TDiscovery and Development of Novel Therapeutic Agents for Advanced Melanoma

Malignant melanoma is the most dangerous form of skin cancer and accounts for about 75% of skin cancer deaths. Once diagnosed at the metastatic stage, it has a very poor prognosis with a median survival rate of 6 months and a 5-year survival rate of less than 5%. In addition, melanoma has become an important public health hazard owing to its rising incidence, which has been well documented over the past 50 years. Currently there is no effective way to treat melanoma. It is highly resistant to existing chemotherapy, radiotherapy, and immunotherapy. Over the past 30 years, only two drugs have been approved by the Food and Drug Administration (FDA) for metastatic melanoma: dacarbazine (DTIC) and interleukin-2 (IL-2). But even with these two drugs, fewer than 15% of patients have a favorable response and fewer than 5% of patients reach complete remission. On the other hand, the toxicity associated with DTIC and IL-2 is often significant, resulting in serious or life threatening side effects in many patients. In recent years, great efforts have been made in fighting metastatic melanoma. But neither combinations of DTIC with other chemotherapy drugs (e.g., cisplatin, vinblastine, and carmustine) nor adding interferon-α2b to DTIC have shown a survival advantage over DTIC treatment alone. Extensive clinical trials with a lot of antibodies and vaccines to treat metastatic melanoma have also failed to demonstrate satisfactory efficacy. Therefore, developing more effective drugs for melanoma is urgently needed. We started our efforts in finding new drugs for melanoma by screening a large compound library. The in vitro cytotoxicity data on several melanoma cell lines led us to the discovery of three active structure scaffolds: serine amides, serine amino alcohols, and arylthiazolidine-4-carboxylic acid amides (ATCAAs). Because ATCAAs showed better selectivity between cancer cells and normal fibroblast cells, the chemists in our group focused on this scaffold and performed extensive structure modifications for structure-activity-relationship (SAR) studies. The SAR results were then used to guide further synthesis in an effort to maximize activity and selectivity. Two active compounds identified during the process were sent to the U.S. National Cancer Institute for anticancer screening using 60 human tumor cell lines. Results showed that these two compounds have extensive cytotoxic activity against all nine types of cancer cells with IC50values ranging from 120 nM (leukemia, CCRF-CEM cell line) to 11 μM (colon cancer, HCC-15 cell line). One compound showed particularly good activity against melanoma cells (IC50=130 nM~1 mM against all eight melanoma cell lines). I then evaluated ATCAAs inhibitory effect on melanoma colony formation and in vivo melanoma tumor growth. The in vivo data were very encouraging. One tested compound significantly inhibited melanoma tumor growth at a dose of 10 mg/kg and showed higher efficacy than did DTIC at a dose of 60 mg/kg. These findings built up a strong basis for the development of novel chemotherapeutic drugs for advanced melanoma. Furthermore, the chemists in our group also synthesized some new imidazole and imidazoline analogs by focusing on the SAR studies of the central five-member ring. Although the current compounds displayed lower potency when compared with our lead thiazolidine analogs, they may have the distinct advantage of being more stable in vivo with the reduced necessity of chiral separations. Some of these new compounds have activity similar to Sorafenib, an FDA-approved drug that has been tested clinically in melanoma patients. To further expand our understanding of SARs and to potentially identify new platforms for active compounds, Dr. Li and Dr. Seibel explored a compound library from the University of Cincinnati’s Drug Discovery Center. This library contains 342,910 small molecules. Based on the structure of our lead molecule, two ligand-based virtual screening approaches were used: 1) similarity search based on atom connectivity by using Scitegic Pipeline Pilot software and 2) similarity search based on molecular shape by using Schrodinger software. Results showed that these two approaches are highly complementary and lead to different active molecular structures. These structures are quite suitable for further structural modification and provide new platforms for our anticancer drug discovery efforts. Subsequently, further lead structure optimization led to the discovery of substituted methoxylbenzoyl-aryl-thiazole (SMART) compounds. To improve solubility and to circumvent the metabolic instability brought by the thiazole ring, our team designed and synthesized a new series of analogs: 2-aryl-4-benzoyl-imidazoles (ABIs). These two classes of compounds showed great in vitro cytotoxicity against melanoma, and the IC50 of the most active compound was below 10 nM. They also showed equal potency against multi-drug resistant melanoma cells and the sensitive parent cells, indicating that these compounds can effectively overcome multi-drug resistance, which is a major cause of cancer chemotherapy failure. In vivo testing on C57BL/6 mice bearing B16-F1 melanoma allograft and on double homozygous SCID (severe combined immunodeficiency) hairless outbred (SHO) mice or athymic nude mice bearing A375 human melanoma xenograft showed these two classes of compounds significantly inhibited melanoma tumor growth. Some compounds even showed substantially better activity than did DTIC, the gold standard anti-melanoma drug. Meanwhile, preliminary toxicity studies suggested that mice can tolerate tested compounds well at effective dose levels. No sign of acute toxicity was observed from the experiments. More importantly, I identified the cellular target for ABI and SMART compounds through a series of biotechniques and molecular modeling studies. Strong experimental evidence has shown that these compounds bind to tubulin at the colchicine binding site in the α/β-tubulin heterodimers to disrupt functional microtubule formation. In the meantime, I also tested the pharmacokinetic properties of some active compounds in mice together with Mr. Chien-ming Li. With their good in vivo anti-melanoma activity and their ability to overcome multi-drug resistance, these new classes of compounds have great potential for melanoma therapy

Analogue-based approaches in anti-cancer compound modelling: the relevance of QSAR models

Background: QSAR is among the most extensively used computational methodology for analogue-based design. The application of various descriptor classes like quantum chemical, molecular mechanics, conceptual density functional theory (DFT)- and docking-based descriptors for predicting anti-cancer activity is well known. Although in vitro assay for anti-cancer activity is available against many different cell lines, most of the computational studies are carried out targeting insufficient number of cell lines. Hence, statistically robust and extensive QSAR studies against 29 different cancer cell lines and its comparative account, has been carried out. Results: The predictive models were built for 266 compounds with experimental data against 29 different cancer cell lines, employing independent and least number of descriptors. Robust statistical analysis shows a high correlation, cross-validation coefficient values, and provides a range of QSAR equations. Comparative performance of each class of descriptors was carried out and the effect of number of descriptors (1-10) on statistical parameters was tested. Charge-based descriptors were found in 20 out of 39 models (approx. 50%), valency-based descriptor in 14 (approx. 36%) and bond order-based descriptor in 11 (approx. 28%) in comparison to other descriptors. The use of conceptual DFT descriptors does not improve the statistical quality of the models in most cases. Conclusion: Analysis is done with various models where the number of descriptors is increased from 1 to 10; it is interesting to note that in most cases 3 descriptor-based models are adequate. The study reveals that quantum chemical descriptors are the most important class of descriptors in modelling these series of compounds followed by electrostatic, constitutional, geometrical, topological and conceptual DFT descriptors. Cell lines in nasopharyngeal (2) cancer average R2 = 0.90 followed by cell lines in melanoma cancer (4) with average R2 = 0.81 gave the best statistical values

Application of Molecular Topology for the Prediction of Reaction Yields and Anti-Inflammatory Activity of Heterocyclic Amidine Derivatives

Topological-mathematical models based on multiple linear regression analyses have been built to predict the reaction yields and the anti-inflammatory activity of a set of heterocylic amidine derivatives, synthesized under environmental friendly conditions, using microwave irradiation. Two models with three variables each were selected. The models were validated by cross-validation and randomization tests. The final outcome demonstrates a good agreement between the predicted and experimental results, confirming the robustness of the method. These models also enabled the screening of virtual libraries for new amidine derivatives predicted to show higher values of reaction yields and anti-inflammatory activity

Lysophospholipid (LPA) receptors in GtoPdb v.2021.3

Lysophosphatidic acid (LPA) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Lysophospholipid Receptors [55, 19, 82, 129]) are activated by the endogenous phospholipid LPA. The first receptor, LPA1, was identified as ventricular zone gene-1 (vzg-1) [40], This discovery represented the beginning of the de-orphanisation of members of the endothelial differentiation gene (edg) family, as other LPA and sphingosine 1-phosphate (S1P) receptors were found. Five additional LPA receptors (LPA2,3,4,5,6) have since been identified [82] and their gene nomenclature codified for human LPAR1, LPAR2, etc. (HUGO Gene Nomenclature Committee, HGNC) and Lpar1, Lpar2, etc. for mice (Mouse Genome Informatics Database, MGI) to reflect species and receptor function of their corresponding proteins. The crystal structure of LPA1 is solved and indicates that LPA accesses the extracellular binding pocket, consistent with its proposed delivery via autotaxin [13]. These studies have also implicated cross-talk with endocannabinoids via phosphorylated intermediates that can also activate these receptors. The binding affinities to LPA1 of unlabeled, natural LPA and anandamide phosphate (AEAp) were measured using backscattering interferometry (pKd = 9) [83, 104]. Utilization of this method indicated affinities that were 77-fold lower than when measured using radioactivity-based protocols [128]. Targeted deletion of LPA receptors has clarified signalling pathways and identified physiological and pathophysiological roles. Multiple groups have independently published validation of all six LPA receptors described in these tables, and further validation was achieved using a distinct read-out via a novel TGFα "shedding* assay [48]. LPA has been proposed to be a ligand for GPR35 [94], supported by a study revealing that LPA modulates macrophage function through GPR35 [54]. However chemokine (C-X-C motif) ligand 17 (CXCL17) is reported to be a ligand for GPR35/CXCR8 [76]. Moreover, LPA has also been described as an agonist for the transient receptor potential (Trp) ion channels TRPV1 [87] and TRPA1 [58]. All of these proposed non-GPCR receptor identities require confirmation and are not currently recognized as bona fide LPA receptors

Lysophospholipid (LPA) receptors in GtoPdb v.2021.2

Lysophosphatidic acid (LPA) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Lysophospholipid Receptors [55, 19, 82, 129]) are activated by the endogenous phospholipid LPA. The first receptor, LPA1, was identified as ventricular zone gene-1 (vzg-1) [40], This discovery represented the beginning of the de-orphanisation of members of the endothelial differentiation gene (edg) family, as other LPA and sphingosine 1-phosphate (S1P) receptors were found. Five additional LPA receptors (LPA2,3,4,5,6) have since been identified [82] and their gene nomenclature codified for human LPAR1, LPAR2, etc. (HUGO Gene Nomenclature Committee, HGNC) and Lpar1, Lpar2, etc. for mice (Mouse Genome Informatics Database, MGI) to reflect species and receptor function of their corresponding proteins. The crystal structure of LPA1 is solved and indicates that LPA accesses the extracellular binding pocket, consistent with its proposed delivery via autotaxin [13]. These studies have also implicated cross-talk with endocannabinoids via phosphorylated intermediates that can also activate these receptors. The binding affinities to LPA1 of unlabeled, natural LPA and anandamide phosphate (AEAp) were measured using backscattering interferometry (pKd = 9) [83, 104]. Utilization of this method indicated affinities that were 77-fold lower than when measured using radioactivity-based protocols [128]. Targeted deletion of LPA receptors has clarified signalling pathways and identified physiological and pathophysiological roles. Multiple groups have independently published validation of all six LPA receptors described in these tables, and further validation was achieved using a distinct read-out via a novel TGFα "shedding* assay [48]. LPA LPA has been proposed to be a ligand for GPCR35 [94], supported by a recent study revealing that LPA modulates macrophage function through GPR35 [54]. However chemokine (C-X-C motif) ligand 17 (CXCL17) is reported to be a ligand for GPR35/CXCR8 [76]. Moreover, LPA has also been described as an agonist for the transient receptor potential (Trp) ion channels TRPV1 [87] and TRPA1 [58]. All of these proposed non-GPCR receptor identities require confirmation and are not currently recognized as bona fide LPA receptors

Lysophospholipid (LPA) receptors (version 2020.5) in the IUPHAR/BPS Guide to Pharmacology Database

Lysophosphatidic acid (LPA) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Lysophospholipid Receptors [54, 18, 80, 125]) are activated by the endogenous phospholipid LPA. The first receptor, LPA1, was identified as ventricular zone gene-1 (vzg-1) [39], leading to deorphanisation of members of the endothelial differentiation gene (edg) family as other LPA receptors along with sphingosine 1-phosphate (S1P) receptors. Additional LPA receptor GPCRs were later identified. Gene names have been codified as LPAR1, etc. to reflect the receptor function of proteins. The crystal structure of LPA1 was solved and demonstrates extracellular LPA access to the binding pocket, consistent with proposed delivery via autotaxin [12]. These studies have also implicated cross-talk with endocannabinoids via phosphorylated intermediates that can also activate these receptors. The identified receptors can account for most, although not all, LPA-induced phenomena in the literature, indicating that a majority of LPA-dependent phenomena are receptor-mediated. Binding affinities of unlabeled, natural LPA and AEAp to LPA1 were measured using backscattering interferometry (pKd = 9) [81, 102]. Binding affinities were 77-fold lower than than values obtained using radioactivity [124]. Targeted deletion of LPA receptors has clarified signalling pathways and identified physiological and pathophysiological roles. Independent validation by multiple groups has been reported in the peer-reviewed literature for all six LPA receptors described in the tables, including further validation using a distinct read-out via a novel TGFα "shedding" assay [47]. LPA LPA has been proposed to be a ligand for GPCR35 [92], supported by a recent study revealing that LPA modulates macrophage function through GPR35 [53]. However CXCL17 is reported to be a ligand for GPR35/CXCR8 [74]. Moreover, LPA has also been described as an agonist for the transient receptor potential (Trp) ion channel TRPV1 [85] and TRPA1 [57]. All of these proposed non-GPCR receptor identities require confirmation and are not currently recognized as bona fide LPA receptors

Synthesis of Novel Sulfonamide-Based Calpain Inhibitors and Their Potential as Anti-Tumor Agents

Calpain is a class of intracellular cytoplasmic cysteine proteases.1 The enzyme participates in different intracellular signaling pathways that are mediated by Ca2+.2 The two major isoforms of calpain universally distributed in most mammalian tissues are calpain 1 (µ-calpain) and calpain 2 (m-calpain). The exact in vivo function of the enzyme is not clear, but calpain has been implicated in a variety of physiological and pathological conditions,3 such as cancer, stroke, cardiac ischaemia, muscular dystrophy, cataract and Alzheimer’s disease. Calpain inhibitors are therefore of interest as therapeutic agents and as biomedical tools. Several potent calpain inhibitors isolated from natural sources as well as synthesized in the laboratory have been reported (Chapter 1.4). Unfortunately, most of the inhibitors show poor calpain selectivity, metabolic stability and cell permeability. In an attempt to develop potential calpain inhibitors based on the X-ray crystal structure of the µ-calpain, NCI compound library was screened by virtual screening method and diazosulfonamide 1 (Ki = 1.0 ± 0.02 µM) was identified as a new nonpeptide competitive inhibitor of µ-calpain. Analogues of 1 were synthesized to explore structure requirements of 1 (Chapter 2). In order to test the hypothesis that derivatives of diazosulfonamide 1 with an electrophilic group can have a covalent interaction with cysteine at the active site of calpain, novel sulfonamide-based peptidomimetic analogues of 1 were synthesized and assayed for their ability to inhibit µ-calpain utilizing a kinetic fluorescence assay and for their anti-tumor ability by SRB colorimetric assay (Chapter 3). Introduction of the electrophilic functionality significantly enhanced calpain inhibition. From 13 target compounds, 7 compounds had better calpain 1 inhibition (Ki ranging from 9 to 500nM) than 1 and 5 showed good anticancer activity (GI50 ranging from 4 to 22µM). Sulfonamide-based peptidomimetic analogue 19 with Ki of 9 nM is over 100-fold more potent than the lead diazosulfonamide 1. Compound 16 was the most effective anticancer agent (GI50 4µM) of the series

Discovery of Novel Tubulin Polymerization Inhibitors and Survivin Inhibitors as Potential Anticancer Agents

Melanoma is the most dangerous form of skin cancer and accounts for the majority of skin cancer death. Although considerable advances have been made in melanoma treatment in recent years, there are many problems associated with current therapies. Drug resistance to targeted therapies is almost inevitable after short term treatment. Immunotherapies generally have low response rate and the effects vary among patients. Therefore, the need to develop new and more effective treatment for melanoma is high. The work presented here focuses on discovery of novel anticancer agents for melanoma by targeting two important cancer targets: tubulin and survivin. Microtubules play important role in mitosis and cell division. Cancer cells divide more rapidly than normal cells, and thus are more sensitive to tubulin targeting agents which disrupt mitosis. Targeting tubulin for anticancer treatment has been very successful. Chapter 3 describes our discovery process of a series of novel tubulin polymerization inhibitors. Our lab previously reported a set of 2-aryl-4-benzoyl- imidazoles (ABI) derivatives with potent anti-proliferative activity against melanoma cells and xenografts. A new series of 4-aryl-2-benzoyl-imidazoles were designed and synthesized after further optimization of the ABI series. The new scaffold reversed the position of the aryl group and benzoyl group on the imidazole ring of ABI structure and thus was named RABI. Those newly synthesized compounds were tested against eight different cancer cells, including multidrug-resistant cancer cell lines. The in vitro results showed that several compounds in this series had excellent anti-proliferative activities. The best compound displayed IC50 value in single-digit nano-molar against several tumor cell lines. In addition, RABI compounds showed advantages in overcoming multiple drug resistance, compared with existing tubulin targeting agents, paclitaxel, colchicine, and vinblastine. Mechanism of action for the RABI compounds was investigated using cell cycle analysis, tubulin polymerization assay, competitive mass spectrometry binding assay, and molecular docking studies. These studies suggested that RABI compounds exerted their anticancer effects by inhibiting tubulin polymerization at the colchicine binding site. The RABI compounds represent promising cancer drug candidates for further development. Another attractive target for anticancer treatment is survivin. The differential expression of survivin between normal differentiated cells and tumor cells and the essential role of survivin in tumor make it an ideal target for cancer. Although survivin is an attractive cancer drug target, the pool of existing survivin inhibitors is quite limited. Thus, it is highly significant to develop new survivin inhibitors. Our attempts to search for new survivin inhibitors were initiated by the identification of UC-112 which turned out to be a potent and selective survivin inhibitor. UC-112 was discovered through virtual screening from a library of compounds. It showed good potency both in vitro and in vivo. Structural modifications of the UC-112 oxyquinoline template generated a number of UC-112 analogs. The new analogs were tested on a panel of cancer cell lines. The results suggested that the new derivatives had strong anticancer activity. Several compounds showed IC50 value in the nano-molar range. The structure-activity relationships were also elucidated through the structural modifications. Our best compounds along with UC-112 were submitted for NCI-60 cancer cell line screening. The results from the screening indicated the best compound in this series improved potency in terms of GI50. The most potent compound also showed good drug-like properties. Mechanism of action for the UC-112 analogs was investigated using Western blot and drug affinity responsive target stability (DARTS) assay. This set of compounds selectively inhibited the expression of survivin over other IAP proteins and also protected survivin from digestion by pronase in the DARTS assay. The most potent compound from the in vitro assay effectively suppressed tumor growth in melanoma xenograft. This novel scaffold represents a new structure template for survivin inhibitors and can be further developed as potential anticancer agents

Investigation into the Anticancer Mechanism of Action of Novel 4-Substituted Phenylthiazoles and Antihelminthic Benzimidazoles

Recently, two novel 4-substituted phenylthiazoles, D1 and D2, have been synthesised based upon the marine bacterial secondary metabolite anithiactin A/thiasporine C, and shown to be cytotoxic towards cancer cells below 100 μM, with suggestions of inhibition of tubulin polymerisation. D1 and D2 were then further screened for cytotoxicity in three cancer cell lines (MDA-MB-231, MCF-7, HT-29) and one non-transformed mesenchymal stem-cell line (RCB2157), using the MTT assay. Their effects on the cell cycle and cell death mechanisms were analysed using flow cytometry and fluorescent microscopy and spectroscopy. Unfortunately, no significant changes in cell death mechanisms were observed and previous cytotoxicity results were unable to be replicated. This was attributed to the degradation of the compounds in DMSO solution, likely to cause a loss in biological activity. Antihelminthic benzimidazole drugs mebendazole and albendazole are commonly used to treat a variety of worm infestations in humans. Their mechanism of action against helminths is well-established and involves the inhibition of microtubule formation. Mebendazole has recently shown promising results in pre-clinical in vitro and in vivo cancer studies and is currently in Phase I trials for treatment of glioma. However, the way in which it causes cell death in cancer cells has not been fully explored. Here, the in vitro analysis of the anticancer mechanism of action of mebendazole and a structural analogue albendazole was undertaken. The two drugs were screened for cytotoxicity in three cancer cell lines (MDA-MB-231, MCF-7, HT-29) and one non-transformed mesenchymal stem-cell line (RCB2157), using the MTT assay. Their effects on the cell cycle and cell death mechanisms were analysed using flow cytometry and fluorescent microscopy and spectroscopy. Mebendazole and albendazole were found to selectively kill cancer cells, being most potent in the colorectal cancer cell line HT-29, with IC50 values of 1.3 ± 0.1 μM and 1.4 ± 0.1 μM, respectively. Both mebendazole and albendazole induced caspase-3 activation. Phosphatidylserine exposure, mitochondrial and lysosomal membrane permeability and reactive oxygen species production were all significantly increased compared to control and peaked at 24 hours, with DNA fragmentation increasing in a time-dependent manner peaking at 48 hours. Using Hoechst 33342 staining, nuclear features of apoptosis such as chromatin condensation were found following treatment with both drugs. Cell cycle arrest in the G2/M phase was found, and tubulin structures were significantly altered. Mebendazole and albendazole appear to cause cancer cell death via a mechanism of classical apoptosis and cell cycle arrest, which may originate from the destabilisation of microtubules