New Trends in Aging Drug Discovery

Aging is considered the main risk factor for many chronic diseases that frequently appear at advanced ages. However, the inevitability of this process is being questioned by recent research that suggests that senescent cells have specific features that differentiate them from younger cells and that removal of these cells ameliorates senescent phenotype and associated diseases. This opens the door to the design of tailored therapeutic interventions aimed at reducing and delaying the impact of senescence in life, that is, extending healthspan and treating aging as another chronic disease. Although these ideas are still far from reaching the bedside, it is conceivable that they will revolutionize the way we understand aging in the next decades. In this review, we analyze the main and well-validated cellular pathways and targets related to senescence as well as their implication in aging-associated diseases. In addition, the most relevant small molecules with senotherapeutic potential, with a special emphasis on their mechanism of action, ongoing clinical trials, and potential limitations, are discussed. Finally, a brief overview of alternative strategies that go beyond the small molecule field, together with our perspectives for the future of the field, is provided.Depto. de Ingeniería Química y de MaterialesDepto. de Química OrgánicaSección Deptal. de Química Orgánica (Óptica y Optometría)Fac. de Ciencias QuímicasFac. de Óptica y OptometríaTRUEMinisterio de Ciencia e Innovación de Españapu

Synthetic inhibitors of bacterial cell division targeting the GTP-binding site of FtsZ

Cell division protein FtsZ is the organizer of the cytokinetic Z-ring in most bacteria and a target for new antibiotics. FtsZ assembles with GTP into filaments that hydrolyze the nucleotide at the association interface between monomers and then disassemble. We have replaced FtsZ's GTP with non-nucleotide synthetic inhibitors of bacterial division. We searched for these small molecules among compounds from the literature, from virtual screening (VS), and from our in-house synthetic library (UCM), employing a fluorescence anisotropy primary assay. From these screens we have identified the polyhydroxy aromatic compound UCM05 and its simplified analogue UCM44 that specifically bind to Bacillus subtilis FtsZ monomers with micromolar affinities and perturb normal assembly, as examined with light scattering, polymer sedimentation, and negative stain electron microscopy. On the other hand, these ligands induce the cooperative assembly of nucleotide-devoid archaeal FtsZ into distinct well-ordered polymers, different from GTP-induced filaments. These FtsZ inhibitors impair localization of FtsZ into the Z-ring and inhibit bacterial cell division. The chlorinated analogue UCM53 inhibits the growth of clinical isolates of antibiotic-resistant Staphylococcus aureus and Enterococcus faecalis. We suggest that these interfacial inhibitors recapitulate binding and some assembly-inducing effects of GTP but impair the correct structural dynamics of FtsZ filaments and thus inhibit bacterial division, possibly by binding to a small fraction of the FtsZ molecules in a bacterial cell, which opens a new approach to FtsZ-based antibacterial drug discovery.This work was supported by grants from Plan Nacional de Investigación BFU 2011-23416 (J.M.A.), BFU2099-09552 (P.C.), and SAF2010-22198 (M.L.L.-R.), grant CM S2010/BMD-2353 (M.L.L.-R, P.C., J.M.A.), and fellowships FPI (L.B.R.-A.), FPU (M.A.) and CSIC-JAE (E.R.-A.)

Targeting bacterial cell division protein FtsZ with small molecules and fluorescent probes

Trabajo presentado en el 248th National Meeting of the American-Chemical-Society (ACS), celebrado en San Francisco, CA (Estados Unidos), del 10 al 14 de agosto de 201

Cannabinoid receptor CB2 drives HER2 pro-oncogenic signaling in breast cancer

Pharmacological activation of cannabinoid receptors elicits antitumoral responses in different models of cancer. However, the biological role of these receptors in tumor physio-pathology is still unknown. We analyzed CB2 cannabinoid receptor protein expression in two series of 166 and 483 breast tumor samples operated in the University Hospitals of Kiel, Tübingen and Freiburg between 1997 and 2010. CB2 mRNA expression was also analyzed in previously published DNA microarray datasets. The role of CB2 in oncogenesis was studied by generating a mouse line that expresses the HER2 rat ortholog (neu) and lacks CB2, and by a variety of biochemical and cell biology approaches in human breast cancer cells in culture and in vivo, upon modulation of CB2 expression by si/shRNAs and overexpression plasmids. CB2-HER2 molecular interaction was studied by co-localization, coimmunoprecipitation and proximity ligation assays. We show an association between elevated CB2 expression in HER2+ breast tumors and poor patient prognosis. We also demonstrate that genetic inactivation of CB2 impairs tumor generation and progression in MMTV-neu mice. Moreover, we show that HER2 upregulates CB2 expression by activating the transcription factor ELK1 via the ERK cascade, and that an increased CB2 expression activates the HER2 prooncogenic signaling machinery at the level of the tyrosine kinase c-SRC. Finally, HER2 and CB2 form heteromers in cancer cells. Our findings reveal an unprecedented role of CB2 as a pivotal regulator of HER2 pro-oncogenic signaling in breast cancer, and suggest that CB2 may be a biomarker with prognostic value in these tumors

The structural assembly switch of cell division protein FtsZ probed with fluorescent allosteric inhibitors

FtsZ is a widely conserved tubulin-like GTPase that directs bacterial cell division and a new target for antibiotic discovery. This protein assembly machine cooperatively polymerizes forming single-stranded filaments, by means of self-switching between inactive and actively associating monomer conformations. The structural switch mechanism was proposed to involve a movement of the C-terminal and N-terminal FtsZ domains, opening a cleft between them, allosterically coupled to the formation of a tight association interface between consecutive subunits along the filament. The effective antibacterial benzamide PC190723 binds into the open interdomain cleft and stabilizes FtsZ filaments, thus impairing correct formation of the FtsZ ring for cell division. We have designed fluorescent analogs of PC190723 to probe the FtsZ structural assembly switch. Among them, nitrobenzoxadiazole probes specifically bind to assembled FtsZ rather than to monomers. Probes with several spacer lengths between the fluorophore and benzamide moieties suggest a binding site extension along the interdomain cleft. These probes label FtsZ rings of live Bacillus subtilis and Staphylococcus aureus, without apparently modifying normal cell morphology and growth, but at high concentrations they induce impaired bacterial division phenotypes typical of benzamide antibacterials. During the FtsZ assembly-disassembly process, the fluorescence anisotropy of the probes changes upon binding and dissociating from FtsZ, thus reporting open and closed FtsZ interdomain clefts. Our results demonstrate the structural mechanism of the FtsZ assembly switch, and suggest that the probes bind into the open clefts in cellular FtsZ polymers preferably to unassembled FtsZ in the bacterial cytosol

Novel Antagonist of the Type 2 Lysophosphatidic Acid Receptor (LPA2), UCM-14216, Ameliorates Spinal Cord Injury in Mice

Spinal cord injuries (SCIs) irreversibly disrupt spinal connectivity, leading to permanent neurological disabilities. Current medical treatments for reducing the secondary damage that follows the initial injury are limited to surgical decompression and anti-inflammatory drugs, so there is a pressing need for new therapeutic strategies. Inhibition of the type 2 lysophosphatidic acid receptor (LPA2) has recently emerged as a new potential pharmacological approach to decrease SCIassociated damage. Toward validating this receptor as a target in SCI, we have developed a new series of LPA2 antagonists, among which compound 54 (UCM14216) stands out as a potent and selective LPA2 receptor antagonist (Emax = 90%, IC50 = 1.9 μM, KD = 1.3 nM; inactive at LPA1,3−6 receptors). This compound shows efficacy in an in vivo mouse model of SCI in an LPA2-dependent manner, confirming the potential of LPA2 inhibition for providing a new alternative for treating SCI

Cancer-Stem-Cell Phenotype-Guided Discovery of a Microbiota-Inspired Synthetic Compound Targeting NPM1 for Leukemia

The human microbiota plays an important role in human health and disease, through the secretion of metabolites that regulate key biological functions. We propose that microbiota metabolites represent an unexplored chemical space of small drug-like molecules in the search of new hits for drug discovery. Here, we describe the generation of a set of complex chemotypes inspired on selected microbiota metabolites, which have been synthesized using asymmetric organocatalytic reactions. Following a primary screening in CSC models, we identified the novel compound UCM-13369 (4b) whose cytotoxicity was mediated by NPM1. This protein is one of the most frequent mutations of AML, and NPM1-mutated AML is recognized by the WHO as a distinct hematopoietic malignancy. UCM-13369 inhibits NPM1 expression, downregulates the pathway associated with mutant NPM1 C+, and specifically recognizes the C-end DNA-binding domain of NPM1 C+, avoiding the nucleus-cytoplasm translocation involved in the AML tumorological process. The new NPM1 inhibitor triggers apoptosis in AML cell lines and primary cells from AML patients and reduces tumor infiltration in a mouse model of AML with NPM1 C+ mutation. The disclosed phenotype-guided discovery of UCM-13369, a novel small molecule inspired on microbiota metabolites, confirms that CSC death induced by NPM1 inhibition represents a promising therapeutic opportunity for NPM1-mutated AML, a high-mortality disease.This work was supported by grants PID2022-138797OB-I00, PGC2018-096049-B-I00 and PID2021-126663NB-I00 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”; grant PID2019-106279RB-I00 funded by MCIN/AEI/10.13039/501100011033; grant PDC2022-133488-I00 funded by MCIN/AEI/10.13039/501100011033 and by the “European Union NextGenerationEU/PRTR”; grants PI21/00191 and CP19/00140 funded by Instituto de Salud Carlos III; CNIO agreements 2017-2020, 2020-2023 funded by Foundation CRIS contra el Cancer; grants BIO-198 and P18-FR-3487 funded by Junta de Andalucía; VI PPIT program funded by Universidad de Sevilla; and by Ramón Areces Foundation. The authors acknowledge technological support from NMR, mass spectrometry and elemental analysis CAIs (Complutense University of Madrid), Biointeractomicts Platform (cicCartuja, Seville), and the Services at CITIUS (University of Seville). S.A., A.S.-M., I.A.-A. and R.L.G.-A. are grateful to Ministerio de Ciencia e Innovación and Complutense University of Madrid for predoctoral fellowships; M.V.-E. to European Union’s Horizon 2020 for Marie Sklodowska-Curie grant; and P.A.-G. to Fundación Española de Hematología y Hemoterapia for grant. The authors thank Dr. Adrián Velázquez-Campoy at the University of Saragossa for helping in fitting ITC analysis and Prof. Miguel A. De la Rosa at the University of Seville for critical reading of the manuscript.Peer reviewe

2-(Fluoromethoxy)-4′-(S-methanesulfonimidoyl)-1,1′-biphenyl (UCM-1306), an Orally Bioavailable Positive Allosteric Modulator of the Human Dopamine D1 Receptor for Parkinson’s Disease

Tolerance development caused by dopamine replacement with L-DOPA and therapeutic drawbacks upon activation of dopaminergic receptors with orthosteric agonists reveal a significant unmet need for safe and effective treatment of Parkinson’s disease. In search for selective modulators of the D1 receptor, the screening of a chemical library and subsequent medicinal chemistry program around an identified hit resulted in new synthetic compound 26 [UCM-1306, 2-(fluoromethoxy)-4′- (S-methanesulfonimidoyl)-1,1′-biphenyl] that increases the dopamine maximal effect in a dose-dependent manner in human and mouse D1 receptors, is inactive in the absence of dopamine, modulates dopamine affinity for the receptor, exhibits subtype selectivity, and displays low binding competition with orthosteric ligands. The new allosteric modulator potentiates cocaine-induced locomotion and enhances L-DOPA recovery of decreased locomotor activity in reserpinized mice after oral administration. The behavior of compound 26 supports the interest of a positive allosteric modulator of the D1 receptor as a promising therapeutic approach for Parkinson’s disease

The status of the lysophosphatidic acid receptor type 1 (LPA1R)

Lysophospholipids are lipid molecules that are receiving growing attention because, in addition to their structural function in the cell membrane, they are now regarded as important regulators for diverse biological functions through activation of specific receptors. These receptors have been characterized during the last two decades as G protein-coupled receptors (GPCRs) and, among them, two families stand out: lysophosphatidic acid (LPA1–6) and sphingosine 1-phoshate (S1P1–5) receptors. Despite their interest, the high structural similarity between them has restrained the development of selective and high affinity ligands and therefore the elucidation of the role of these receptors in the central nervous system (CNS). This review provides an overview about the different LPA receptors with a special focus on the LPA1 subtype from a medicinal chemistry perspective. It summarizes the most recent developments in the search for selective and specific agonists and antagonists of the LPA1 receptor and highlights their current status in the drug development pipeline

The Search for Antibacterial Inhibitors Targeting Cell Division Protein FtsZ at Its Nucleotide and Allosteric Binding Sites

The global spread of bacterial antimicrobial resistance is associated to millions of deaths from bacterial infections per year, many of which were previously treatable. This, combined with slow antibiotic deployment, has created an urgent need for developing new antibiotics. A still clinically unexploited mode of action consists in suppressing bacterial cell division. FtsZ, an assembling GTPase, is the key protein organizing division in most bacteria and an attractive target for antibiotic discovery. Nevertheless, developing effective antibacterial inhibitors targeting FtsZ has proven challenging. Here we review our decade-long multidisciplinary research on small molecule inhibitors of bacterial division, in the context of global efforts to discover FtsZ-targeting antibiotics. We focus on methods to characterize synthetic inhibitors that either replace bound GTP from the FtsZ nucleotide binding pocket conserved across diverse bacteria or selectively bind into the allosteric site at the interdomain cleft of FtsZ from Bacillus subtilis and the pathogen Staphylococcus aureus. These approaches include phenotype screening combined with fluorescence polarization screens for ligands binding into each site, followed by detailed cytological profiling, and biochemical and structural studies. The results are analyzed to design an optimized workflow to identify effective FtsZ inhibitors, and new approaches for the discovery of FtsZ-targeting antibiotics are discussed