Structural Evaluation of a Nitroreductase Engineered for Improved Activation of the 5-Nitroimidazole PET Probe SN33623.

Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell ablation models. We recently engineered a nitroreductase (E. coli NfsB F70A/F108Y) for the substantially enhanced reduction of the 5-nitroimidazole PET-capable probe, SN33623, which permits the theranostic imaging of vectors labeled with oxygen-insensitive bacterial nitroreductases. This mutant enzyme also shows improved activation of the DNA-alkylation prodrugs CB1954 and metronidazole. To elucidate the mechanism behind these enhancements, we resolved the crystal structure of the mutant enzyme to 1.98 Å and compared it to the wild-type enzyme. Structural analysis revealed an expanded substrate access channel and new hydrogen bonding interactions. Additionally, computational modeling of SN33623, CB1954, and metronidazole binding in the active sites of both the mutant and wild-type enzymes revealed key differences in substrate orientations and interactions, with improvements in activity being mirrored by reduced distances between the N5-H of isoalloxazine and the substrate nitro group oxygen in the mutant models. These findings deepen our understanding of nitroreductase substrate specificity and catalytic mechanisms and have potential implications for developing more effective theranostic imaging strategies in cancer treatment.

Clostridium Bacteria: Harnessing Tumour Necrosis for Targeted Gene Delivery.

Necrosis is a common feature of solid tumours that offers a unique opportunity for targeted cancer therapy as it is absent from normal healthy tissues. Tumour necrosis provides an ideal environment for germination of the anaerobic bacterium Clostridium from endospores, resulting in tumour-specific colonisation. Two main species, Clostridium novyi-NT and Clostridium sporogenes, are at the forefront of this therapy, showing promise in preclinical models. However, anti-tumour activity is modest when used as a single agent, encouraging development of Clostridium as a tumour-selective gene delivery system. Various methods, such as allele-coupled exchange and CRISPR-cas9 technology, can facilitate the genetic modification of Clostridium, allowing chromosomal integration of transgenes to ensure long-term stability of expression. Strains of Clostridium can be engineered to express prodrug-activating enzymes, resulting in the generation of active drug selectively in the tumour microenvironment (a concept termed Clostridium-directed enzyme prodrug therapy). More recently, Clostridium strains have been investigated in the context of cancer immunotherapy, either in combination with immune checkpoint inhibitors or with engineered strains expressing immunomodulatory molecules such as IL-2 and TNF-α. Localised expression of these molecules using tumour-targeting Clostridium strains has the potential to improve delivery and reduce systemic toxicity. In summary, Clostridium species represent a promising platform for cancer therapy, with potential for localised gene delivery and immunomodulation selectively within the tumour microenvironment. The ongoing clinical progress being made with C. novyi-NT, in addition to developments in genetic modification techniques and non-invasive imaging capabilities, are expected to further progress Clostridium as an option for cancer treatment.

Tumor-targeting bacteria as immune stimulants - the future of cancer immunotherapy?

Cancer immunotherapies have been widely hailed as a breakthrough for cancer treatment in the last decade, epitomized by the unprecedented results observed with checkpoint blockade. Even so, only a minority of patients currently achieve durable remissions. In general, responsive patients appear to have either a high number of tumor neoantigens, a preexisting immune cell infiltrate in the tumor microenvironment, or an 'immune-active' transcriptional profile, determined in part by the presence of a type I interferon gene signature. These observations suggest that the therapeutic efficacy of immunotherapy can be enhanced through strategies that release tumor neoantigens and/or produce a pro-inflammatory tumor microenvironment. In principle, exogenous tumor-targeting bacteria offer a unique solution for improving responsiveness to immunotherapy. This review discusses how tumor-selective bacterial infection can modulate the immunological microenvironment of the tumor and the potential for combination with cancer immunotherapy strategies to further increase therapeutic efficacy. In addition, we provide a perspective on the clinical translation of replicating bacterial therapies, with a focus on the challenges that must be resolved to ensure a successful outcome.

Discovery of 6-Formylpyridyl Urea Derivatives as Potent Reversible-Covalent Fibroblast Growth Factor Receptor 4 Inhibitors with Improved Anti-Hepatocellular Carcinoma Activity.

Fibroblast growth factor receptor 4 (FGFR4) has been considered as a potential anticancer target due to FGF19/FGFR4 mediated aberrant signaling in hepatocellular carcinoma (HCC). Several FGFR4 inhibitors have been reported, but none have gained approval. Herein, a series of 5-formyl-pyrrolo[3,2-b]pyridine-3-carboxamides and a series of 6-formylpyridyl ureas were characterized as selective reversible-covalent FGFR4 inhibitors. The representative 6-formylpyridyl urea 8z exhibited excellent potency against FGFR4WT, FGFR4V550L, and FGFR4V550M with IC50 values of 16.3, 12.6, and 57.3 nM, respectively. It also potently suppressed proliferation of Ba/F3 cells driven by FGFR4WT, FGFR4V550L, and FGFR4V550M, and FGFR4-dependent Hep3B and Huh7 HCC cells, with IC50 values of 1.2, 13.5, 64.5, 15.0, and 20.4 nM, respectively. Furthermore, 8z displayed desirable microsomal stability and significant in vivo efficacy in the Huh7 HCC cancer xenograft model in nude mice. The study provides a promising new lead for anticancer drug discovery directed toward overcoming FGFR4 gatekeeper mutation mediated resistance in HCC patients.

Design and Biological Evaluation of Piperazine-Bearing Nitrobenzamide Hypoxia/GDEPT Prodrugs: The Discovery of CP-506.

Off-target aerobic activation of PR-104A by human aldo-keto reductase 1C3 (AKR1C3) has confounded the development of this dual hypoxia/gene therapy prodrug. Previous attempts to design prodrugs resistant to AKR1C3 activation have resulted in candidates that require further optimization. Herein we report the evaluation of a lipophilic series of PR-104A analogues in which a piperazine moiety has been introduced to improve drug-like properties. Octanol-water partition coefficients (LogD7.4) spanned >2 orders of magnitude. 2D antiproliferative and 3D multicellular clonogenic assays using isogenic HCT116 and H1299 cells confirmed that all examples were resistant to AKR1C3 metabolism while producing an E. coli NfsA nitroreductase-mediated bystander effect. Prodrugs 16, 17, and 20 demonstrated efficacy in H1299 xenografts where only a minority of tumor cells express NfsA. These prodrugs and their bromo/mesylate counterparts (25-27) were also evaluated for hypoxia-selective cell killing in vitro. These results in conjunction with stability assays recommended prodrug 26 (CP-506) for Phase I/II clinical trial.

Structural Mechanism and Inhibitors Targeting EGFR Exon 20 Insertion (Ex20ins) Mutations.

Epidermal growth factor receptor (EGFR) targeted therapy is one of the most important and effective strategies to combat EGFR mutant nonsmall-cell lung cancer (NSCLC). However, a substantial number of patients bearing EGFR exon 20 insertion (Ex20ins) mutations respond poorly to common EGFR targeted therapies. This clinical need remained unmet until recently, when the EGFR Ex20ins mutation inhibitor mobocertinib was approved by the FDA. Despite this progress, the structural mechanisms of EGFR Ex20ins mutation resistance and characterization of inhibitor binding modes have not been systematically summarized. Herein, we analyze the structural mechanisms for ligand binding and resistance and summarize recent developments for the reported inhibitors of EGFR Ex20ins mutations. Furthermore, this Perspective aims to provide insights for the design of the next generation of EGFR Ex20ins inhibitors.

Beyond Residents-as-Teachers: The Development of an Advanced Medical Education Pilot Elective for Pediatric Residents.

Introduction An advanced medical education elective can encompass themes that transcend traditional residents-as-teachers curricula. The literature is scarce regarding the development of such a curriculum for pediatric residents. Objectives To develop and implement an advanced medical education elective for pediatric residents and evaluate the effectiveness of the educational strategies and curriculum. Methods Kern's Six Steps were applied to create a two-week-long elective for pediatric residents. Residents worked through Kern's model to collaboratively develop the elective. Faculty were recruited based on expertise and content previously created. Residents developed teaching sessions for fourth-year medical students and received feedback. The curriculum was evaluated using quantitative and qualitative feedback with a five-point Likert scale and open-ended questions, group discussions, elective evaluations, and the New World Kirkpatrick Model. Results Five residents, 17 students, and 22 faculty participated. Lectures, expert panels, group discussions, and teaching sessions were seen as effective instructional methods. All residents were satisfied with the elective and its strategies and developed useful skills. Resident-led teaching sessions and interactive learning strategies were cited as a strength, while some redundancy was noted as a weakness. Faculty recommended more formal feedback on resident-led teaching sessions in the future. Conclusions Our medical education elective was designed collaboratively with residents on a medical education track. Strong faculty participation, asynchronous learning, and resident-led teaching sessions were strengths of the curriculum. The curriculum's reproducible components may serve as a foundation for institutions interested in improving their medical education didactics for residents. More research is needed to determine the external validity of this novel curriculum.

Design, Synthesis, and Biological Evaluation of 5-Formyl-pyrrolo[3,2-b]pyridine-3-carboxamides as New Selective, Potent, and Reversible-Covalent FGFR4 Inhibitors.

The FGF19-FGFR4 signaling pathway has been extensively studied as a promising target for the treatment of hepatocellular carcinoma (HCC). Several FGFR4-selective inhibitors have been developed, but none of them receives approval. Additionally, acquired resistance caused by FGFR4 gatekeeper mutations is emerging as a serious limitation for these targeted therapies. Herein, we report a novel series of 5-formyl-pyrrolo[3,2-b]pyridine derivatives as new reversible-covalent inhibitors targeting wild-type and gatekeeper mutant variants of FGFR4 kinase. The representative compound 10z exhibited single-digit nanomolar activity against wild-type FGFR4 and the FGFR4V550L/M mutant variants in biochemical and Ba/F3 cellular assays, while sparing FGFR1/2/3. Furthermore, 10z showed significant antiproliferative activity against Hep3B, JHH-7, and HuH-7 HCC cells with IC50 values of 37, 32, and 94 nM, respectively. MALDI-TOF-MS and X-ray protein crystallography studies were consistent with 10z acting as a reversible-covalent inhibitor of FGFR4, serving as a promising lead compound for further anticancer drug development.

Discovery of Isoform-Selective Akt3 Degraders Overcoming Osimertinib-Induced Resistance in Non-Small Cell Lung Cancer Cells.

EGFR inhibitor therapies have brought significant benefit to NSCLC patients. However, all patients gradually progress to acquired resistance via diverse mechanisms. Akt3 overexpression but not Akt1/2 is one of the found molecular events that mediate osimertinib (1) resistance in NSCLC patients. Here, we report 12l as the first bona fide isoform-selective Akt3 degrader which potently induced proteasomal degradation of the target both in vitro and in vivo, whereas its effects on Akt1/2 were minimal. Using 12l as a tool, non-canonical function of Akt3 was validated to contribute greatly to survival of 1-resistant H1975OR NSCLC cells. Degrader 12l potently suppressed the growth of H1975OR as well as several NSCLC cell lines with low nanomolar IC50 values and demonstrated promising in vivo antitumor efficacy in nude mice bearing H1975OR or PC9 NSCLC xenograft models. Selective degradation of Akt3 may be considered as a novel strategy for human cancer therapy.

Prevalence of serum immunoglobulin E against cross-reactive carbohydrate determinants in allergic and nonallergic horses, and its impact on polysensitisation in serum allergen tests.

BACKGROUND: The existence of antibodies against cross-reactive carbohydrate determinants (CCDs) has been studied extensively in humans, and more recently, in dogs and cats. These antibodies can reduce the specificity of in vitro serum allergen tests. OBJECTIVES: To investigate the prevalence of anti-CCD immunoglobulin (Ig)E in both allergic and nonallergic horses as well as evaluate its potential impact on serum allergen testing. ANIMALS: Twenty-one allergic and 21 nonallergic horses. METHODS AND MATERIALS: Sera were analysed for anti-CCD IgE utilising a commercial CHO enzyme-linked immunosorbent assay (ELISA). An allergen specific Fc-ε receptor ELISA then was performed to evaluate polysensitisation, both with and without the addition of a proprietary anti-CCD blocking solution. RESULTS: Antibodies against CCD were detected in 30 of 42 horses. There was no statistically significant difference (p = 0.18) between the allergic and healthy groups in regard to anti-CCD prevalence. Horses with anti-CCD IgE exhibited more polysensitisation on serum allergen tests than horses without anti-CCD IgE in all allergen groups except mites. Polysensitisation was statistically significant at the 95% confidence interval for grasses (p <0.03), weeds (p = 0.02) and stinging insects (p = 0.0005). This was found to be true across both study groups. Inhibition with an anti-CCD blocking solution resulted in a 43% average reduction in polysensitisation. CONCLUSION AND CLINICAL IMPORTANCE: The prevalence of anti-CCD IgE of horses in this study coincides with the prevalence detected in pollen-sensitised people. Horses with anti-CCD IgE exhibited more positive reactions on serum allergen tests. By minimising potential artifactual polysensitisation, inclusion of an anti-CCD blocker may facilitate identification of allergen-specific IgE.

Contexte - L'existence d'anticorps contre les déterminants glucidiques à réaction croisée (CCD) a été largement étudiée chez l'homme et, plus récemment, chez le chien et le chat. Ces anticorps peuvent réduire la spécificité des tests d'allergènes sériques in vitro. Objectifs - Étudier la prévalence de l'immunoglobuline anti-CCD (Ig)E chez les chevaux allergiques et non allergiques et évaluer son impact potentiel sur les tests d'allergènes sériques. Animaux - Vingt et un chevaux allergiques et 21 chevaux non allergiques. Matériels et méthodes - Les sera ont été analysés pour les IgE anti-CCD en utilisant un dosage immuno-enzymatique CHO commercial (ELISA). Un ELISA du récepteur Fc-ε spécifique à l'allergène a ensuite été réalisé pour évaluer la polysensibilisation, avec et sans l'ajout d'une solution exclusive de blocage anti-CCD. Résultats - Des anticorps anti-CCD ont été détectés chez 30 des 42 chevaux. Il n'y avait pas de différence statistiquement significative (P = 0,18) entre les groupes allergiques et sains en ce qui concerne la prévalence anti-CCD. Les chevaux avec des IgE anti-CCD ont montré plus de polysensibilisation aux tests d'allergènes sériques que les chevaux sans IgE anti-CCD dans tous les groupes d'allergènes à l'exception des acariens. La polysensibilisation était statistiquement significative à l'intervalle de confiance de 95 % pour les graminées (P < 0,03), les herbacées (P = 0,02) et les insectes piqueurs (P = 0,0005). Cela s'est avéré vrai dans les deux groupes d'étude. L'inhibition avec une solution de blocage anti-CCD a entraîné une réduction moyenne de 43 % de la polysensibilisation. Conclusion et importance clinique - La prévalence des IgE anti-CCD des chevaux dans cette étude coïncide avec la prévalence détectée chez les personnes sensibilisées au pollen. Les chevaux avec des IgE anti-CCD ont présenté des réactions plus positives aux tests d'allergènes sériques. En minimisant la polysensibilisation artificielle potentielle, l'inclusion d'un bloqueur anti-CCD peut faciliter l'identification des IgE spécifiques de l'allergène.

Introducción- la existencia de anticuerpos contra los determinantes de carbohidratos de reacción cruzada (CCD) se ha estudiado ampliamente en humanos y, más recientemente, en perros y gatos. Estos anticuerpos pueden reducir la especificidad de las pruebas de alérgenos séricos in vitro. Objetivos - Investigar la prevalencia de inmunoglobulina (Ig)E anti-CCD tanto en caballos alérgicos como no alérgicos, así como evaluar su impacto potencial en las pruebas de alérgenos séricos. Animales- veintiún caballos alérgicos y 21 no alérgicos. Métodos y materiales- los sueros se analizaron para detectar IgE anti-CCD utilizando un ensayo comercial inmunoabsorbente ligado a enzimas (ELISA) para CHO. A continuación, se realizó un ELISA del receptor Fc-ε específico del alérgeno para evaluar la polisensibilización, con y sin la adición de una solución de bloqueo anti-CCD patentada. Resultados- se detectaron anticuerpos contra CCD en 30 de 42 caballos. No hubo diferencia estadísticamente significativa (P = 0,18) entre los grupos alérgicos y sanos con respecto a la prevalencia de anti-CCD. Los caballos con IgE anti-CCD exhibieron más polisensibilización en las pruebas de alérgenos séricos que los caballos sin IgE anti-CCD en todos los grupos de alérgenos excepto los ácaros. La polisensibilización fue estadísticamente significativa en el intervalo de confianza del 95 % para pastos (P < 0,03), malas hierbas (P = 0,02) e insectos picadores (P = 0,0005). Se encontró que esto fue similar en ambos grupos de estudio. La inhibición con una solución de bloqueo anti-CCD resultó en una reducción promedio del 43 % en la polisensibilización. Conclusión e importancia clínica - La prevalencia de IgE anti-CCD de los caballos en este estudio coincide con la prevalencia detectada en personas sensibilizadas al polen. Los caballos con IgE anti-CCD exhibieron más reacciones positivas en las pruebas de alérgenos en suero. La inclusión de un bloqueador anti-CCD puede facilitar la identificación de IgE específica de alérgeno al minimizar la posible polisensibilización artificial.

Contexto - A existência de anticorpos contra determinantes de carboidratos de reação cruzada (CCDs) tem sido estudada extensivamente em humanos, e, mas recentemente, em cães e gatos. Estes anticorpos podem reduzir a especificidade de testes alérgicos sorológicos in vitro. Objetivos - Investigar a prevalência de imunoglobulina (Ig)E tanto em cavalos alérgicos quanto em cavalos não alérgicos e avaliar o seu potencial impacto no teste alérgico sorológico. Animais - Vinte e um cavalos alérgicos e 21 não alérgicos. Métodos e materiais - O soro foi testado para IgE anti-CCD utilizando um ensaio imunoenzimático CHO comercial (ELISA). Um ELISA alérgeno-específico para receptor Fc-ε foi realizado para avaliar polissensibilização, com e sem a adição de uma solução de bloqueio anti-CCD. Resultados - Anticorpos anti-CCD foram encontrados em 30 de 42 cavalos. Não houve diferença estatística significativa (P = 0,18) entre os grupos alérgico e saudável quanto à prevalência de anti-CCD. Cavalos com IgE anti-CCD exibiram mais polissensibilização no teste alérgico sorológico que cavalos sem IgE anti-CCD em todos os grupos alergênicos, exceto ácaros. Polissensibilização foi estatisticamente significativa em um intervalo de confiança de 95% para gramíneas (P < 0,03), herbáceas (P = 0,02) e insetos que picam (P = 0,0005). Isto foi verdadeiro para os dois grupos estudados. Inibição com uma solução bloqueadora de anti-CCD resultou em uma redução média de 43% na polissensibilização. Conclusão e importância clínica - A prevalência de IgE anti-CCD em cavalos nesse estudo coincide com a prevalência detectada em pessoas sensibilizadas a pólen. Equinos com IgE anti-CCD apresentaram mais reações positivas nos testes alérgicos sorológicos. Minimizando uma potencial polissensibilização a partir da utilização de um bloqueador de anti-CCD pode facilitar a identificação de IgE alérgeno-específica.

Design, Synthesis, and Biological Evaluation of Aminoindazole Derivatives as Highly Selective Covalent Inhibitors of Wild-Type and Gatekeeper Mutant FGFR4.

Aberrant FGF19/FGFR4 signaling has been shown to be an oncogenic driver of growth and survival in human hepatocellular carcinoma (HCC) with several pan-FGFR inhibitors and FGFR4-selective inhibitors currently being evaluated in the clinic. However, FGFR4 gatekeeper mutation induced acquired resistance remains an unmet clinical challenge for HCC treatment. Thus, a series of aminoindazole derivatives were designed and synthesized as new irreversible inhibitors of wild-type and gatekeeper mutant FGFR4. One representative compound (7v) exhibited excellent potency against FGFR4, FGFR4V550L, and FGFR4V550M with nanomolar activity in both the biochemical and cellular assays while sparing FGFR1/2/3. While compound 7v demonstrated modest in vivo antitumor efficacy in nude mice bearing the Huh-7 xenograft model consistent with its unfavorable pharmacokinetic properties, it provides a promising new starting point for future drug discovery combating FGFR4 gatekeeper mediated resistance in HCC patients.

Tissue Pharmacokinetic Properties and Bystander Potential of Hypoxia-Activated Prodrug CP-506 by Agent-Based Modelling.

Hypoxia-activated prodrugs are bioactivated in oxygen-deficient tumour regions and represent a novel strategy to exploit this pharmacological sanctuary for therapeutic gain. The approach relies on the selective metabolism of the prodrug under pathological hypoxia to generate active metabolites with the potential to diffuse throughout the tumour microenvironment and potentiate cell killing by means of a "bystander effect". In the present study, we investigate the pharmacological properties of the nitrogen mustard prodrug CP-506 in tumour tissues using in silico spatially-resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) modelling. The approach employs a number of experimental model systems to define parameters for the cellular uptake, metabolism and diffusion of both the prodrug and its metabolites. The model predicts rapid uptake of CP-506 to high intracellular concentrations with its long plasma half-life driving tissue diffusion to a penetration depth of 190 µm, deep within hypoxic activating regions. While bioreductive metabolism is restricted to regions of severe pathological hypoxia (<1 µM O2), its active metabolites show substantial bystander potential with release from the cell of origin into the extracellular space. Model predictions of bystander efficiency were validated using spheroid co-cultures, where the clonogenic killing of metabolically defective "target" cells increased with the proportion of metabolically competent "activator" cells. Our simulations predict a striking bystander efficiency at tissue-like densities with the bis-chloro-mustard amine metabolite (CP-506M-Cl2) identified as a major diffusible metabolite. Overall, this study shows that CP-506 has favourable pharmacological properties in tumour tissue and supports its ongoing development for use in the treatment of patients with advanced solid malignancies.

Interrogation of the Structure-Activity Relationship of a Lipophilic Nitroaromatic Prodrug Series Designed for Cancer Gene Therapy Applications.

PR-104A is a dual hypoxia/nitroreductase gene therapy prodrug by virtue of its ability to undergo either one- or two-electron reduction to its cytotoxic species. It has been evaluated extensively in pre-clinical GDEPT studies, yet off-target human aldo-keto reductase AKR1C3-mediated activation has limited its use. Re-evaluation of this chemical scaffold has previously identified SN29176 as an improved hypoxia-activated prodrug analogue of PR-104A that is free from AKR1C3 activation. However, optimization of the bystander effect of SN29176 is required for use in a GDEPT setting to compensate for the non-uniform distribution of therapeutic gene transfer that is often observed with current gene therapy vectors. A lipophilic series of eight analogues were synthesized from commercially available 3,4-difluorobenzaldehyde. Calculated octanol-water partition coefficients (LogD7.4) spanned > 2 orders of magnitude. 2D anti-proliferative and 3D multicellular layer assays were performed using isogenic HCT116 cells expressing E. coli NfsA nitroreductase (NfsA_Ec) or AKR1C3 to determine enzyme activity and measure bystander effect. A variation in potency for NfsA_Ec was observed, while all prodrugs appeared AKR1C3-resistant by 2D assay. However, 3D assays indicated that increasing prodrug lipophilicity correlated with increased AKR1C3 activation and NfsA_Ec activity, suggesting that metabolite loss from the cell of origin into media during 2D monolayer assays could mask cytotoxicity. Three prodrugs were identified as bono fide AKR1C3-negative candidates whilst maintaining activity with NfsA_Ec. These were converted to their phosphate ester pre-prodrugs before being taken forward into in vivo therapeutic efficacy studies. Ultimately, 2-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)ethyl dihydrogen phosphate possessed a significant 156% improvement in median survival in mixed NfsA_Ec/WT tumors compared to untreated controls (p = 0.005), whilst still maintaining hypoxia selectivity comparable to PR-104A.

Metabolomics identifies shared lipid pathways in independent amyotrophic lateral sclerosis cohorts.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease lacking effective treatments. This is due, in part, to a complex and incompletely understood pathophysiology. To shed light, we conducted untargeted metabolomics on plasma from two independent cross-sectional ALS cohorts versus control participants to identify recurrent dysregulated metabolic pathways. Untargeted metabolomics was performed on plasma from two ALS cohorts (cohort 1, n = 125; cohort 2, n = 225) and healthy controls (cohort 1, n = 71; cohort 2, n = 104). Individual differential metabolites in ALS cases versus controls were assessed by Wilcoxon, adjusted logistic regression and partial least squares-discriminant analysis, while group lasso explored sub-pathway level differences. Adjustment parameters included age, sex and body mass index. Metabolomics pathway enrichment analysis was performed on metabolites selected using the above methods. Additionally, we conducted a sex sensitivity analysis due to sex imbalance in the cohort 2 control arm. Finally, a data-driven approach, differential network enrichment analysis (DNEA), was performed on a combined dataset to further identify important ALS metabolic pathways. Cohort 2 ALS participants were slightly older than the controls (64.0 versus 62.0 years, P = 0.009). Cohort 2 controls were over-represented in females (68%, P < 0.001). The most concordant cohort 1 and 2 pathways centred heavily on lipid sub-pathways, including complex and signalling lipid species and metabolic intermediates. There were differences in sub-pathways that were enriched in ALS females versus males, including in lipid sub-pathways. Finally, DNEA of the merged metabolite dataset of both ALS and control cohorts identified nine significant subnetworks; three centred on lipids and two encompassed a range of sub-pathways. In our analysis, we saw consistent and important shared metabolic sub-pathways in both ALS cohorts, particularly in lipids, further supporting their importance as ALS pathomechanisms and therapeutics targets.

Discovery of Cysteine-targeting Covalent Protein Kinase Inhibitors.

Small molecule covalent kinase inhibitors (CKIs) have entered a new era in drug discovery, which have the advantage for sustained target inhibition and high selectivity. An increased understanding of binding kinetics of CKIs and discovery of additional irreversible and reversible-covalent cysteine-targeted warheads has inspired the development of this area. Herein, we summarize the major medicinal chemistry strategies employed in the discovery of these representative CKIs, which are categorized by the location of the target cysteine within seven main regions of the kinase: the front region, the glycine rich loop (P-loop), the hinge region, the DFG region, the activation loop (A-loop), the catalytic loop (C-loop), and the remote loop. The emphasis is placed on the design and optimization strategies of CKIs that are generated by addition of a warhead to a reversible lead/inhibitor scaffold. In addition, we address the challenges facing this area of drug discovery.

Use of an optimised enzyme/prodrug combination for Clostridia directed enzyme prodrug therapy induces a significant growth delay in necrotic tumours.

Necrosis is a typical histological feature of solid tumours that provides a selective environment for growth of the non-pathogenic anaerobic bacterium Clostridium sporogenes. Modest anti-tumour activity as a single agent encouraged the use of C. sporogenes as a vector to express therapeutic genes selectively in tumour tissue, a concept termed Clostridium Directed Enzyme Prodrug Therapy (CDEPT). Here, we examine the ability of a recently identified Neisseria meningitidis type I nitroreductase (NmeNTR) to metabolise the prodrug PR-104A in an in vivo model of CDEPT. Human HCT116 colon cancer cells stably over-expressing NmeNTR demonstrated significant sensitivity to PR-104A, the imaging agent EF5, and several nitro(hetero)cyclic anti-infective compounds. Chemical induction of necrosis in human H1299 xenografts by the vascular disrupting agent vadimezan promoted colonisation by NmeNTR-expressing C. sporogenes, and efficacy studies demonstrated moderate but significant anti-tumour activity of spores when compared to untreated controls. Inclusion of the pre-prodrug PR-104 into the treatment schedule provided significant additional activity, indicating proof-of-principle. Successful preclinical evaluation of a transferable gene that enables metabolism of both PET imaging agents (for vector visualisation) and prodrugs (for conditional enhancement of efficacy) is an important step towards the prospect of CDEPT entering clinical evaluation.

Bioreductive prodrug PR-104 improves the tumour distribution and titre of the nitroreductase-armed oncolytic adenovirus ONYX-411NTR leading to therapeutic benefit.

Advances in the field of cancer immunotherapy have stimulated renewed interest in adenoviruses as oncolytic agents. Clinical experience has shown that oncolytic adenoviruses are safe and well tolerated but possess modest single-agent activity. One approach to improve the potency of oncolytic viruses is to utilise their tumour selectivity to deliver genes encoding prodrug-activating enzymes. These enzymes can convert prodrugs into cytotoxic species within the tumour; however, these cytotoxins can interfere with viral replication and limit utility. In this work, we evaluated the activity of a nitroreductase (NTR)-armed oncolytic adenovirus ONYX-411NTR in combination with the clinically tested bioreductive prodrug PR-104. Both NTR-expressing cells in vitro and xenografts containing a minor population of NTR-expressing cells were highly sensitive to PR-104. Pharmacologically relevant prodrug exposures did not interfere with ONYX-411NTR replication in vitro. In vivo, prodrug administration increased virus titre and improved virus distribution within tumour xenografts. Colonisation of tumours with high ONYX-411NTR titre resulted in NTR expression and prodrug activation. The combination of ONYX-411NTR with PR-104 was efficacious against HCT116 xenografts, whilst neither prodrug nor virus were active as single agents. This work highlights the potential for future clinical development of NTR-armed oncolytic viruses in combination with bioreductive prodrugs.

Restoring Tumour Selectivity of the Bioreductive Prodrug PR-104 by Developing an Analogue Resistant to Aerobic Metabolism by Human Aldo-Keto Reductase 1C3.

PR-104 is a phosphate ester pre-prodrug that is converted in vivo to its cognate alcohol, PR-104A, a latent alkylator which forms potent cytotoxins upon bioreduction. Hypoxia selectivity results from one-electron nitro reduction of PR-104A, in which cytochrome P450 oxidoreductase (POR) plays an important role. However, PR-104A also undergoes 'off-target' two-electron reduction by human aldo-keto reductase 1C3 (AKR1C3), resulting in activation in oxygenated tissues. AKR1C3 expression in human myeloid progenitor cells probably accounts for the dose-limiting myelotoxicity of PR-104 documented in clinical trials, resulting in human PR-104A plasma exposure levels 3.4- to 9.6-fold lower than can be achieved in murine models. Structure-based design to eliminate AKR1C3 activation thus represents a strategy for restoring the therapeutic window of this class of agent in humans. Here, we identified SN29176, a PR-104A analogue resistant to human AKR1C3 activation. SN29176 retains hypoxia selectivity in vitro with aerobic/hypoxic IC50 ratios of 9 to 145, remains a substrate for POR and triggers γH2AX induction and cell cycle arrest in a comparable manner to PR-104A. SN35141, the soluble phosphate pre-prodrug of SN29176, exhibited superior hypoxic tumour log cell kill (>4.0) to PR-104 (2.5-3.7) in vivo at doses predicted to be achievable in humans. Orthologues of human AKR1C3 from mouse, rat and dog were incapable of reducing PR-104A, thus identifying an underlying cause for the discrepancy in PR-104 tolerance in pre-clinical models versus humans. In contrast, the macaque AKR1C3 gene orthologue was able to metabolise PR-104A, indicating that this species may be suitable for evaluating the toxicokinetics of PR-104 analogues for clinical development. We confirmed that SN29176 was not a substrate for AKR1C3 orthologues across all four pre-clinical species, demonstrating that this prodrug analogue class is suitable for further development. Based on these findings, a prodrug candidate was subsequently identified for clinical trials.