumor cells as well as tumor-associated blood vessels, although the level of CXCR7 in normal blood cells is low. Recently, many studies have suggested that CXCR7 promotes cell growth and metastasis in various cancers, including lymphoma and leukemia, hepatocecullar, ovarian, colorectal, breast and lung cancer. Compared to CXCR4, CXCR7 is a non-classical GPCR that is unable to activate G proteins. The function of CXCR7 is generally considered to be mediated by: (a) recruiting ß-arrestin-2; (b) heterodimerizing with CXCR4; and (c) acting as a "scavenger" of CXCL12, thus lowering the level of CXCL12 to weaken the activity of CXCR4. However, the crosstalk between CXCL12/CXCR7/CXCR4 and other signaling pathways (such as the p38 MAPK pathway, the PI3K/mTOR pathway, the STAT3 signaling, and metalloproteinases MMP-9 and MMP-2) is more complicated. The function of CXCR7 is also involved in modulating tumor microenvironment, tumor cell migration and apoptosis. Understanding these complex interactions will provide insight in drug design targeting the CXCR7 as potential anticancer therapy.

Marinopyrroles: Unique Drug Discoveries Based on Marine Natural Products.

Natural products provide a successful supply of new chemical entities (NCEs) for drug discovery to treat human diseases. Approximately half of the NCEs are based on natural products and their derivatives. Notably, marine natural products, a largely untapped resource, have contributed to drug discovery and development with eight drugs or cosmeceuticals approved by the U.S. Food and Drug Administration and European Medicines Agency, and ten candidates undergoing clinical trials. Collaborative efforts from drug developers, biologists, organic, medicinal, and natural product chemists have elevated drug discoveries to new levels. These efforts are expected to continue to improve the efficiency of natural product-based drugs. Marinopyrroles are examined here as a case study for potential anticancer and antibiotic agents.

Rho Kinase (ROCK) Inhibitors and Their Therapeutic Potential.

Rho kinases (ROCKs) belong to the serine-threonine family, the inhibition of which affects the function of many downstream substrates. As such, ROCK inhibitors have potential therapeutic applicability in a wide variety of pathological conditions including asthma, cancer, erectile dysfunction, glaucoma, insulin resistance, kidney failure, neuronal degeneration, and osteoporosis. To date, two ROCK inhibitors have been approved for clinical use in Japan (fasudil and ripasudil) and one in China (fasudil). In 1995 fasudil was approved for the treatment of cerebral vasospasm, and more recently, ripasudil was approved for the treatment of glaucoma in 2014. In this Perspective, we present a comprehensive review of the physiological and biological functions for ROCK, the properties and development of over 170 ROCK inhibitors as well as their therapeutic potential, the current status, and future considerations.

Design, synthesis and evaluation of marinopyrrole derivatives as selective inhibitors of Mcl-1 binding to pro-apoptotic Bim and dual Mcl-1/Bcl-xL inhibitors.

Inhibition of anti-apoptotic Mcl-1 is a promising anticancer strategy to overcome the survival and chemoresistance of a broad spectrum of human cancers. We previously reported on the identification of a natural product marinopyrrole A (1) that induces apoptosis in Mcl-1-dependent cells through Mcl-1 degradation. Here, we report the design and synthesis of novel marinopyrrole-based analogs and their evaluation as selective inhibitors of Mcl-1 as well as dual Mcl-1/Bcl-xL inhibitors. The most selective Mcl-1 antagonists were 34, 36 and 37 with 16-, 13- and 9-fold more selectivity for disrupting Mcl-1/Bim over Bcl-xL/Bim binding, respectively. Among the most potent dual inhibitors is 42 which inhibited Mcl-1/Bim and Bcl-xL/Bim binding 15-fold (IC50 = 600 nM) and 33-fold (500 nM) more potently than (±)-marinopyrrole A (1), respectively. Fluorescence quenching, NMR analysis and molecular docking indicated binding of marinopyrroles to the BH3 binding site of Mcl-1. Several marinopyrroles potently decreased Mcl-1 cellular levels and induced caspase 3 activation in human breast cancer cells. Our studies provide novel "lead" marinopyrroles for further optimization as selective Mcl-1 inhibitors and dual Mcl-1 and Bcl-xL inhibitors.

Structural analysis of a novel small molecule ligand bound to the CXCL12 chemokine.

CXCL12 binds to CXCR4, promoting both chemotaxis of lymphocytes and metastasis of cancer cells. We previously identified small molecule ligands that bind CXCL12 and block CXCR4-mediated chemotaxis. We now report a 1.9 Å resolution X-ray structure of CXCL12 bound by such a molecule at a site normally bound by sY21 of CXCR4. The complex structure reveals binding hot spots for future inhibitor design and suggests a new approach to targeting CXCL12-CXCR4 signaling in drug discovery.

Marinopyrrole derivatives with sulfide spacers as selective disruptors of Mcl-1 binding to pro-apoptotic protein Bim.

A series of novel marinopyrroles with sulfide and sulphone spacers were designed and synthesized. Their activity to disrupt the binding of the pro-apoptotic protein, Bim, to the pro-survival proteins, Mcl-1 and Bcl-xL, was evaluated using ELISA assays. Fluorescence-quenching (FQ) assays confirmed the direct binding of marinopyrroles to Mcl-1. Benzyl- and benzyl methoxy-containing sulfide derivatives 4 and 5 were highly potent dual Mcl-1/Bim and Bcl-xL/Bim disruptors (IC50 values of 600 and 700 nM), whereas carboxylate-containing sulfide derivative 9 exhibited 16.4-fold more selectivity for disrupting Mcl-1/Bim over Bcl-xL/Bim binding. In addition, a nonsymmetrical marinopyrrole 12 is as equally potent as the parent marinopyrrole A (1) for disrupting both Mcl-1/Bim and Bcl-xL/Bim binding. Some of the derivatives were also active in intact human breast cancer cells where they reduced the levels of Mcl-1, induced programd cell death (apoptosis) and inhibited cell proliferation.

Cyclic marinopyrrole derivatives as disruptors of Mcl-1 and Bcl-x(L) binding to Bim.

A series of novel cyclic marinopyrroles were designed and synthesized. Their activity to disrupt the binding of the pro-apoptotic protein, Bim, to the pro-survival proteins, Mcl-1 and Bcl-x(L), was evaluated using ELISA assays. Both atropisomers of marinopyrrole A (1) show similar potency. A tetrabromo congener 9 is two-fold more potent than 1. Two novel cyclic marinopyrroles (3 and 4) are two- to seven-fold more potent than 1.

Discovery of marinopyrrole A (maritoclax) as a selective Mcl-1 antagonist that overcomes ABT-737 resistance by binding to and targeting Mcl-1 for proteasomal degradation.

The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-X(L) and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-X(L) with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-X(L), and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-X(L)-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ∼60- to 2000-fold at 1-2 µM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation.

Fragment-based and structure-guided discovery and optimization of Rho kinase inhibitors.

Using high concentration biochemical assays and fragment-based screening assisted by structure-guided design, we discovered a novel class of Rho-kinase inhibitors. Compound 18 was equipotent for ROCK1 (IC(50) = 650 nM) and ROCK2 (IC(50) = 670 nM), whereas compound 24 was more selective for ROCK2 (IC(50) = 100 nM) over ROCK1 (IC(50) = 1690 nM). The crystal structure of the compound 18-ROCK1 complex revealed that 18 is a type 1 inhibitor that binds the hinge region in the ATP binding site. Compounds 18 and 24 inhibited potently the phosphorylation of the ROCK substrate MLC2 in intact human breast cancer cells.

Fragment-based optimization of small molecule CXCL12 inhibitors for antagonizing the CXCL12/CXCR4 interaction.

The chemokine CXCL12 and its G protein-coupled receptor (GPCR) CXCR4 are high-priority clinical targets because of their involvement in metastatic cancers (also implicated in autoimmune disease and cardiovascular disease). Because chemokines interact with two distinct sites to bind and activate their receptors, both the GPCRs and chemokines are potential targets for small molecule inhibition. A number of chemokines have been validated as targets for drug development, but virtually all drug discovery efforts focus on the GPCRs. However, all CXCR4 receptor antagonists with the exception of MSX-122 have failed in clinical trials due to unmanageable toxicities, emphasizing the need for alternative strategies to interfere with CXCL12/CXCR4-guided metastatic homing. Although targeting the relatively featureless surface of CXCL12 was presumed to be challenging, focusing efforts at the sulfotyrosine (sY) binding pockets proved successful for procuring initial hits. Using a hybrid structure-based in silico/NMR screening strategy, we recently identified a ligand that occludes the receptor recognition site. From this initial hit, we designed a small fragment library containing only nine tetrazole derivatives using a fragment-based and bioisostere approach to target the sY binding sites of CXCL12. Compound binding modes and affinities were studied by 2D NMR spectroscopy, X-ray crystallography, molecular docking and cell-based functional assays. Our results demonstrate that the sY binding sites are conducive to the development of high affinity inhibitors with better ligand efficiency (LE) than typical protein-protein interaction inhibitors (LE ≤ 0.24). Our novel tetrazole-based fragment 18 was identified to bind the sY21 site with a K(d) of 24 µM (LE = 0.30). Optimization of 18 yielded compound 25 which specifically inhibits CXCL12-induced migration with an improvement in potency over the initial hit 9. The fragment from this library that exhibited the highest affinity and ligand efficiency (11: K(d) = 13 µM, LE = 0.33) may serve as a starting point for development of inhibitors targeting the sY12 site.

Total synthesis of (+/-)-marinopyrrole A and its library as potential antibiotic and anticancer agents.

The first total synthesis of marine natural product, (+/-)-marinopyrrole A, has been accomplished via a nine-step synthesis in an overall yield of 30%. A small focused library based on marinopyrrole has been designed and synthesized. The scope of chemistry was investigated, and a robust chemistry suitable for library synthesis has been developed in the current study. The method that we have developed has made it possible to generate diverse analogues based on structurally novel marinopyrroles for study of potential antibiotic and anticancer activities.

Design, synthesis, cytoselective toxicity, structure-activity relationships, and pharmacophore of thiazolidinone derivatives targeting drug-resistant lung cancer cells.

Ten cytoselective compounds have been identified from 372 thiazolidinone analogues by applying iterative library approaches. These compounds selectively killed both non-small cell lung cancer cell line H460 and its paclitaxel-resistant variant H460 taxR at an IC 50 between 0.21 and 2.93 microM while showing much less toxicity to normal human fibroblasts at concentrations up to 195 microM. Structure-activity relationship studies revealed that (1) the nitrogen atom on the 4-thiazolidinone ring (ring B in Figure 1) cannot be substituted, (2) several substitutions on ring A are tolerated at various positions, and (3) the substitution on ring C is restricted to the -NMe 2 group at the 4-position. A pharmacophore derived from active molecules suggested that two hydrogen bond acceptors and three hydrophobic regions were common features. Activities against P-gp-overexpressing and paclitaxel-resistant cell line H460 taxR and modeling using a previously validated P-gp substrate pharmacophore suggested that active compounds were not likely P-gp substrates.

Development of anaplastic lymphoma kinase (ALK) small-molecule inhibitors for cancer therapy.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) involved in the genesis of several human cancers; indeed, ALK was initially identified in constitutively activated and oncogenic fusion forms--the most common being nucleophosmin (NPM)-ALK--in a non-Hodgkin's lymphoma (NHL) known as anaplastic large-cell lymphoma (ALCL) and subsequent studies identified ALK fusions in the human sarcomas called inflammatory myofibroblastic tumors (IMTs). In addition, two recent reports have suggested that the ALK fusion, TPM4-ALK, may be involved in the genesis of a subset of esophageal squamous cell carcinomas. While the cause-effect relationship between ALK fusions and malignancies such as ALCL and IMT is very well established, more circumstantial links implicate the involvement of the full-length, normal ALK receptor in the genesis of additional malignancies including glioblastoma, neuroblastoma, breast cancer, and others; in these instances, ALK is believed to foster tumorigenesis following activation by autocrine and/or paracrine growth loops involving the reported ALK ligands, pleiotrophin (PTN) and midkine (MK). There are no currently available ALK small-molecule inhibitors approved for clinical cancer therapy; however, recognition of the variety of malignancies in which ALK may play a causative role has recently begun to prompt developmental efforts in this area. This review provides a succinct summary of normal ALK biology, the confirmed and putative roles of ALK fusions and the full-length ALK receptor in the development of human cancers, and efforts to target ALK using small-molecule kinase inhibitors.

Application of a novel design paradigm to generate general nonpeptide combinatorial templates mimicking beta-turns: synthesis of ligands for melanocortin receptors.

We report the further application of a novel approach to template and ligand design by the synthesis of agonists of the melanocortin receptor. This design method uses the conserved structural data from the three-dimensional conformations of beta-turn peptides to design rigid nonpeptide templates that mimic the orientation of the main chain C-alpha atoms in a peptide beta-turn. We report details on a new synthesis of derivatives of template 1 that are useful for the synthesis of exploratory libraries. The utility of this technique is further exemplified by several iterative rounds of high-throughput synthesis and screening, which result in new partially optimized nonpeptide agonists for several melanocortin receptors.