Covalent isothiocyanate inhibitors of macrophage migration inhibitory factor as potential colorectal cancer treatments.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with roles in innate and adaptive human immune responses, as well as inflammation. MIF exerts its biological activity by binding to the cell surface receptor CD74 as well as intracellular signalling proteins. MIF also possesses keto-enol tautomerase activity. Inhibition of the tautomerase activity has been associated with loss of biological activity of MIF and a potential anticancer target. Isothiocyanates (ITCs) are a class of compounds present in cruciferous vegetables that inhibit the MIF tautomerase activity via covalent modification of the N-terminal proline. A range of substituted ITCs featuring benzyl, phenethyl and phenyl propyl isothiocyanates were designed, synthesised and tested to determine any structure activity relationship for inhibiting MIF. Crystal structures of covalent compounds 8 and 9 in complex with rhMIF revealed key hydrogen bonding and edge-to-face π stacking interactions. Compound 9 and 11 with sub micromolar activity were tested in the NCI60 cancer cell lines panel. Both compounds showed tissue-specific reduced growth in colon and renal cancer cell lines, while one of these showed potent, dose-dependent inhibition of growth against all seven colon cancer cell lines (GI50 < 2.5 µM) and all eight renal cancer cell lines (GI50 < 2.2 µM).

Design, synthesis and biological evaluation of P2-modified proline analogues targeting the HtrA serine protease in Chlamydia.

High temperature requirement A (HtrA) serine proteases have emerged as a novel class of antibacterial target, which are crucial in protein quality control and are involved in the pathogenesis of a wide array of bacterial infections. Previously, we demonstrated that HtrA in Chlamydia is essential for bacterial survival, replication and virulence. Here, we report a new series of proline (P2)-modified inhibitors of Chlamydia trachomatis HtrA (CtHtrA) developed by proline ring expansion and Cγ-substitutions. The structure-based drug optimization process was guided by molecular modelling and in vitro pharmacological evaluation of inhibitory potency, selectivity and cytotoxicity. Compound 25 from the first-generation 4-substituted proline analogues increased antiCtHtrA potency and selectivity over human neutrophil elastase (HNE) by approximately 6- and 12-fold, respectively, relative to the peptidic lead compound 1. Based on this compound, second-generation substituted proline residues containing 1,2,3-triazole moieties were synthesized by regioselective azide-alkyne click chemistry. Compound 49 demonstrated significantly improved antichlamydial activity in whole cell assays, diminishing the bacterial infectious progeny below the detection limit at the lowest dose tested. Compound 49 resulted in approximately 9- and 22-fold improvement in the inhibitory potency and selectivity relative to 1, respectively. To date, compound 49 is the most potent HtrA inhibitor developed against Chlamydia spp.

EGFR-targeted prodrug activation using bioorthogonal alkene-azide click-and-release chemistry.

Epidermal growth factor receptor (EGFR) is overexpressed in many cancers and therefore serves as an excellent target for prodrug activation. Functionalised trans-cyclooctenes (TCO) were conjugated to an EGFR antibody (cetuximab), providing a reagent for pre-targeting and localisation of the bioorthogonal reagent. The TCOs react with a 4-azidobenzyl carbamate doxorubicin prodrug via a [3 + 2]-cycloaddition and subsequent self-immolation leads to release of doxorubicin (click-and-release). In vitro cell-based assays demonstrated proof-of-concept, that cetuximab conjugated to highly strained TCO (AB-d-TCO) could bind to the EGFR in a melanoma cell line, and selectively activate the doxorubicin prodrug. In a non-EGFR expressing melanoma cell line, no significant prodrug activation was observed. In vivo experiments using this combination of AB-d-TCO and the azido-doxorubicin prodrug in a murine melanoma model revealed no significant anti-tumour activity or increased survival, suggesting there was insufficient prodrug activation and drug release at the tumour site.

Optimization of peptide-based inhibitors targeting the HtrA serine protease in Chlamydia: Design, synthesis and biological evaluation of pyridone-based and N-Capping group-modified analogues.

The obligate intracellular bacterium Chlamydia trachomatis (C. trachomatis) is responsible for the most common bacterial sexually transmitted infection and is the leading cause of preventable blindness, representing a major global health burden. While C. trachomatis infection is currently treatable with broad-spectrum antibiotics, there would be many benefits of a chlamydia-specific therapy. Previously, we have identified a small-molecule lead compound JO146 [Boc-Val-Pro-ValP(OPh)2] targeting the bacterial serine protease HtrA, which is essential in bacterial replication, virulence and survival, particularly under stress conditions. JO146 is highly efficacious in attenuating infectivity of both human (C. trachomatis) as well as koala (C. pecorum) species in vitro and in vivo, without host cell toxicity. Herein, we present our continuing efforts on optimizing JO146 by modifying the N-capping group as well as replacing the parent peptide structure with the 2-pyridone scaffold at P3/P2. The drug optimization process was guided by molecular modelling, enzyme and cell-based assays. Compound 18b from the pyridone series showed improved inhibitory activity against CtHtrA by 5-fold and selectivity over human neutrophil elastase (HNE) by 109-fold compared to JO146, indicating that 2-pyridone is a suitable bioisostere of the P3/P2 amide/proline for developing CtHtrA inhibitors. Most pyridone-based inhibitors showed superior anti-chlamydial potency to JO146 especially at lower doses (25 and 50 µM) in C. trachomatis and C. pecorum cell culture assays. Modifications of the N-capping group of the peptidyl inhibitors did not have much influence on the anti-chlamydial activities, providing opportunities for more versatile alterations and future optimization. In summary, we present 2-pyridone based analogues as a new generation of non-peptidic CtHtrA inhibitors, which hold better promise as anti-chlamydial drug candidates.

Self-immolative Linkers in Prodrugs and Antibody Drug Conjugates in Cancer Treatment.

BACKGROUND: The design of anti-cancer therapies with high anti-tumour efficacy and reduced toxicity continues to be challenging. Anti-cancer prodrug and antibody-drug-conjugate (ADC) strategies that can specifically and efficiently deliver cytotoxic compounds to cancer cells have been used to overcome some of the challenges. The key to the success of many of these strategies is a self-immolative linker, which after activation can release the drug payload. Various types of triggerable self-immolative linkers are used in prodrugs and ADCs to improve their efficacy and safety. OBJECTIVE: Numerous patents have reported the significance of self-immolative linkers in prodrugs and ADCs in cancer treatment. Based on the recent patent literature, we summarise methods for designing the site-specific activation of non-toxic prodrugs and ADCs in order to improve selectivity for killing cancer cells. METHODS: In this review, an integrated view of the potential use of prodrugs and ADCs in cancer treatment are provided. This review presents recent patents and related publications over the past ten years uptill 2020. RESULTS: The recent patent literature has been summarised for a wide variety of self-immolative PABC linkers, which are cleaved by factors including responding to the difference between the extracellular and intracellular environments (pH, ROS, glutathione) through over-expressed enzymes (cathepsin, plasmin, ß-glucuronidase) or bioorthogonal activation. The mechanism for self-immolation involves the linker undergoing a 1,4- or 1,6-elimination (via electron cascade) or intramolecular cyclisation to release cytotoxic drug at the targeted site. CONCLUSION: This review provides the commonly used strategies from recent patent literature in the development of prodrugs based on targeted cancer therapy and antibody-drug conjugates, which show promise in therapeutic applications.

Synthesis and Biological Evaluation of (-) and (+)-Spiroleucettadine and Analogues.

A second-generation enantiospecific synthesis of spiroleucettadine is described. The original reported antibacterial activity was not observed when the experiment was repeated on the synthetic samples; however, significant anti-proliferative activity was uncovered for both enantiomers of spiroleucettadine. Comparison of the optical rotational data and ORD-CD spectra of both enantiomers and the reported spectrum from the natural source have not provided a definitive answer regarding the absolute stereochemistry of naturally occurring spiroleucettadine. Efforts then focussed on alteration at the C-4 and C-5 positions of the slightly more active (-)-spiroleucettadine. Ten analogues were synthesised, with three analogues found to possess similar anti-proliferative profiles to spiroleucettadine against the H522 lung cancer cell line.

Controlled Delivery of Nitric Oxide for Cancer Therapy.

Nitric oxide (NO) is a short-lived, endogenously produced, signaling molecule which plays multiple roles in mammalian physiology. Underproduction of NO is associated with several pathological processes; hence a broad range of NO donors have emerged as potential therapeutics for cardiovascular and respiratory disorders, wound healing, the immune response to infection, and cancer. However, short half-lives, chemical reactivity, rapid systemic clearance, and cytotoxicity have hindered the clinical development of most low molecular weight NO donors. Hence, for controlled NO delivery, there has been extensive effort to design novel NO-releasing biomaterials for tumor targeting. This review covers the effects of NO in cancer biology, NO releasing moieties which can be used for NO delivery, and current advances in the design of NO releasing biomaterials focusing on their applications for tumor therapy.

Structure-activity analysis of peptidic Chlamydia HtrA inhibitors.

Chlamydia trachomatis high temperature requirement A (CtHtrA) is a serine protease that performs proteolytic and chaperone functions in pathogenic Chlamydiae; and is seen as a prospective drug target. This study details the strategies employed in optimizing the irreversible CtHtrA inhibitor JO146 [Boc-Val-Pro-ValP(OPh)2] for potency and selectivity. A series of adaptations both at the warhead and specificity residues P1 and P3 yielded 23 analogues, which were tested in human neutrophil elastase (HNE) and CtHtrA enzyme assays as well as Chlamydia cell culture assays. Trypsin and chymotrypsin inhibition assays were also conducted to measure off-target selectivity. Replacing the phosphonate moiety with α-ketobenzothiazole produced a reversible analogue with considerable CtHtrA inhibition and cell culture activity. Tertiary leucine at P3 (8a) yielded approximately 33-fold increase in CtHtrA inhibitory activity, with an IC50 = 0.68 ±â€¯0.02 µM against HNE, while valine at P1 retained the best anti-chlamydial activity. This study provides a pathway for obtaining clinically relevant inhibitors.

Alkene-Azide 1,3-Dipolar Cycloaddition as a Trigger for Ultrashort Peptide Hydrogel Dissolution.

An alkene-azide 1,3-dipolar cycloaddition between trans-cyclooctene (TCO) and an azide-capped hydrogel that promotes rapid gel dissolution is reported. Using an ultrashort aryl azide-capped peptide hydrogel (PhePhe), we have demonstrated proof-of-concept where upon reaction with TCO, the hydrogel undergoes a gel-sol transition via 1,2,3-triazoline degradation and 1,6-self-immolation of the generated aniline. The potential application of this as a general trigger in sustained drug delivery is demonstrated through release of encapsulated cargo (doxorubicin). Administration of TCO resulted in 87 % of the cargo being released in 10 h, compared to 13-14 % in the control gels. This is the first example of a potential bioorthogonal-triggered hydrogel dissolution using a traditional click-type reaction. This type of stimulus could be extended to other aryl azide-capped hydrogels.

Honey reduces the metastatic characteristics of prostate cancer cell lines by promoting a loss of adhesion.

Honey has been shown to have a range of therapeutic effects in humans, with anti-inflammatory and anti-bacterial effects among those previously characterised. Here, we examine the possibility of New Zealand thyme, manuka and honeydew honeys, and their major sugar and phenolic components, reducing the development of metastatic cancer. Their activity was examined in vitro, in PC3 and DU145 prostate cancer cell lines, through measuring the compounds' effects on the metastatic characteristics of migration, invasion and adhesion. First, the phenolic compounds gallic acid, caffeic acid, quercetin, kaempferol and chrysin were quantified in the honeys using high performance liquid chromatography, and found in nanomolar concentrations. In a Boyden chamber-based migration assay, non-toxic concentrations of thyme and honeydew honeys reduced cell migration by 20%, and all phenolic compounds except caffeic acid also lowered migration, although a mixture of only the sugars found in honey had no effect. All of the honeys, phenolics and the sugar-only mixture reduced invasive movement of cells through extracellular matrix by up to 75%. Most notably, each of the three honeys and the sugar-only mixture reduced cell adhesion to collagen I by 90%. With the exception of quercetin, phenolic compounds did not reduce adhesion. Therefore, honey and its sugar and phenolic components can lower the metastatic properties of cancer cells, and may do this by preventing effective cell adhesion to the extracellular matrix. The sugars and phenol compounds of honey are much more effective in combination than individually.

Poloxamer 407-chitosan grafted thermoresponsive hydrogels achieve synchronous and sustained release of antigen and adjuvant from single-shot vaccines.

Sustained-release vaccine delivery systems may enhance the immunogenicity of subunit vaccines and reduce the need for multiple vaccinations. The aim of this study was to develop a thermoresponsive hydrogel using poloxamer 407-chitosan (CP) grafted copolymer as a delivery system for single-shot sustained-release vaccines. The CP copolymer was synthesized using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide chemistry. The CP copolymer was a free flowing solution at ambient temperature and transformed rapidly into a gel at body temperature. The hydrogels were loaded with vaccine antigen and adjuvants or the vaccine components were encapsulated in poly (lactic-co-glycolic acid) nanoparticles in order to ensure synchronous release. The CP hydrogels were stable for up to 18 days in vitro. Release of both nanoparticles and the individual components was complete, with release of the individual components being modulated by incorporation into nanoparticles. In vivo, a single dose of CP hydrogel vaccine induced strong, long lasting, cellular and humoral responses that could protect against the development of tumors in a murine melanoma model.

Mechanistic Evaluation of Bioorthogonal Decaging with trans-Cyclooctene: The Effect of Fluorine Substituents on Aryl Azide Reactivity and Decaging from the 1,2,3-Triazoline.

Bioorthogonal prodrug activation/decaging strategies need to be selective, rapid and release the drug from the masking group upon activation. The rates of the 1,3-dipolar cycloaddition between a trans-cyclooctene (TCO) and a series of fluorine-substituted azido-PABC self-immolative spacers caging two model drugs, and subsequent release from the 1,2,3-triazoline are reported. As the number of fluorine substituents on the PABC linker increases from one to four, the rate of cycloaddition increases by almost one order of magnitude. Using a combination of fluorescence, 1H/19F NMR, and computational experiments, we have been able to determine how substituents on the PABC ring can influence the degradation rates and also the product distribution of the 1,2,3-triazoline. We have also been able to determine how these substituents influence the rate of imine hydrolysis and 1,6-self-immolation decaging rates of the generated anilines. The NMR and computational studies demonstrate that fluorine substituents on the aromatic ring lower the transition state energy required for converting the triazoline to the imine or aziridine intermediates via extrusion of diatomic nitrogen, and that in the case of a tetrafluoro substituted aromatic ring, it is the imine hydrolysis and 1,6-self-immolation that is rate-limiting. This knowledge further enhances the understanding of factors which influence the stability of triazolines, and enables potential applications of fluorinated aromatics, in particular, perfluorinated aromatics, in synthetic chemistry and sustained-release drug delivery systems.

Detection of Biosynthetic Precursors, Discovery of Glycosylated Forms, and Homeostasis of Calcitonin in Human Cancer Cells.

The peptide hormone calcitonin is intimately connected with human cancer development and proliferation. Its biosynthesis is reasoned to proceed via glycine-, α-hydroxyglycine-, glycyllysine-, and glycyllysyllysine-extended precursors; however, as a result of the limitations of current analytical methods, until now, there has been no procedure capable of detecting these individual species in cell or tissue samples. Therefore, their presence and dynamics in cancer had not been established. Here, we report the first methodology for the separation, detection, and quantification of calcitonin and each of its precursors in human cancer cells. We also report the discovery and characterization of O-glycosylated calcitonin and its analogous biosynthetic precursors. Through direct and simultaneous analysis of the glycosylated and nonglycosylated species, we interrogate the hormone biosynthesis. This shows that the cellular calcitonin level is maintained to mitigate effects of biosynthetic enzyme inhibitors that substantially change the proportions of calcitonin-related species released into the culture medium.

Stability, Kinetic, and Mechanistic Investigation of 1,8-Self-Immolative Cinnamyl Ether Spacers for Controlled Release of Phenols and Generation of Resonance and Inductively Stabilized Methides.

Three cinnamyl ether spacers (non-methyl, α-methyl, and γ-methyl) for caging of phenols have been synthesized and are physiologically stable. When triggered, the γ-methyl spacer releases phenols (pKa 7.8 and 9.8) with a t1/2 < 30 s and <2 min in aqueous and aqueous-organic solvent, respectively. The α-methyl spacer releases a phenol (pKa 7.8) with a t1/2 = 27 and 54 min. For the γ-methyl spacer, the results suggest the presence of a resonance and inductively stabilized aza-cinnamyl methide.

Hypoxia Responsive Drug Delivery Systems in Tumor Therapy.

Hypoxia is a common characteristic of solid tumors. It is mainly determined by low levels of oxygen resulting from imperfect vascular networks supplying most tumors. In an attempt to improve the present chemotherapeutic treatment and reduce associated side effects, several prodrug strategies have been introduced to achieve hypoxia-specific delivery of cytotoxic anticancer agents. With the advances in nanotechnology, novel delivery systems activated by the consequent outcomes of hypoxia have been developed. However, developing hypoxia responsive drug delivery systems (which only depend on low oxygen levels) is currently naïve. This review discusses four main hypoxia responsive delivery systems: polymeric based drug delivery systems, oxygen delivery systems combined with radiotherapy and chemotherapy, anaerobic bacteria which are used for delivery of genes to express anticancer proteins such as tumor necrosis alpha (TNF-α) and hypoxia-inducible transcription factors 1 alpha (HIF1α) responsive gene delivery systems.

Prohormone-substrate peptide sequence recognition by peptidylglycine α-amidating monooxygenase and its reflection in increased glycolate inhibitor potency.

The interactions of nineteen peptide substrates and fifteen analogous peptidomimetic glycolate inhibitors with human peptidylglycine α-amidating monooxygenase (PAM) have been investigated. The substrates and inhibitors are the prohormones of calcitonin and oxytocin and their analogues. PAM both secreted into the medium by and extracted from DMS53 small lung carcinoma cells has been studied. The results show that recognition of the prooxytocin and procalcitonin peptide sequences by the enzyme extends more than four and five amino acid residues, respectively, from their C-termini. This substrate sequence recognition is mirrored by increased inhibitor potency with increased peptide length in the glycolate peptidomimetics. Substitution of the C-terminal penultimate glycine and proline residues of prooxytocin and procalcitonin and their analogues with phenylalanine increases the enzyme binding affinity. However, this changes the binding mode from one that depends on peptide sequence recognition to another primarily determined by the phenylalanine moiety, for both the substrates and analogous glycolate inhibitors.

Development and assessment of novel all-in-one parenteral formulations with integrated anticoagulant properties for the concomitant delivery of 5-fluorouracil and calcium folinate.

5-Fluorouracil in combination with its biomodulator folinic acid maintains a pivotal position in current anticancer treatment regimens. However, limitations in clinical management persist with the administration of these drugs. These limitations are associated with the use of a high pH to maintain 5-fluorouracil in solution, resulting in high rates of phlebitis and catheter blockages. Herein, we describe and compare initial studies on novel all-in-one formulations of 5-fluorouracil and folinic acid incorporating either sulfated or hydroxypropyl beta-cyclodextrins at physiological pH that potentially address these issues. All formulations markedly improved the stability of supersaturated solutions of 5-fluorouracil in the presence of folinic acid. In-vitro evaluation of the PC-3, HCT-116, MDA-MB-231, PC-14, and COLO-201 human carcinoma cell lines showed that all formulations exhibited equivalent or better cytotoxicity compared with cells exposed to 5-fluorouracil and folinic acid. Thus, these cyclodextrins do not compromise the cytotoxicity of 5-fluorouracil. Preliminary in-vivo dose tolerance profiles of the formulations were also equivalent to 5-fluorouracil and folinic acid administered separately. Furthermore, given the association between thrombosis and cancer, the potentially beneficial anticoagulant activity of the sulfated cyclodextrin-based formulations was also confirmed in vitro. Extended activated partial thromboplastin times and prothrombin times were observed for the sulfated cyclodextrins in human plasma both as individual compounds and as components of the formulations. In conclusion, these novel all-in-one formulations maintain the in-vitro potency while overcoming the accepted incompatibility of 5-fluorouracil and folinic acid, and represent improved injectable forms of 5-fluorouracil that may reduce phlebitis, catheter blockages, and thromboembolic events.