Design, Synthesis and Anticancer Evaluation of Nitroimidazole Radiosensitisers.

The role of hypoxic tumour cells in resistance to radiotherapy, and in suppression of immune response, continues to endorse tumour hypoxia as a bona fide, yet largely untapped, drug target. Radiotherapy innovations such as stereotactic body radiotherapy herald new opportunities for classical oxygen-mimetic radiosensitisers. Only nimorazole is used clinically as a radiosensitiser, and there is a dearth of new radiosensitisers in development. In this report, we augment previous work to present new nitroimidazole alkylsulfonamides and we document their cytotoxicity and ability to radiosensitise anoxic tumour cells in vitro. We compare radiosensitisation with etanidazole and earlier nitroimidazole sulfonamide analogues and we identify 2-nitroimidazole and 5-nitroimidazole analogues with marked tumour radiosensitisation in ex vivo assays of surviving clonogens and with in vivo tumour growth inhibition.

Chemical Space Mimicry for Drug Discovery.

We describe a new library generation method, Machine-based Identification of Molecules Inside Characterized Space (MIMICS), that generates sets of molecules inspired by a text-based input. MIMICS-generated libraries were found to preserve distributions of properties while simultaneously increasing structural diversity. Newly identified MIMICS-generated compounds were found to be bioactive as inhibitors of specific components of the unfolded protein response (UPR) and the VEGFR2 pathway in cell-based assays, thus confirming the applicability of this methodology toward drug design applications. Wider application of MIMICS could facilitate the efficient utilization of chemical space.

Identification of 6-Anilino Imidazo[4,5-c]pyridin-2-ones as Selective DNA-Dependent Protein Kinase Inhibitors and Their Application as Radiosensitizers.

The dominant role of non-homologous end-joining in the repair of radiation-induced double-strand breaks identifies DNA-dependent protein kinase (DNA-PK) as an excellent target for the development of radiosensitizers. We report the discovery of a new class of imidazo[4,5-c]pyridine-2-one DNA-PK inhibitors. Structure-activity studies culminated in the identification of 78 as a nM DNA-PK inhibitor with excellent selectivity for DNA-PK compared to related phosphoinositide 3-kinase (PI3K) and PI3K-like kinase (PIKK) families and the broader kinome, and displayed DNA-PK-dependent radiosensitization of HAP1 cells. Compound 78 demonstrated robust radiosensitization of a broad range of cancer cells in vitro, displayed high oral bioavailability, and sensitized colorectal carcinoma (HCT116/54C) and head and neck squamous cell carcinoma (UT-SCC-74B) tumor xenografts to radiation. Compound 78 also provided substantial tumor growth inhibition of HCT116/54C tumor xenografts in combination with radiation. Compound 78 represents a new, potent, and selective class of DNA-PK inhibitors with significant potential as radiosensitizers for cancer treatment.

Discovery and preliminary structure-activity relationship analysis of 1,14-sperminediphenylacetamides as potent and selective antimalarial lead compounds.

Screening of synthesized and isolated marine natural products for in vitro activity against four parasitic protozoa has identified the ascidian metabolite 1,14-sperminedihomovanillamide (orthidine F, 1) as being a non-toxic, moderate growth inhibitor of Plasmodium falciparum (IC(50) 0.89 µM). Preliminary structure-activity relationship investigation identified essentiality of the spermine polyamine core and the requirement for 1,14-disubstitution for potent activity. One analogue, 1,14-spermine-di-(2-hydroxyphenylacetamide) (3), exhibited two orders of magnitude increased anti-P. f activity (IC(50) 8.6 nM) with no detectable in vitro toxicity. The ease of synthesis of phenylacetamido-polyamines, coupled with potent nM levels of activity towards dual drug resistant strains of P. falciparum makes this compound class of interest in the development of new antimalarial therapeutics.

Identification of Small-Molecule Positive Modulators of Calcitonin-like Receptor-Based Receptors.

Class B G protein-coupled receptors are highly therapeutically relevant but challenges remain in identifying suitable small-molecule drugs. The calcitonin-like receptor (CLR) in particular is linked to conditions such as migraine, cardiovascular disease, and inflammatory bowel disease. The CLR cannot act as a cell-surface receptor alone but rather must couple to one of three receptor activity-modifying proteins (RAMPs), forming heterodimeric receptors for the peptides adrenomedullin and calcitonin gene-related peptide. These peptides have extended binding sites across their receptors. This is one reason why there are few small-molecule ligands that can modulate these receptors. Here we describe small molecules that are able to positively modulate the signaling of the CLR with all three RAMPs but are not active at the related calcitonin receptor. These compounds were selected from a ß-arrestin recruitment screen, coupled with rounds of medicinal chemistry to improve their activity. Translational potential is shown as the compounds can positively modulate cAMP signaling in a vascular cell line model. Binding experiments do not support an extracellular domain binding site; however, molecular modeling reveals potential allosteric binding sites in multiple receptor regions. These are the first small-molecule positive modulators described for the CLR:RAMP complexes.

Hypoxia-Activated Prodrugs of PERK Inhibitors.

Tumour hypoxia plays an important role in tumour progression and resistance to therapy. Under hypoxia unfolded proteins accumulate in the endoplasmic reticulum (ER) and this stress is relieved through the protein kinase R-like ER kinase (PERK) signalling arm of the unfolded protein response (UPR). Targeting the UPR through PERK kinase inhibitors provides tumour growth inhibition, but also elicits on-mechanism normal tissue toxicity. Hypoxia presents a target for tumour-selective drug delivery using hypoxia-activated prodrugs. We designed and prepared hypoxia-activated prodrugs of modified PERK inhibitors using a 2-nitroimidazole bioreductive trigger. The new inhibitors retained PERK kinase inhibitory activity and the corresponding prodrugs were strongly deactivated. The prodrugs were able to undergo fragmentation following radiolytic reduction, or bioreduction in HCT116 cells, to release their effectors, albeit inefficiently. We examined the effects of the prodrugs on PERK signalling in hypoxic HCT116 cells. This study has identified a 2-substituted nitroimidazole carbamate prodrug with potential to deliver PERK inhibitors in a hypoxia-selective manner.

Hypoxia-selective radiosensitisation by SN38023, a bioreductive prodrug of DNA-dependent protein kinase inhibitor IC87361.

DNA-dependent protein kinase (DNA-PK) plays a key role in repair of radiation-induced DNA double strand breaks (DSB) by non-homologous end-joining. DNA-PK inhibitors (DNA-PKi) are therefore efficient radiosensitisers, but normal tissue radiosensitisation represents a risk for their use in radiation oncology. Here we describe a novel prodrug, SN38023, that is metabolised to a potent DNA-PKi (IC87361) selectively in radioresistant hypoxic cells. DNA-PK inhibitory potency of SN38023 was 24-fold lower than IC87361 in cell-free assays, consistent with molecular modelling studies suggesting that SN38023 is unable to occupy one of the predicted DNA-PK binding modes of IC87361. One-electron reduction of the prodrug by radiolysis of anoxic formate solutions, and by metabolic reduction in anoxic HCT116/POR cells that overexpress cytochrome P450 oxidoreductase (POR), generated IC87361 efficiently as assessed by LC-MS. SN38023 inhibited radiation-induced Ser2056 autophosphorylation of DNA-PK catalytic subunit and radiosensitised HCT116/POR and UT-SCC-54C cells selectively under anoxia. SN38023 was an effective radiosensitiser in anoxic HCT116 spheroids, demonstrating potential for penetration into hypoxic tumour tissue, but in spheroid co-cultures of high-POR and POR-null cells it showed no evidence of bystander effects resulting from local diffusion of IC87361. Pharmacokinetics of IC87361 and SN38023 at maximum achievable doses in NIH-III mice demonstrated sub-optimal exposure of UT-SCC-54C tumour xenografts and did not provide significant tumour radiosensitisation. In conclusion, SN38023 has potential for exploiting hypoxia for selective delivery of a potent DNA-PKi to the most radioresistant subpopulation of cells in tumours. However, prodrugs providing improved systemic pharmacokinetics and that release DNA-PKi that elicit bystander effects are needed to maximise therapeutic utility.

Next-Generation Hypoxic Cell Radiosensitizers: Nitroimidazole Alkylsulfonamides.

Innovations in the field of radiotherapy such as stereotactic body radiotherapy, along with the advent of radio-immuno-oncology, herald new opportunities for classical oxygen-mimetic radiosensitizers. The role of hypoxic tumor cells in resistance to radiotherapy and in suppression of immune response continues to endorse tumor hypoxia as a bona fide, yet largely untapped, drug target. Only nimorazole is used clinically as a radiosensitizer, and there is a dearth of new radiosensitizers in development. Here we present a survey of novel nitroimidazole alkylsulfonamides and document their cytotoxicity and ability to radiosensitize anoxic tumor cells in vitro. We use a phosphate prodrug approach to increase aqueous solubility and to improve tumor drug delivery. A 2-nitroimidazole and a 5-nitroimidazole analogue demonstrated marked tumor radiosensitization in either ex vivo assays of surviving clonogens or tumor regrowth delay.

Synthesis and in vitro and in vivo evaluation of antimalarial polyamines.

We recently reported that 1,14-diphenylacetamide derivatives of spermine exhibit potent nM in vitro growth inhibition properties of Plasmodium falciparum. In an effort to expand the structure-activity relationship of this compound class towards malaria, we have prepared and biologically tested a library that includes benzamide and 3-phenylpropanamide 'capping acid' groups, and polyamines that include spermine (PA3-4-3) and chain extended analogues PA3-8-3 and PA3-12-3. 2-Hydroxy and 2,5-dimethoxy analogues were typically found to exhibit the most potent activity towards the dual drug resistant strain K1 of P. falciparum with IC50's in the range of 1.3-9.5 nM, and selectivity indices (SI) of 42,300 to 4880. In vivo evaluation of three analogues against Plasmodium berghei was undertaken, with one demonstrating a modest 27.9% reduction in parasitaemia.