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.

Spin Trapping Hydroxyl and Aryl Radicals of One-Electron Reduced Anticancer Benzotriazine 1,4-Dioxides.

Hypoxia in tumors results in resistance to both chemotherapy and radiotherapy treatments but affords an environment in which hypoxia-activated prodrugs (HAP) are activated upon bioreduction to release targeted cytotoxins. The benzotriazine 1,4-di-N-oxide (BTO) HAP, tirapazamine (TPZ, 1), has undergone extensive clinical evaluation in combination with radiotherapy to assist in the killing of hypoxic tumor cells. Although compound 1 did not gain approval for clinical use, it has spurred on the development of other BTOs, such as the 3-alkyl analogue, SN30000, 2. There is general agreement that the cytotoxin(s) from BTOs arise from the one-electron reduced form of the compounds. Identifying the cytotoxic radicals, and whether they play a role in the selective killing of hypoxic tumor cells, is important for continued development of the BTO class of anticancer prodrugs. In this study, nitrone spin-traps, combined with electron spin resonance, give evidence for the formation of aryl radicals from compounds 1, 2 and 3-phenyl analogues, compounds 3 and 4, which form carbon C-centered radicals. In addition, high concentrations of DEPMPO (5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide) spin-trap the •OH radical. The combination of spin-traps with high concentrations of DMSO and methanol also give evidence for the involvement of strongly oxidizing radicals. The failure to spin-trap methyl radicals with PBN (N-tert-butylphenylnitrone) on the bioreduction of compound 2, in the presence of DMSO, implies that free •OH radicals are not released from the protonated radical anions of compound 2. The spin-trapping of •OH radicals by high concentrations of DEPMPO, and the radical species arising from DMSO and methanol give both direct and indirect evidence for the scavenging of •OH radicals that are involved in an intramolecular process. Hypoxia-selective cytotoxicity is not related to the formation of aryl radicals from the BTO compounds as they are associated with high aerobic cytotoxicity.

Subcellular Location of Tirapazamine Reduction Dramatically Affects Aerobic but Not Anoxic Cytotoxicity.

Hypoxia is an adverse prognostic feature of solid cancers that may be overcome with hypoxia-activated prodrugs (HAPs). Tirapazamine (TPZ) is a HAP which has undergone extensive clinical evaluation in this context and stimulated development of optimized analogues. However the subcellular localization of the oxidoreductases responsible for mediating TPZ-dependent DNA damage remains unclear. Some studies conclude only nuclear-localized oxidoreductases can give rise to radical-mediated DNA damage and thus cytotoxicity, whereas others identify a broader role for endoplasmic reticulum and cytosolic oxidoreductases, indicating the subcellular location of TPZ radical formation is not a critical requirement for DNA damage. To explore this question in intact cells we engineered MDA-231 breast cancer cells to express the TPZ reductase human NADPH: cytochrome P450 oxidoreductase (POR) harboring various subcellular localization sequences to guide this flavoenzyme to the nucleus, endoplasmic reticulum, cytosol or inner surface of the plasma membrane. We show that all POR variants are functional, with differences in rates of metabolism reflecting enzyme expression levels rather than intracellular TPZ concentration gradients. Under anoxic conditions, POR expression in all subcellular compartments increased the sensitivity of the cells to TPZ, but with a fall in cytotoxicity per unit of metabolism (termed 'metabolic efficiency') when POR is expressed further from the nucleus. However, under aerobic conditions a much larger increase in cytotoxicity was observed when POR was directed to the nucleus, indicating very high metabolic efficiency. Consequently, nuclear metabolism results in collapse of hypoxic selectivity of TPZ, which was further magnified to the point of reversing O2 dependence (oxic > hypoxic sensitivity) by employing a DNA-affinic TPZ analogue. This aerobic hypersensitivity phenotype was partially rescued by cellular copper depletion, suggesting the possible involvement of Fenton-like chemistry in generating short-range effects mediated by the hydroxyl radical. In addition, the data suggest that under aerobic conditions reoxidation strictly limits the TPZ radical diffusion range resulting in site-specific cytotoxicity. Collectively these novel findings challenge the purported role of intra-nuclear reductases in orchestrating the hypoxia selectivity of TPZ.

Studies Towards Hypoxia-Activated Prodrugs of PARP Inhibitors.

Poly(ADP-ribose)polymerase (PARP) inhibitors (PARPi) have recently been approved for the treatment of breast and ovarian tumors with defects in homologous recombination repair (HRR). Although it has been demonstrated that PARPi also sensitize HRR competent tumors to cytotoxic chemotherapies or radiotherapy, normal cell toxicity has remained an obstacle to their use in this context. Hypoxia-activated prodrugs (HAPs) provide a means to limit exposure of normal cells to active drug, thus adding a layer of tumor selectivity. We have investigated potential HAPs of model PARPi in which we attach a bioreducible "trigger" to the amide nitrogen, thereby blocking key binding interactions. A representative example showed promise in abrogating PARPi enzymatic activity in a biochemical assay, with a ca. 160-fold higher potency of benzyl phthalazinone 4 than the corresponding model HAP 5, but these N-alkylated compounds did not release the PARPi upon one-electron reduction by radiolysis. Therefore, we extended our investigation to include NU1025, a PARPi that contains a phenol distal to the core binding motif. The resulting 2-nitroimidazolyl ether provided modest abrogation of PARPi activity with a ca. seven-fold decrease in potency, but released the PARPi efficiently upon reduction. This investigation of potential prodrug approaches for PARPi has identified a useful prodrug strategy for future exploration.

Benzotriazine Di-Oxide Prodrugs for Exploiting Hypoxia and Low Extracellular pH in Tumors.

Extracellular acidification is an important feature of tumor microenvironments but has yet to be successfully exploited in cancer therapy. The reversal of the pH gradient across the plasma membrane in cells that regulate intracellular pH (pHi) has potential to drive the selective uptake of weak acids at low extracellular pH (pHe). Here, we investigate the dual targeting of low pHe and hypoxia, another key feature of tumor microenvironments. We prepared eight bioreductive prodrugs based on the benzotriazine di-oxide (BTO) nucleus by appending alkanoic or aminoalkanoic acid sidechains. The BTO acids showed modest selectivity for both low pHe (pH 6.5 versus 7.4, ratios 2 to 5-fold) and anoxia (ratios 2 to 8-fold) in SiHa and FaDu cell cultures. Related neutral BTOs were not selective for acidosis, but had greater cytotoxic potency and hypoxic selectivity than the BTO acids. Investigation of the uptake and metabolism of representative BTO acids confirmed enhanced uptake at low pHe, but lower intracellular concentrations than expected for passive diffusion. Further, the modulation of intracellular reductase activity and competition by the cell-excluded electron acceptor WST-1 suggests that the majority of metabolic reductions of BTO acids occur at the cell surface, compromising the engagement of the resulting free radicals with intracellular targets. Thus, the present study provides support for designing bioreductive prodrugs that exploit pH-dependent partitioning, suggesting, however, that that the approach should be applied to prodrugs with obligate intracellular activation.

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.

Radical Chemistry and Cytotoxicity of Bioreductive 3-Substituted Quinoxaline Di-N-Oxides.

The radical chemistry and cytotoxicity of a series of quinoxaline di-N-oxide (QDO) compounds has been investigated to explore the mechanism of action of this class of bioreductive drugs. A series of water-soluble 3-trifluoromethyl (4-10), 3-phenyl (11-19), and 3-methyl (20-21) substituted QDO compounds were designed to span a range of electron affinities consistent with bioreduction. The stoichiometry of loss of QDOs by steady-state radiolysis of anaerobic aqueous formate buffer indicated that one-electron reduction of QDOs generates radicals able to initiate chain reactions by oxidation of formate. The 3-trifluoromethyl analogues exhibited long chain reactions consistent with the release of the HO(•), as identified in EPR spin trapping experiments. Several carbon-centered radical intermediates, produced by anaerobic incubation of the QDO compounds with N-terminal truncated cytochrome P450 reductase (POR), were characterized using N-tert-butyl-α-phenylnitrone (PBN) and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) spin traps and were observed by EPR. Experimental data were well simulated for the production of strongly oxidizing radicals, capable of H atom abstraction from methyl groups. The kinetics of formation and decay of the radicals produced following one-electron reduction of the parent compounds, both in oxic and anoxic solutions, were determined using pulse radiolysis. Back oxidation of the initially formed radical anions by molecular oxygen did not compete effectively with the breakdown of the radical anions to form oxidizing radicals. The QDO compounds displayed low hypoxic selectivity when tested against oxic and hypoxic cancer cell lines in vitro. The results from this study form a kinetic description and explanation of the low hypoxia-selective cytotoxicity of QDOs against cancer cells compared to the related benzotriazine 1,4-dioxide (BTO) class of compounds.

Efficient Protocol for the Identification of Hypoxic Cell Radiosensitisers.

An evolution in radiotherapy practice is leading to greater use of stereotactic body radiotherapy (SBRT), raising the prospect of increased hypoxic cell radioresistance. New clinical interest in nitroimidazole radiosensitisers, combined with appropriate biomarkers, signals a revival for radiosensitisers in the context of SBRT. Our interest in modifiers of radiation therapy led us to revisit this area and we have identified a new class of nitroimidazole radiosensitiser. We have developed an abbreviated screening protocol suitable for an academic drug discovery laboratory which allows expeditious triage of compounds with poor physicochemical and in vitro properties and combines in vitro radiosensitisation data with tumour pharmacokinetic data to efficiently select candidates for further evaluation.

Characterisation of radicals formed by the triazine 1,4-dioxide hypoxia-activated prodrug, SN30000.

The radical species underlying the activity of the bioreductive anticancer prodrug, SN30000, have been identified by electron paramagnetic resonance and pulse radiolysis techniques. Spin-trapping experiments indicate both an aryl-type radical and an oxidising radical, trapped as a carbon-centred radical, are formed from the protonated radical anion of SN30000. The carbon-centred radical, produced upon the one-electron oxidation of the 2-electron reduced metabolite of SN30000, oxidises 2-deoxyribose, a model for the site of damage on DNA which leads to double strand breaks. Calculations using density functional theory support the assignments made.

Identifying novel targets in renal cell carcinoma: design and synthesis of affinity chromatography reagents.

Two novel scaffolds, 4-pyridylanilinothiazoles (PAT) and 3-pyridylphenylsulfonyl benzamides (PPB), previously identified as selective cytotoxins for von Hippel-Lindau-deficient Renal Carcinoma cells, were used as templates to prepare affinity chromatography reagents to aid the identification of the molecular targets of these two classes. Structure-activity data and computational models were used to predict possible points of attachment for linker chains. In the PAT class, Click coupling of long chain azides with 2- and 3-pyridylanilinothiazoleacetylenes gave triazole-linked pyridylanilinothiazoles which did not retain the VHL-dependent selectivity of parent analogues. For the PPB class, Sonagashira coupling of 4-iodo-(3-pyridylphenylsulfonyl)benzamide with a propargyl hexaethylene glycol carbamate gave an acetylene which was reduced to the corresponding alkyl 3-pyridylphenylsulfonylbenzamide. This reagent retained the VHL-dependent selectivity of the parent analogues and was successfully utilized as an affinity reagent.

Novel nitroimidazole alkylsulfonamides as hypoxic cell radiosensitisers.

A novel class of nitroimidazole alkylsulfonamides have been prepared and evaluated as hypoxia-selective cytotoxins and radiosensitisers. The sulfonamide side chain markedly influences the physicochemical properties of the analogues: lowering aqueous solubility and raising the electron affinity of the nitroimidazole group. The addition of hydroxyl or basic amine groups increased aqueous solubility, with charged amine groups contributing to increased electron affinity. The analogues covered the range of electron affinity for effective radiosensitisation with one-electron reduction potentials ranging from -503 to -342mV. Cytotoxicity under normoxia or anoxia against a panel of human tumour cell lines was determined using a proliferation assay. 2-Nitroimidazole sulfonamides displayed significant hypoxia-selective cytotoxicity (6 to 64-fold), while 4- and 5-nitroimidazole analogues did not display hypoxia-selective cytotoxicity. All analogues sensitised anoxic HCT-116 human colorectal cells to radiation at non-toxic concentrations. 2-Nitroimidazole analogues provided modest sensitisation due to the relatively low concentrations used while several 5-nitroimidazole analogues provided equivalent sensitisation to misonidazole and etanidazole at similar molar concentrations.

Hypoxia-directed drug strategies to target the tumor microenvironment.

Hypoxia is an important component of the tumor microenvironment and has been the target of drug discovery efforts for almost half a century. These efforts have evolved from offsetting the impact of hypoxia on radiotherapy with oxygen-mimetic radiosensitizers to using hypoxia as a means to selectively target tumors. The more recent description of hypoxia-inducible factors and their role in the hypoxia response network has revealed a host of new drug targets to selectively target tumors. We are developing hypoxia-directed drugs in each of the following areas: novel radiosensitizers for hypofractionated radiotherapy, a second-generation benzotriazine di-N-oxide hypoxia-activated prodrug, and a hypoxia-inducible factor-1-dependent cytotoxin that targets glucose transport. These projects are discussed in the context of hypoxia-directed drug discovery.

Pseudomonas aeruginosa NfsB and nitro-CBI-DEI--a promising enzyme/prodrug combination for gene directed enzyme prodrug therapy.

BACKGROUND: The nitro-chloromethylbenzindoline prodrug nitro-CBI-DEI appears a promising candidate for the anti-cancer strategy gene-directed enzyme prodrug therapy, based on its ability to be converted to a highly cytotoxic cell-permeable derivative by the nitroreductase NfsB from Escherichia coli. However, relative to some other nitroaromatic prodrugs, nitro-CBI-DEI is a poor substrate for E. coli NfsB. To address this limitation we evaluated other nitroreductase candidates from E. coli and Pseudomonas aeruginosa. FINDINGS: Initial screens of candidate genes in the E. coli reporter strain SOS-R2 identified two additional nitroreductases, E. coli NfsA and P. aeruginosa NfsB, as being more effective activators of nitro-CBI-DEI than E. coli NfsB. In monolayer cytotoxicity assays, human colon carcinoma (HCT-116) cells transfected with P. aeruginosa NfsB were >4.5-fold more sensitive to nitro-CBI-DEI than cells expressing either E. coli enzyme, and 23.5-fold more sensitive than untransfected HCT-116. In three dimensional mixed cell cultures, not only were the P. aeruginosa NfsB expressing cells 540-fold more sensitive to nitro-CBI-DEI than pure cultures of untransfected HCT-116, the activated drug that they generated also displayed an unprecedented local bystander effect. CONCLUSION: We posit that the discrepancy in the fold-sensitivity to nitro-CBI-DEI between the two and three dimensional cytotoxicity assays stems from loss of activated drug into the media in the monolayer cultures. This emphasises the importance of evaluating high-bystander GDEPT prodrugs in three dimensional models. The high cytotoxicity and bystander effect exhibited by the NfsB_Pa/nitro-CBI-DEI combination suggest that further preclinical development of this GDEPT pairing is warranted.

Hypoxia-activated prodrugs of phenolic olaparib analogues for tumour-selective chemosensitisation.

Poly(ADP-ribose)polymerase inhibitors (PARPi) are used for treatment of tumours with a defect in homologous recombination (HR) repair. Combination with radio- or chemotherapy could broaden their applicability but a major hurdle is enhancement of normal tissue toxicity. Development of hypoxia-activated prodrugs (HAPs) of PARPi has potential to restrict PARP inhibition to tumours thereby avoiding off-target toxicity. We have designed and synthesised phenolic derivatives of olaparib (termed phenolaparibs) and corresponding ether-linked HAPs. Phenolaparib cytotoxicity in HR-proficient and deficient cell lines was consistent with inhibition of PARP-1. Prodrugs were deactivated relative to phenolaparibs in biochemical PARP-1 inhibition assays, and cell culture. Prodrug 7 was selectively converted to phenolaparib 4 under hypoxia and demonstrated hypoxia-selective cytotoxicity, including chemosensitisation of HR-proficient cells in combination with temozolomide. This work demonstrates the feasibility of a HAP approach to PARPi for use in combination therapies.

Characterisation of enzyme prodrug gene therapy combinations in coated spheroids and vascular networks in vitro.

BACKGROUND: Enzyme prodrug gene therapy is designed as a targeted cancer treatment, destroying gene-modified and bystander cells via exogenous enzyme-generated cytotoxins. Targeting of tumour blood vessels using gene therapy is attractive, although optimal enzyme prodrug combinations have yet to be identified. METHODS: Seven enzyme prodrug combinations were ranked in two endothelial (HUVEC, HMEC-1) and one tumour cell line (T24) for their ability to reduce proliferation and viability. The ability to destroy bystander cells in two dimensions (2D) and three dimensions (3D), mode of cell kill, and the ability to disrupt vascular networks were measured. RESULTS: Endothelial cell proliferation (bromodeoxyuridine uptake) was reduced most effectively by Herpes simplex virus thymidine kinase (TK) with ganciclovir (GCV), followed by Escherichia coli nitroreductase NfsB (NTR) with CB1954; viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay] was reduced most efficiently by NTR/CB1954 followed by TK/GCV. Of the seven combinations, only NTR/CB1954 displayed measurable bystander effects in 2D monolayers, and none demonstrated bystander killing in coated spheroids, a 3D spatially distinct model with tissue-like cell density. NTR-expressing endothelial cells displayed increased apoptosis, necrosis and caspase-3 activity after CB1954 treatment. Despite good antiproliferative activity, TK/GCV was ineffective at disrupting vascular network-like structures of endothelial cells, whereas NTR/CB1954 was efficient. NTR/metronidazole and the vascular disrupting agent, combretastatin A-4 phosphate, were the only other effective agents. CONCLUSIONS: Collectively, these data demonstrate that cytotoxic rather than cytostatic activity is necessary for efficient vascular disruption in vitro, and bystander killing is not essential. We identify NTR/CB1954 and NTR/metronidazole as candidates for in vivo investigation of vascular-targeted gene therapy.

Radiosensitisation of SCCVII tumours and normal tissues in mice by the DNA-dependent protein kinase inhibitor AZD7648.

BACKGROUND AND PURPOSE: Inhibitors of DNA-dependent protein kinase (DNA-PK) are effective radiation sensitisers in preclinical tumours, but little is known about risks of normal tissue radiosensitisation. Here, we evaluate radiosensitisation of head and neck squamous cell carcinoma (HNSCC) cells by DNA-PK inhibitor AZD7648 under oxia and anoxia in vitro, and tumour (SCCVII), oral mucosa and small intestine in mice. MATERIALS AND METHODS: Radiosensitisation of human (UT-SCC-54C) and murine (SCCVII) HNSCC cells by AZD7648 under oxia and anoxia was evaluated by clonogenic assay. Radiosensitisation of SCCVII tumours in C3H mice by oral AZD7648 (75 mg/kg) was determined by ex vivo clonogenic assay 3.5 days post-irradiation, with evaluation of normal tissue surrogate endpoints using 5-ethynyl-2'-deoxyuridine to facilitate detection of regenerating crypts in the ileum and repopulating S-phase cells in the ileum and oral mucosa of the same animals. RESULTS: AZD7648 potently radiosensitised both cell lines, with similar sensitiser enhancement ratios for 10% survival (SER10) under oxia and anoxia. AZD7648 diffused rapidly through multicellular layers, suggesting rapid equilibration between plasma and hypoxic zones in tumours. SCCVII tumours were radiosensitised by AZD7648 (SER10 2.5). AZD7648 also enhanced radiation-induced body weight loss and suppressed regenerating intestinal crypts and repopulating S-phase cells in the ileum and tongue epithelium with SER values similar to SCCVII tumours. CONCLUSION: AZD7648 is a potent radiation sensitiser of both oxic and anoxic tumour cells, but also markedly radiosensitises stem cells in the small intestine and oral mucosa.

SAR studies of 4-pyridyl heterocyclic anilines that selectively induce autophagic cell death in von Hippel-Lindau-deficient renal cell carcinoma cells.

We recently identified a class of pyridyl aniline thiazoles (PAT) that displayed selective cytotoxicity for von Hippel-Lindau (VHL) deficient renal cell carcinoma (RCC) cells in vitro and in vivo. Structure-activity relationship (SAR) studies were used to develop a comparative molecular field analysis (CoMFA) model that related VHL-selective potency to the three-dimensional arrangement of chemical features of the chemotype. We now report the further molecular alignment-guided exploration of the chemotype to discover potent and selective PAT analogues. The contribution of the central thiazole ring was explored using a series of five- and six-membered ring heterocyclic replacements to vary the electronic and steric interactions in the central unit. We also explored a positive steric CoMFA contour adjacent to the pyridyl ring using Pd-catalysed cross-coupling Suzuki-Miyaura, Sonogashira and nucleophilic displacement reactions to prepare of a series of aryl-, alkynyl-, alkoxy- and alkylamino-substituted pyridines, respectively. In vitro potency and selectivity were determined using paired RCC cell lines: the VHL-null cell line RCC4 and the VHL-positive cell line RCC4-VHL. Active analogues selectively induced autophagy in RCC4 cells. We have used the new SAR data to further develop the CoMFA model, and compared this to a 2D-QSAR method. Our progress towards realising the therapeutic potential of this chemotype as a targeted cytotoxic therapy for the treatment of RCC by exploiting the absence of the VHL tumour suppressor gene is reported.

Characterization of radicals formed following enzymatic reduction of 3-substituted analogues of the hypoxia-selective cytotoxin 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine).

The mechanism by which the 1,2,4-benzotriazine 1,4-dioxide (BTO) class of bioreductive hypoxia-selective prodrugs (HSPs) form reactive radicals that kill cancer cells has been investigated by steady-state radiolysis, pulse radiolysis (PR), electron paramagnetic resonance (EPR), and density functional theory (DFT) calculations. Tirapazamine (TPZ, 3-amino BTO, 1) and a series of 3-substituted analogues, -H (2), -methyl (3), -ethyl (4), -methoxy (5), -ethoxymethoxy (6), and -phenyl (7), were reduced in aqueous solution under anaerobic steady-state radiolysis conditions, and their radicals were found to remove the substrates by short chain reactions of different lengths in the presence of formate ions. Multiple carbon-centered radical intermediates, produced upon anaerobic incubation of the compounds with cytochrome P(450) reductase enriched microsomes, were trapped by N-tert-butyl-alpha-phenylnitrone and observed using EPR. The highly oxidizing oxymethyl radical, from compound 5, was identified, and experimental spectra obtained for compounds 1, 2, 3, and 7 were well simulated after the inclusion of aryl radicals. The identification of a range of oxidizing radicals in the metabolism of the BTO compounds gives a new insight into the mechanism by which these HSPs can cause a wide variety of damage to biological targets such as DNA.

Patient-Derived Xenograft and Organoid Models for Precision Medicine Targeting of the Tumour Microenvironment in Head and Neck Cancer.

Patient survival from head and neck squamous cell carcinoma (HNSCC), the seventh most common cause of cancer, has not markedly improved in recent years despite the approval of targeted therapies and immunotherapy agents. Precision medicine approaches that seek to individualise therapy through the use of predictive biomarkers and stratification strategies offer opportunities to improve therapeutic success in HNSCC. To enable precision medicine of HNSCC, an understanding of the microenvironment that influences tumour growth and response to therapy is required alongside research tools that recapitulate the features of human tumours. In this review, we highlight the importance of the tumour microenvironment in HNSCC, with a focus on tumour hypoxia, and discuss the fidelity of patient-derived xenograft and organoids for modelling human HNSCC and response to therapy. We describe the benefits of patient-derived models over alternative preclinical models and their limitations in clinical relevance and how these impact their utility in precision medicine in HNSCC for the discovery of new therapeutic agents, as well as predictive biomarkers to identify patients' most likely to respond to therapy.

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.