Deep Transfer Learning-Based Approach for Glucose Transporter-1 (GLUT1) Expression Assessment.

Glucose transporter-1 (GLUT-1) expression level is a biomarker of tumour hypoxia condition in immunohistochemistry (IHC)-stained images. Thus, the GLUT-1 scoring is a routine procedure currently employed for predicting tumour hypoxia markers in clinical practice. However, visual assessment of GLUT-1 scores is subjective and consequently prone to inter-pathologist variability. Therefore, this study proposes an automated method for assessing GLUT-1 scores in IHC colorectal carcinoma images. For this purpose, we leverage deep transfer learning methodologies for evaluating the performance of six different pre-trained convolutional neural network (CNN) architectures: AlexNet, VGG16, GoogleNet, ResNet50, DenseNet-201 and ShuffleNet. The target CNNs are fine-tuned as classifiers or adapted as feature extractors with support vector machine (SVM) to classify GLUT-1 scores in IHC images. Our experimental results show that the winning model is the trained SVM classifier on the extracted deep features fusion Feat-Concat from DenseNet201, ResNet50 and GoogLeNet extractors. It yields the highest prediction accuracy of 98.86%, thus outperforming the other classifiers on our dataset. We also conclude, from comparing the methodologies, that the off-the-shelf feature extraction is better than the fine-tuning model in terms of time and resources required for training.

In vitro radiosensitization of breast cancer with hypoxia-activated prodrugs.

KP167 is a novel hypoxia-activated prodrug (HAP), targeting cancer cells via DNA intercalating and alkylating properties. The single agent and radiosensitizing efficacy of KP167 and its parental comparator, AQ4N, were evaluated in 2D and 3D cultures of luminal and triple negative breast cancer (TNBC) cell lines and compared against DNA damage repair inhibitors. 2D normoxic treatment with the DNA repair inhibitors, Olaparib or KU-55933 caused, as expected, substantial radiosensitization (sensitiser enhancement ratio, SER0.01 of 1.60-3.42). KP167 induced greater radiosensitization in TNBC (SER0.01 2.53 in MDAMB-231, 2.28 in MDAMB-468, 4.55 in MDAMB-436) and luminal spheroids (SER0.01 1.46 in MCF-7 and 1.76 in T47D cells) compared with AQ4N. Significant radiosensitization was also obtained using KP167 and AQ4N in 2D normoxia. Although hypoxia induced radioresistance, radiosensitization by KP167 was still greater under 2D hypoxia, yielding SER0.01 of 1.56-2.37 compared with AQ4N SER0.01 of 1.13-1.94. Such data show KP167 as a promising single agent and potent radiosensitiser of both normoxic and hypoxic breast cancer cells, with greater efficacy in TNBCs.

Cytochrome P450 Binding and Bioactivation of Tumor-Targeted Duocarmycin Agents.

Duocarmycin natural products are promising anticancer cytotoxins but too potent for systemic use. Re-engineering of the duocarmycin scaffold has enabled the discovery of prodrugs designed for bioactivation by tissue-specific cytochrome P450 (P450) enzymes. Lead prodrugs bioactivated by both P450 isoforms CYP1A1 and CYP2W1 have shown promising results in xenograft studies; however, to fully understand the potential of these agents it is desirable to compare dual-targeting compounds with isoform-selective analogs. Such redesign requires insight into the molecular interactions with these P450 enzymes. Herein binding and metabolism of the individual stereoisomers of the indole-based duocarmycin prodrug ICT2700 and a nontoxic benzofuran analog ICT2726 were evaluated with CYP1A1 and CYP2W1, revealing differences exploitable for drug design. Although enantiomers of both compounds bound to and were metabolized by CYP1A1, the stereochemistry of the chloromethyl fragment was critical for CYP2W1 interactions. CYP2W1 differentially binds the S enantiomer of ICT2726, and its metabolite profile could potentially be used as a biomarker to identify CYP2W1 functional activity. In contrast to benzofuran-based ICT2726, CYP2W1 differentially binds the R isomer of the indole-based ICT2700 over the S stereoisomer. Thus the ICT2700 R configuration warrants further investigation as a scaffold to favor CYP2W1-selective bioactivation. Furthermore, structures of both duocarmycin S enantiomers with CYP1A1 reveal orientations correlating with nontoxic metabolites, and further drug design optimization could lead to a decrease of CYP1A1 bioactivation. Overall, distinctive structural features present in the two P450 active sites can be useful for improving P450-and thus tissue-selective-bioactivation. SIGNIFICANCE STATEMENT: Prodrug versions of the natural product duocarmycin can be metabolized by human tissue-specific cytochrome P450 (P450) enzymes 1A1 and 2W1 to form an ultrapotent cytotoxin and/or high affinity 2W1 substrates to potentially probe functional activity in situ. The current work defines the binding and metabolism by both P450 enzymes to support the design of duocarmycins selectively activated by only one human P450 enzyme.

Genetic variation in the ADIPOQ gene and serum adiponectin increase the risk of bladder cancer.

Bladder cancer (BC) is the 10th most common cancer worldwide. Genetic studies estimated 30% heritability in BC risk. Adiponectin is an adipocytokine that has important roles in the regulation of energy metabolism. Recent evidence suggests dysregulation of adiponectin levels in BC tissues. Serum level of adiponectin is influenced by single nucleotide polymorphisms (SNPs) in the ADIPOQ gene. However, limited evidence is available regarding the association between adiponectin serum levels or SNPs in ADIPOQ and BC risk. This study aimed to assess whether adiponectin serum levels or SNPs in ADIPOQ may modify BC risk. In this case-control study, 114 BC patients were recruited along with 114 controls. Study subjects were genotyped for variations in ADIPOQ SNPs, namely rs17300539, rs266729, rs2241766, and rs1501299. Adiponectin levels were measured from the serum of study subjects. Our analysis showed that the G allele and the GG genotype of rs1501299 were significantly more frequent in BC patients compared to those in the control group (p-value < 0.05). Moreover, two ADIPOQ haplotypes containing the above G allele were associated with increased BC risk (p-value < 0.05). Multivariate analysis showed that increased serum adiponectin, smoking or age were all significant predictors of BC (p-value < 0.05). The data supports use of serum adiponectin and the G allele of rs1501299 SNP in ADIPOQ as potential biomarkers and/or targets in BC. To further validate findings in this study, larger populations of various ethnicities and/or genetic backgrounds are required. More investigations on the functional role of adiponectin in BC will also provide better understanding of potential targeting adiponectin for BC treatment.

Probing cytochrome P450 (CYP) bioactivation with chloromethylindoline bioprecursors derived from the duocarmycin family of compounds.

The duocarmycins belong to a class of agent which has great potential for use in cancer therapy. Their exquisite potency means they are too toxic for systemic use, and targeted approaches are required to unlock their clinical potential. In this study, we have explored seco-OH-chloromethylindoline (CI) duocarmycin-based bioprecursors for their potential for cytochrome P450 (CYP)-mediated cancer cell kill. We report on synthetic and biological explorations of racemic seco-CI-MI, where MI is a 5-methoxy indole motif, and dehydroxylated analogues. We show up to a 10-fold bioactivation of de-OH CI-MI and a fluoro bioprecursor analogue in CYP1A1-transfected cells. Using CYP bactosomes, we also demonstrate that CYP1A2 but not CYP1B1 or CYP3A4 has propensity for potentiating these compounds, indicating preference for CYP1A bioactivation.

Design, Synthesis, Biological Evaluation and In Silico Study of Benzyloxybenzaldehyde Derivatives as Selective ALDH1A3 Inhibitors.

Aldehyde dehydrogenase 1A3 (ALDH1A3) has recently gained attention from researchers in the cancer field. Several studies have reported ALDH1A3 overexpression in different cancer types, which has been found to correlate with poor treatment recovery. Therefore, finding selective inhibitors against ALDH1A3 could result in new treatment options for cancer treatment. In this study, ALDH1A3-selective candidates were designed based on the physiological substrate resemblance, synthesized and investigated for ALDH1A1, ALDH1A3 and ALDH3A1 selectivity and cytotoxicity using ALDH-positive A549 and ALDH-negative H1299 cells. Two compounds (ABMM-15 and ABMM-16), with a benzyloxybenzaldehyde scaffold, were found to be the most potent and selective inhibitors for ALDH1A3, with IC50 values of 0.23 and 1.29 µM, respectively. The results also show no significant cytotoxicity for ABMM-15 and ABMM-16 on either cell line. However, a few other candidates (ABMM-6, ABMM-24, ABMM-32) showed considerable cytotoxicity on H1299 cells, when compared to A549 cells, with IC50 values of 14.0, 13.7 and 13.0 µM, respectively. The computational study supported the experimental results and suggested a good binding for ABMM-15 and ABMM-16 to the ALDH1A3 isoform. From the obtained results, it can be concluded that benzyloxybenzaldehyde might be considered a promising scaffold for further drug discovery aimed at exploiting ALDH1A3 for therapeutic intervention.

Probing cytochrome P450 bioactivation and fluorescent properties with morpholinyl-tethered anthraquinones.

Structural features from the anticancer prodrug nemorubicin (MMDX) and the DNA-binding molecule DRAQ5™ were used to prepare anthraquinone-based compounds, which were assessed for their potential to interrogate cytochrome P450 (CYP) functional activity and localisation. 1,4-disubstituted anthraquinone 8 was shown to be 5-fold more potent in EJ138 bladder cancer cells after CYP1A2 bioactivation. In contrast, 1,5-bis((2-morpholinoethyl)amino) substituted anthraquinone 10 was not CYP-bioactivated but was shown to be fluorescent and subsequently photo-activated by a light pulse (at a bandwidth 532-587 nm), resulting in punctuated foci accumulation in the cytoplasm. It also showed low toxicity in human osteosarcoma cells. These combined properties provide an interesting prospective approach for opto-tagging single or a sub-population of cells and seeking their location without the need for continuous monitoring.

Synthesis and biological properties of aryl methyl sulfones.

A novel group of aryl methyl sulfones based on nonsteroidal anti-inflammatory compounds exhibiting a methyl sulfone instead of the acetic or propionic acid group was designed, synthesized and evaluated in vitro for inhibition against the human cyclooxygenase of COX-1 and COX-2 isoenzymes and in vivo for anti-inflammatory activity using the carrageenan induced rat paw edema model in rats. Also, in vitro chemosensitivity and in vivo analgesic and intestinal side effects were determined for defining the therapeutic and safety profile. Molecular modeling assisted the design of compounds and the interpretation of the experimental results. Biological assay results showed that methyl sulfone compounds 2 and 7 were the most potent COX inhibitors of this series and best than the corresponding carboxylic acids (methyl sulfone 2: IC50 COX-1 = 0.04 and COX-2 = 0.10 µM, and naproxen: IC50 COX-1 = 11.3 and COX-2 = 3.36 µM). Interestingly, the inhibitory activity of compound 2 represents a significant improvement compared to that of the parent carboxylic compound, naproxen. Further support to the results were gained by the docking studies which suggested the ability of compound 2 and 7 to bind into COX enzyme with low binding free energies. The improvement of the activity of some sulfones compared to the carboxylic analogues would be performed through a change of the binding mode or mechanism compared to the standard binding mode displayed by ibuprofen, as disclosed by molecular modeling studies. So, this study paves the way for further attention in investigating the participation of these new compounds in the pain inhibitory mechanisms. The most promising compounds 2 and 7 possess a therapeutical profile that enables their chemical scaffolds to be utilized for development of new NSAIDs.

Therapeutic antibodies against cancer stem cells: a promising approach.

Monoclonal antibodies have been extensively used to treat malignancy along with routine chemotherapeutic drugs. Chemotherapy for metastatic cancer has not been successful in securing long-term remission of disease. This is in part due to the resistance of cancer cells to drugs. One aspect of the drug resistance is the inability of conventional drugs to eliminate cancer stem cells (CSCs) which often constitute less than 1-2% of the whole tumor. In some tumor types, it is possible to identify these cells using surface markers. Monoclonal antibodies targeting these CSCs are an attractive option for a new therapeutic approach. Although administering antibodies has not been effective, when combined with chemotherapy they have proved synergistic. This review highlights the potential of improving treatment efficacy using functional antibodies against CSCs, which could be combined with chemotherapy in the future.

Solid-Phase Synthesis of Duocarmycin Analogues and the Effect of C-Terminal Substitution on Biological Activity.

The duocarmycins are potent antitumor agents with potential for use in the development of antibody-drug conjugates (ADCs) as well as being clinical candidates in their own right. In this article, we describe the synthesis of a duocarmycin monomer (DSA) that is suitably protected for utilization in solid-phase synthesis. The synthesis was performed on a large scale, and the resulting racemic protected Fmoc-DSA subunit was separated by supercritical fluid chromatography (SFC) into the single enantiomers; its application to solid-phase synthesis methodology gave a series of monomeric and extended duocarmycin analogues with amino acid substituents. The DNA sequence selectivity was similar to that in previous reports for both the monomeric and extended compounds. Substitution at the C-terminus of duocarmycin caused a decrease in antiproliferative activity for all of the compounds studied. An extended compound containing an alanine at the C-terminus was converted to the primary amide or to an extended structure containing a terminal tertiary amine, but this had no beneficial effects on biological activity.

Quantitative analysis of cytochrome P450 isoforms in human liver microsomes by the combination of proteomics and chemical probe-based assay.

Cytochrome P450 (CYP) is one of the most important drug-metabolizing enzyme families, which participates in the biotransformation of many endogenous and exogenous compounds. Quantitative analysis of CYP expression levels is important when studying the efficacy of new drug molecules and assessing drug-drug interactions in drug development. At present, chemical probe-based assay is the most widely used approach for the evaluation of CYP activity although there are cross-reactions between the isoforms with high sequence homologies. Therefore, quantification of each isozyme is highly desired in regard to meeting the ever-increasing requirements for carrying out pharmacokinetics and personalized medicine in the academic, pharmaceutical, and clinical setting. Herein, an absolute quantification method was employed for the analysis of the seven isoforms CYP1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1 using a proteome-derived approach in combination with stable isotope dilution assay. The average absolute amount measured from twelve human liver microsomes samples were 39.3, 4.3, 54.0, 4.6, 10.3, 3.0, and 9.3 (pmol/mg protein) for 1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1, respectively. Importantly, the expression level of CYP3A4 showed high correlation (r = 0.943, p < 0.0001) with the functional activity, which was measured using bufalin-a highly selective chemical probe we have developed. The combination of MRM identification and analysis of the functional activity, as in the case of CYP3A4, provides a protocol which can be extended to other functional enzyme studies with wide application in pharmaceutical research.

The Capacity of Drug-Metabolising Enzymes in Modulating the Therapeutic Efficacy of Drugs to Treat Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a rare soft tissue sarcoma (STS) that predominantly affects children and teenagers. It is the most common STS in children (40%) and accounts for 5-8% of total childhood malignancies. Apart from surgery and radiotherapy in eligible patients, standard chemotherapy is the only therapeutic option clinically available for RMS patients. While survival rates for this childhood cancer have considerably improved over the last few decades for low-risk and intermediate-risk cases, the mortality rate remains exceptionally high in high-risk RMS patients with recurrent and/or metastatic disease. The intensification of chemotherapeutic protocols in advanced-stage RMS has historically induced aggravated toxicity with only very modest therapeutic gain. In this review, we critically analyse what has been achieved so far in RMS therapy and provide insight into how a diverse group of drug-metabolising enzymes (DMEs) possess the capacity to modify the clinical efficacy of chemotherapy. We provide suggestions for new therapeutic strategies that exploit the presence of DMEs for prodrug activation, targeted chemotherapy that does not rely on DMEs, and RMS-molecular-subtype-targeted therapies that have the potential to enter clinical evaluation.

Insight into the liposomal encapsulation of mono and bis-naphthalimides.

Mitonafide-loaded liposomes are a promising strategy to overcome the neurotoxicity observed in clinical trials for this drug. This study investigates the influence of loaded mitonafide or a dimer analogue on different liposomal formulations and their therapeutic efficacy in vitro. Physicochemical properties of the liposomes were manipulated using different loading methods (namely bilayer or core loading) and varying the rigidity of the bilayer using distinct phospholipid compositions. Our results demonstrated that the mitonafide dimer analogue had a comparable encapsulation efficiency (EE%) into the liposomes when loaded into rigid or flexible bilayers in contrast to the low mitonafide monomer EE%. A pH gradient core loading method resulted in a more efficient mechanism to load the monomer into the liposomes. DOSY NMR and spectrofluorometric studies revealed key differences in the structure of the vesicles and the arrangement of the monomer or the dimer in the bilayer or the core of the liposomes. The in vitro assessment of the formulations using MDA-MB-231 and RT-112 cells revealed that a flexible lipid bilayer allows a faster drug release, which correlated well with the spectroscopy studies. This study investigated for the first time that the characteristics of the lipid bilayer and the loading method influence the encapsulation efficacy, colloidal properties, photoactivity and stability of mono and bis-naphthalimides loaded in a liposomal carrier, essential factors that will impact the performance of the formulation in a biological scenario.

Structure-guided optimization of 3-hydroxybenzoisoxazole derivatives as inhibitors of Aldo-keto reductase 1C3 (AKR1C3) to target prostate cancer.

AKR1C3 is an enzyme that is overexpressed in several types of radiotherapy- and chemotherapy-resistant cancers. Despite AKR1C3 is a validated target for drug development, no inhibitor has been approved for clinical use. In this manuscript, we describe our study of a new series of potent AKR1C3-targeting 3-hydroxybenzoisoxazole based inhibitors that display high selectivity over the AKR1C2 isoform and low micromolar activity in inhibiting 22Rv1 prostate cancer cell proliferation. In silico studies suggested proper substituents to increase compound potency and provided with a mechanistic explanation that could clarify their different activity, later confirmed by X-ray crystallography. Both the in-silico studies and the crystallographic data highlight the importance of 90° rotation around the single bond of the biphenyl group, in ensuring that the inhibitor can adopt the optimal binding mode within the active pocket. The p-biphenyls that bear the meta-methoxy, and the ortho- and meta-trifluoromethyl substituents (in compounds 6a, 6e and 6f respectively) proved to be the best contributors to cellular potency as they provided the best IC50 values in series (2.3, 2.0 and 2.4 µM respectively) and showed no toxicity towards human MRC-5 cells. Co-treatment with scalar dilutions of either compound 6 or 6e and the clinically used drug abiraterone led to a significant reduction in cell proliferation, and thus confirmed that treatment with both CYP171A1-and AKR1C3-targeting compounds possess the potential to intervene in key steps in the steroidogenic pathway. Taken together, the novel compounds display desirable biochemical potency and cellular target inhibition as well as good in-vitro ADME properties, which highlight their potential for further preclinical studies.

Kinetic analysis of intracellular Hoechst 33342--DNA interactions by flow cytometry: misinterpretation of side population status?

We outline a simple approach involving instrument setup and calibration for the analysis of Hoechst dye 33342-loading in human cell lines for exploring heterogeneity in dye efflux efficiency and the status of side population (SP) A549 lung cancer cells. Dual excitation 488 nm/multiline UV (351-364 nm) flow cytometry was used to confirm ABCG2-specific inhibition of dye efflux using Fumitremorgin C. Transporter gene expression, assayed by qRT-PCR, confirmed higher expression of ABCG2 versus ABCB1, reiterated in a cloned subline. Coexpression of aldehyde dehydrogenase genes ranked as aldehyde dehydrogenase class 1A1 (ALDH1A1) > ALDH3A1 > ALDH3, relative expression of all genes was again reiterated in a cloned subline. Permeabilized cells were used to create red:violet (660:405 nm Em wavelengths) ratiometric references for mapping temporal changes in Hoechst 33342-DNA fluorescence in live cells. A live cell "kinetic SP gate" tracked progressive dye loading of the whole population and coapplication of the far red (>695 nm wavelength) fluorescing dye DRAQ7 enabled viable cell gating. Kinetic gating revealed a continuum for dye accumulation suggesting that SP enumeration is critically dependent upon the nonlinear relationship of the spectral shift with progressive dye-DNA binding and thus requires accurate definition. To this end, permeabilized cell reference samples permit reproducible instrument setup, guide gate boundaries for SP and compromised cells, and offer a simple means of comparing SP enumeration across laboratory sites/platforms. Our approach reports the dynamic range for the spectral shift, revealing noninformative staining conditions and explaining a source of variability for SP enumeration. We suggest that live cell kinetic sorting of all cells with the same dye:DNA load but with differences in efflux capacity can be used to explore drug resistance capability without prejudice. The SP phenotype should be regarded as a kinetic parameter and not a fixed characteristic--critical for functional assay design and the interpretation of heterogeneity.

Synthesis and biological evaluation of colchicine C-ring analogues tethered with aliphatic linkers suitable for prodrug derivatisation.

Colchicine was modified at the 10-OCH(3) position of the C-ring by reaction with heterocyclic amines or commercially available amines to afford a library of target colchicinoids in high yields (62-99%). Molecular modeling revealed that the incorporation of the linker groups led to a reduction in entropy and therefore binding affinity when compared with colchicine. Some colchicinoids were shown to be equicytotoxic with colchicine when evaluated in the DLD-1 colon cancer cells and retained activity in resistant A2780AD or HeLa cells with mutant Class III ß-tubulin. Importantly, unlike colchicine, the analogues in this study are amenable for prodrug derivatisation and with potential for tumor-selective delivery.

The Chemerin-CMKLR1 Axis is Functionally important for Central Regulation of Energy Homeostasis.

Chemerin is an adipokine involved in inflammation, adipogenesis, angiogenesis and energy metabolism, and has been hypothesized as a link between obesity and type II diabetes. In humans affected by obesity, chemerin gene expression in peripheral tissues and circulating levels are elevated. In mice, plasma levels of chemerin are upregulated by high-fat feeding and gain and loss of function studies show an association of chemerin with body weight, food intake and glucose homeostasis. Therefore, chemerin is an important blood-borne mediator that, amongst its other functions, controls appetite and body weight. Almost all studies of chemerin to date have focused on its release from adipose tissue and its effects on peripheral tissues with the central effects largely overlooked. To demonstrate a central role of chemerin, we manipulated chemerin signaling in the hypothalamus, a brain region associated with appetite regulation, using pharmacological and genetic manipulation approaches. Firstly, the selective chemerin receptor CMKLR1 antagonist α-NETA was administered i.c.v. to rats to test for an acute physiological effect. Secondly, we designed a short-hairpin-RNA (shRNA) lentivirus construct targeting expression of CMKLR1. This shRNA construct, or a control construct was injected bilaterally into the arcuate nucleus of male Sprague Dawley rats on high-fat diet (45%). After surgery, rats were maintained on high-fat diet for 2 weeks and then switched to chow diet for a further 2 weeks. We found a significant weight loss acutely and inhibition of weight gain chronically. This difference became apparent after diet switch in arcuate nucleus-CMKLR1 knockdown rats. This was not accompanied by a difference in blood glucose levels. Interestingly, appetite-regulating neuropeptides remained unaltered, however, we found a significant reduction of the inflammatory marker TNF-α suggesting reduced expression of CMKLR1 protects from high-fat diet induced neuroinflammation. In white and brown adipose tissue, mRNA expression of chemerin, its receptors and markers of adipogenesis, lipogenesis and brown adipocyte activation remained unchanged confirming that the effects are driven by the brain. Our behavioral analyses suggest that knockdown of CMKLR1 had an impact on object recognition. Our data demonstrate that CMKLR1 is functionally important for the central effects of chemerin on body weight regulation and neuroinflammation.

New aldo-keto reductase 1C3 (AKR1C3) inhibitors based on the hydroxytriazole scaffold.

The aldo-keto reductase 1C3 (AKR1C3) enzyme is considered an attractive target in Castration Resistant Prostate Cancer (CRPC) because of its role in the biosynthesis of androgens. Flufenamic acid, a non-selective AKR1C3 inhibitor, has previously been subjected to bioisosteric modulation to give rise to a series of compounds with the hydroxytriazole core. In this work, the hit compound of the previous series has been modulated further, and new, more potent, and selective derivatives have been obtained. The poor solubility of the most active compound (cpd 5) has been improved by substituting the triazole core with an isoxazole heteronucleous, with similar enzymatic activity being retained. Potent AKR1C3 inhibition is translated into antiproliferative effects against the 22RV1 CRPC cellular model, and the in-silico design, synthesis and biological activity of new compounds are described herein. Compounds have also been assayed in combination with two approved antitumor drugs, abiraterone and enzalutamide.

Expansion of the 4-(Diethylamino)benzaldehyde Scaffold to Explore the Impact on Aldehyde Dehydrogenase Activity and Antiproliferative Activity in Prostate Cancer.

Aldehyde dehydrogenases (ALDHs) are overexpressed in various tumor types including prostate cancer and considered a potential target for therapeutic intervention. 4-(Diethylamino)benzaldehyde (DEAB) has been extensively reported as a pan-inhibitor of ALDH isoforms, and here, we report on the synthesis, ALDH isoform selectivity, and cellular potencies in prostate cancer cells of 40 DEAB analogues; three analogues (14, 15, and 16) showed potent inhibitory activity against ALDH1A3, and two analogues (18 and 19) showed potent inhibitory activity against ALDH3A1. Significantly, 16 analogues displayed increased cytotoxicity (IC50 = 10-200 µM) compared with DEAB (>200 µM) against three different prostate cancer cell lines. Analogues 14 and 18 were more potent than DEAB against patient-derived primary prostate tumor epithelial cells, as single agents or in combination treatment with docetaxel. In conclusion, our study supports the use of DEAB as an ALDH inhibitor but also reveals closely related analogues with increased selectivity and potency.

How can the potential of the duocarmycins be unlocked for cancer therapy?

The duocarmycins belong to a class of agent that has fascinated scientists for over four decades. Their exquisite potency, unique mechanism of action, and efficacy in multidrug-resistant tumour models makes them attractive to medicinal chemists and drug hunters. However, despite great advances in fine-tuning biological activity through structure-activity relationship studies (SARS), no duocarmycin-based therapeutic has reached clinical approval. In this review, we provide an overview of the most promising strategies currently used and include both tumour-targeted prodrug approaches and antibody-directed technologies.