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Nature Chemical Biology Oct 2022Aldehyde dehydrogenases (ALDHs) are promising cancer drug targets, as certain isoforms are required for the survival of stem-like tumor cells. We have discovered...
Aldehyde dehydrogenases (ALDHs) are promising cancer drug targets, as certain isoforms are required for the survival of stem-like tumor cells. We have discovered selective inhibitors of ALDH1B1, a mitochondrial enzyme that promotes colorectal and pancreatic cancer. We describe bicyclic imidazoliums and guanidines that target the ALDH1B1 active site with comparable molecular interactions and potencies. Both pharmacophores abrogate ALDH1B1 function in cells; however, the guanidines circumvent an off-target mitochondrial toxicity exhibited by the imidazoliums. Our lead isoform-selective guanidinyl antagonists of ALDHs exhibit proteome-wide target specificity, and they selectively block the growth of colon cancer spheroids and organoids. Finally, we have used genetic and chemical perturbations to elucidate the ALDH1B1-dependent transcriptome, which includes genes that regulate mitochondrial metabolism and ribosomal function. Our findings support an essential role for ALDH1B1 in colorectal cancer, provide molecular probes for studying ALDH1B1 functions and yield leads for developing ALDH1B1-targeting therapies.
Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Aldehyde Dehydrogenase, Mitochondrial; Aldehydes; Colonic Neoplasms; Colorectal Neoplasms; Guanidines; Humans; Molecular Probes; Proteome
PubMed: 35788181
DOI: 10.1038/s41589-022-01048-w -
Cell Chemical Biology Aug 2020Targeted protein degradation (TPD) has emerged as an exciting new era in chemical biology and drug discovery. PROteolysis TArgeting Chimera (PROTAC) technology targets... (Review)
Review
Targeted protein degradation (TPD) has emerged as an exciting new era in chemical biology and drug discovery. PROteolysis TArgeting Chimera (PROTAC) technology targets cellular proteins for degradation by co-opting the ubiquitin-proteasome system. Over the last 5 years, numerous studies have expanded our understanding of the unique mode of action and advantages of PROTACs, which has in turn spurred interest in both academia and industry to explore PROTACs as a novel therapeutic strategy. In this review, we first highlight the key advantages of PROTACs and then discuss the spatiotemporal regulation of protein degradation. Next, we explore current chemically tractable E3 ligases focusing on expanding the existing repertoire with novel E3 ligases to uncover the full potential of TPD. Collectively, these studies are guiding the development of the PROTAC technology as it emerges as a new modality in precision medicine.
Topics: Ligands; Light; Molecular Probes; Precision Medicine; Proteasome Endopeptidase Complex; Protein Interaction Domains and Motifs; Protein Isoforms; Proteins; Proteolysis; Ubiquitin
PubMed: 32795419
DOI: 10.1016/j.chembiol.2020.07.020 -
Biomolecules Feb 2021Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by the deficiency of α-galactosidase A (α-GalA) and the consequent accumulation of toxic... (Review)
Review
Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by the deficiency of α-galactosidase A (α-GalA) and the consequent accumulation of toxic metabolites such as globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3). Early diagnosis and appropriate timely treatment of FD patients are crucial to prevent tissue damage and organ failure which no treatment can reverse. LSDs might profit from four main therapeutic strategies, but hitherto there is no cure. Among the therapeutic possibilities are intravenous administered enzyme replacement therapy (ERT), oral pharmacological chaperone therapy (PCT) or enzyme stabilizers, substrate reduction therapy (SRT) and the more recent gene/RNA therapy. Unfortunately, FD patients can only benefit from ERT and, since 2016, PCT, both always combined with supportive adjunctive and preventive therapies to clinically manage FD-related chronic renal, cardiac and neurological complications. Gene therapy for FD is currently studied and further strategies such as substrate reduction therapy (SRT) and novel PCTs are under investigation. In this review, we discuss the molecular basis of FD, the pathophysiology and diagnostic procedures, together with the current treatments and potential therapeutic avenues that FD patients could benefit from in the future.
Topics: Animals; Enzyme Inhibitors; Enzyme Replacement Therapy; Fabry Disease; Female; Humans; Male; Molecular Probes; Mutation; alpha-Galactosidase
PubMed: 33673160
DOI: 10.3390/biom11020271 -
Pharmacology & Therapeutics May 2020Transient receptor potential canonical (TRPC) channels constitute a group of receptor-operated calcium-permeable nonselective cation channels of the TRP superfamily. The... (Review)
Review
Transient receptor potential canonical (TRPC) channels constitute a group of receptor-operated calcium-permeable nonselective cation channels of the TRP superfamily. The seven mammalian TRPC members, which can be further divided into four subgroups (TRPC1, TRPC2, TRPC4/5, and TRPC3/6/7) based on their amino acid sequences and functional similarities, contribute to a broad spectrum of cellular functions and physiological roles. Studies have revealed complexity of their regulation involving several components of the phospholipase C pathway, G and G proteins, and internal Ca stores. Recent advances in cryogenic electron microscopy have provided several high-resolution structures of TRPC channels. Growing evidence demonstrates the involvement of TRPC channels in diseases, particularly the link between genetic mutations of TRPC6 and familial focal segmental glomerulosclerosis. Because TRPCs were discovered by the molecular identity first, their pharmacology had lagged behind. This is rapidly changing in recent years owning to great efforts from both academia and industry. A number of potent tool compounds from both synthetic and natural products that selective target different subtypes of TRPC channels have been discovered, including some preclinical drug candidates. This review will cover recent advancements in the understanding of TRPC channel regulation, structure, and discovery of novel TRPC small molecular probes over the past few years, with the goal of facilitating drug discovery for the study of TRPCs and therapeutic development.
Topics: Animals; Drug Discovery; Glomerulosclerosis, Focal Segmental; Humans; Molecular Probes; Phosphoinositide Phospholipase C; Protein Structure, Secondary; Receptors, G-Protein-Coupled; TRPC Cation Channels
PubMed: 32004513
DOI: 10.1016/j.pharmthera.2020.107497 -
Molecules (Basel, Switzerland) Apr 2020Fluorescence in situ hybridization (FISH) is a standard technique used in routine diagnostics of genetic aberrations. Thanks to simple FISH procedure is possible to... (Review)
Review
Fluorescence in situ hybridization (FISH) is a standard technique used in routine diagnostics of genetic aberrations. Thanks to simple FISH procedure is possible to recognize tumor-specific abnormality. Its applications are limited to designed probe type. Gene rearrangements e.g., , reflecting numerous translocational partners, deletions of critical regions e.g., 1p and 19q, gene fusions e.g., , genomic imbalances e.g., 6p, 6q, 11q and amplifications e.g., are targets in personalized oncology. Confirmation of genetic marker is frequently a direct indication to start specific, targeted treatment. In other cases, detected aberration helps pathologists to better distinguish soft tissue sarcomas, or to state a final diagnosis. Our main goal is to show that applying FISH to formalin-fixed paraffin-embedded tissue sample (FFPE) enables assessing genomic status in the population of cells deriving from a primary tumor or metastasis. Although many more sophisticated techniques are available, like Real-Time PCR or new generation sequencing, FISH remains a commonly used method in many genetic laboratories.
Topics: Biomarkers, Tumor; Chromosome Painting; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Molecular Probes; Neoplasms; Precision Medicine; Reproducibility of Results
PubMed: 32316657
DOI: 10.3390/molecules25081864 -
Nature Chemical Biology May 2020We recently described glutathione peroxidase 4 (GPX4) as a promising target for killing therapy-resistant cancer cells via ferroptosis. The onset of therapy resistance...
We recently described glutathione peroxidase 4 (GPX4) as a promising target for killing therapy-resistant cancer cells via ferroptosis. The onset of therapy resistance by multiple types of treatment results in a stable cell state marked by high levels of polyunsaturated lipids and an acquired dependency on GPX4. Unfortunately, all existing inhibitors of GPX4 act covalently via a reactive alkyl chloride moiety that confers poor selectivity and pharmacokinetic properties. Here, we report our discovery that masked nitrile-oxide electrophiles, which have not been explored previously as covalent cellular probes, undergo remarkable chemical transformations in cells and provide an effective strategy for selective targeting of GPX4. The new GPX4-inhibiting compounds we describe exhibit unexpected proteome-wide selectivity and, in some instances, vastly improved physiochemical and pharmacokinetic properties compared to existing chloroacetamide-based GPX4 inhibitors. These features make them superior tool compounds for biological interrogation of ferroptosis and constitute starting points for development of improved inhibitors of GPX4.
Topics: Animals; Cell Line, Tumor; Enzyme Inhibitors; Ferroptosis; Humans; Lipid Peroxidation; Mice, SCID; Molecular Probes; Molecular Targeted Therapy; Nitriles; Oxides; Phospholipid Hydroperoxide Glutathione Peroxidase; Prodrugs; Rats, Wistar; Selenocysteine; Small Molecule Libraries; Structure-Activity Relationship
PubMed: 32231343
DOI: 10.1038/s41589-020-0501-5 -
ACS Chemical Biology Dec 2019Vitamin D metabolites are capable of controlling gene expression in mammalian cells through two independent pathways: vitamin D receptor (VDR) and sterol regulatory...
Vitamin D metabolites are capable of controlling gene expression in mammalian cells through two independent pathways: vitamin D receptor (VDR) and sterol regulatory element-binding protein (SREBP) pathways. In the present study, we dissect the complex biological activity of vitamin D by designing synthetic vitamin D analogs specific for VDR or SREBP pathway, i.e., a VDR activator that lacks SREBP inhibitory activity, or an SREBP inhibitor devoid of VDR activity. These synthetic vitamin D probes permitted identification of one of the vitamin D-responsive genes, , as an SREBP-suppressed gene. The chemical probes developed in the present study may prove useful in dissecting the intricate interplay of vitamin D actions, thereby providing insights into how vitamin D target genes are regulated.
Topics: Drug Discovery; Humans; Molecular Probes; Receptors, Calcitriol; Sterol Regulatory Element Binding Proteins; Vitamin D
PubMed: 31618573
DOI: 10.1021/acschembio.9b00718 -
Molecular Oncology Sep 2020Human papillomavirus (HPV) is a clear etiology of cervical cancer (CC). However, the associations between HPV infection and DNA methylation have not been thoroughly...
Human papillomavirus (HPV) is a clear etiology of cervical cancer (CC). However, the associations between HPV infection and DNA methylation have not been thoroughly investigated. Additionally, it remains unknown whether HPV-related methylation signatures can identify subtypes of CC and stratify the prognosis of CC patients. DNA methylation profiles were obtained from The Cancer Genome Atlas to identify HPV-related methylation sites. Unsupervised clustering analysis of HPV-related methylation sites was performed to determine the different CC subtypes. CC patients were categorized into cluster 1 (Methylation-H), cluster 2 (Methylation-M), and cluster 3 (Methylation-L). Compared to Methylation-M and Methylation-L, Methylation-H exhibited a significantly improved overall survival (OS). Gene set enrichment analysis (GSEA) was conducted to investigate the functions that correlated with different CC subtypes. GSEA indicated that the hallmarks of tumors, including KRAS signaling, TNFα signaling via NF-κB, inflammatory response, epithelial-mesenchymal transition, and interferon-gamma response, were enriched in Methylation-M and Methylation-L. Based on mutation and copy number variation analyses, we found that aberrant mutations, amplifications, and deletions among the MYC, Notch, PI3K-AKT, and RTK-RAS pathways were most frequently detected in Methylation-H. Additionally, mutations, amplifications, and deletions within the Hippo, PI3K-AKT, and TGF-β pathways were presented in Methylation-M. Genes within the cell cycle, Notch, and Hippo pathways possessed aberrant mutations, amplifications, and deletions in Methylation-L. Moreover, the analysis of tumor microenvironments revealed that Methylation-H was characterized by a relatively low degree of immune cell infiltration. Finally, a prognostic signature based on six HPV-related methylation sites was developed and validated. Our study revealed that CC patients could be classified into three heterogeneous clusters based on HPV-related methylation signatures. Additionally, we derived a prognostic signature using six HPV-related methylation sites that stratified the OS of patients with CC into high- and low-risk groups.
Topics: Alphapapillomavirus; DNA Methylation; DNA Mutational Analysis; Female; Humans; Kaplan-Meier Estimate; Middle Aged; Molecular Probes; Mutation; Prognosis; ROC Curve; Risk Assessment; Survival Analysis; Uterine Cervical Neoplasms
PubMed: 32408396
DOI: 10.1002/1878-0261.12709 -
Methods in Molecular Biology (Clifton,... 2022High-throughput DNA fluorescence in situ hybridization (hiFISH) combines multicolor combinatorial DNA FISH staining with automated image acquisition and analysis to...
High-throughput DNA fluorescence in situ hybridization (hiFISH) combines multicolor combinatorial DNA FISH staining with automated image acquisition and analysis to visualize and localize tens to hundreds of genomic loci in up to millions of cells. hiFISH can be used to measure physical distances between pairs of genomic loci, radial distances from genomic loci to the nuclear edge or center, and distances between genomic loci and nuclear structures defined by protein or RNA markers. The resulting large datasets of 3D spatial distances can be used to study cellular heterogeneity in genome architecture and the molecular mechanisms underlying this phenomenon in a variety of cellular systems. In this chapter we provide detailed protocols for hiFISH to measure distances between genomic loci, including all steps involved in DNA FISH probe design and preparation, cell culture, DNA FISH staining in 384-well imaging plates, automated image acquisition and analysis, and, finally, statistical analysis.
Topics: Cell Nucleus; DNA; DNA Probes; Genome; In Situ Hybridization, Fluorescence
PubMed: 35867253
DOI: 10.1007/978-1-0716-2497-5_12 -
Cell Chemical Biology Aug 2020Dynamic proteins perform critical roles in cellular machines, including those that control proteostasis, transcription, translation, and signaling. Thus, dynamic... (Review)
Review
Dynamic proteins perform critical roles in cellular machines, including those that control proteostasis, transcription, translation, and signaling. Thus, dynamic proteins are prime candidates for chemical probe and drug discovery but difficult targets because they do not conform to classical rules of design and screening. Selectivity is pivotal for candidate probe molecules due to the extensive interaction network of these dynamic hubs. Recognition that the traditional rules of probe discovery are not necessarily applicable to dynamic proteins and their complexes, as well as technological advances in screening, have produced remarkable results in the last 2-4 years. Particularly notable are the improvements in target selectivity for small-molecule modulators of dynamic proteins, especially with techniques that increase the discovery likelihood of allosteric regulatory mechanisms. We focus on approaches to small-molecule screening that appear to be more suitable for highly dynamic targets and have the potential to streamline identification of selective modulators.
Topics: Allosteric Regulation; CREB-Binding Protein; HSP70 Heat-Shock Proteins; Models, Molecular; Molecular Probes; Protein Binding; Proteins; Small Molecule Libraries
PubMed: 32783965
DOI: 10.1016/j.chembiol.2020.07.019