Paralog-dependent isogenic cell assay cascade generates highly selective SLC16A3 inhibitors.

Despite being considered druggable and attractive therapeutic targets, most of the solute carrier (SLC) membrane transporters remain pharmacologically underexploited. One of the reasons for this is a lack of reliable chemical screening assays, made difficult by functional redundancies among SLCs. In this study we leveraged synthetic lethality between the lactate transporters SLC16A1 and SLC16A3 in a screening strategy that we call paralog-dependent isogenic cell assay (PARADISO). The system involves five isogenic cell lines, each dependent on various paralog genes for survival/fitness, arranged in a screening cascade tuned for the identification of SLC16A3 inhibitors. We screened a diversity-oriented library of ∼90,000 compounds and further developed our hits into slCeMM1, a paralog-selective and potent SLC16A3 inhibitor. By implementing chemoproteomics, we showed that slCeMM1 is selective also at the proteome-wide level, thus fulfilling an important criterion for chemical probes. This study represents a framework for the development of specific cell-based drug discovery assays.

Phosphatidylinositol 4,5-bisphosphate (PIP2) facilitates norepinephrine transporter dimerization and modulates substrate efflux.

The plasmalemmal norepinephrine transporter (NET) regulates cardiovascular sympathetic activity by clearing extracellular norepinephrine in the synaptic cleft. Here, we investigate the subunit stoichiometry and function of NET using single-molecule fluorescence microscopy and flux assays. In particular, we show the effect of phosphatidylinositol 4,5-bisphosphate (PIP2) on NET oligomerization and efflux. NET forms monomers (~60%) and dimers (~40%) at the plasma membrane. PIP2 depletion results in a decrease in the average oligomeric state and decreases NET-mediated substrate efflux while not affecting substrate uptake. Mutation of the putative PIP2 binding residues R121, K334, and R440 to alanines does not affect NET dimerization but results in decreased substrate efflux that is not altered upon PIP2 depletion; this indicates that PIP2 interactions with these residues affect NET-mediated efflux. A dysregulation of norepinephrine and PIP2 signaling have both been implicated in neuropsychiatric and cardiovascular diseases. This study provides evidence that PIP2 directly regulates NET organization and function.

Methylphenidate Analogues as a New Class of Potential Disease-Modifying Agents for Parkinson's Disease: Evidence from Cell Models and Alpha-Synuclein Transgenic Mice.

Parkinson's disease (PD) is characterized by dopaminergic nigrostriatal neurons degeneration and Lewy body pathology, mainly composed of α-synuclein (αSyn) fibrillary aggregates. We recently described that the neuronal phosphoprotein Synapsin III (Syn III) participates in αSyn pathology in PD brains and is a permissive factor for αSyn aggregation. Moreover, we reported that the gene silencing of Syn III in a human αSyn transgenic (tg) mouse model of PD at a pathological stage, manifesting marked insoluble αSyn deposits and dopaminergic striatal synaptic dysfunction, could reduce αSyn aggregates, restore synaptic functions and motor activities and exert neuroprotective effects. Interestingly, we also described that the monoamine reuptake inhibitor methylphenidate (MPH) can recover the motor activity of human αSyn tg mice through a dopamine (DA) transporter-independent mechanism, which relies on the re-establishment of the functional interaction between Syn III and α-helical αSyn. These findings support that the pathological αSyn/Syn III interaction may constitute a therapeutic target for PD. Here, we studied MPH and some of its analogues as modulators of the pathological αSyn/Syn III interaction. We identified 4-methyl derivative I-threo as a lead candidate modulating αSyn/Syn III interaction and having the ability to reduce αSyn aggregation in vitro and to restore the motility of αSyn tg mice in vivo more efficiently than MPH. Our results support that MPH derivatives may represent a novel class of αSyn clearing agents for PD therapy.

Illuminating the norepinephrine transporter: fluorescent probes based on nisoxetine and talopram.

The utilization of fluorescent ligands to study the monoamine transporters (MATs) has increased our knowledge of their function and distribution in live cell systems. In this study, we extend SAR for nisoxetine and talopram as parent compounds, to identify high affinity rhodamine-labeled fluorescent probes for the norepinephrine transporter (NET). Nisoxetine-based fluorescent probe 6 demonstrated high binding affinity (K i = 43 nM) for NET and an overall selectivity compared to the other transporters for dopamine (DAT; K i = 1540 nM) and serotonin (SERT; K i = 785 nM) in competitive radioligand binding assays. Using confocal microscopy, compound 6 was shown to stain both NET and SERT, but not DAT, at low nanomolar concentrations, in transporter-expressing cells.

Staphylococcus aureus RnpA Inhibitors: Computational-Guided Design, Synthesis and Initial Biological Evaluation.

Antibiotic resistance is spreading worldwide and it has become one of the most important issues in modern medicine. In this context, the bacterial RNA degradation and processing machinery are essential processes for bacterial viability that may be exploited for antimicrobial therapy. In Staphylococcus aureus, RnpA has been hypothesized to be one of the main players in these mechanisms. S. aureus RnpA is able to modulate mRNA degradation and complex with a ribozyme (rnpB), facilitating ptRNA maturation. Corresponding small molecule screening campaigns have recently identified a few classes of RnpA inhibitors, and their structure activity relationship (SAR) has only been partially explored. Accordingly, in the present work, using computational modeling of S. aureus RnpA we identified putative crucial interactions of known RnpA inhibitors, and we used this information to design, synthesize, and biologically assess new potential RnpA inhibitors. The present results may be beneficial for the overall knowledge about RnpA inhibitors belonging to both RNPA2000-like thiosemicarbazides and JC-like piperidine carboxamides molecular classes. We evaluated the importance of the different key moieties, such as the dichlorophenyl and the piperidine of JC2, and the semithiocarbazide, the furan, and the i-propylphenyl ring of RNPA2000. Our efforts could provide a foundation for further computational-guided investigations.

Computational Design and Development of Benzodioxane-Benzamides as Potent Inhibitors of FtsZ by Exploring the Hydrophobic Subpocket.

Multidrug resistant Staphylococcus aureus is a severe threat, responsible for most of the nosocomial infections globally. This resistant strain is associated with a 64% increase in death compared to the antibiotic-susceptible strain. The prokaryotic protein FtsZ and the cell division cycle have been validated as potential targets to exploit in the general battle against antibiotic resistance. Despite the discovery and development of several anti-FtsZ compounds, no FtsZ inhibitors are currently used in therapy. This work further develops benzodioxane-benzamide FtsZ inhibitors. We seek to find more potent compounds using computational studies, with encouraging predicted drug-like profiles. We report the synthesis and the characterization of novel promising derivatives that exhibit very low MICs towards both methicillin-susceptible and -resistant S. aureus, as well as another Gram positive species, Bacillus subtilis, while possessing good predicted physical-chemical properties in terms of solubility, permeability, and chemical and physical stability. In addition, we demonstrate by fluorescence microscopy that Z ring formation and FtsZ localization are strongly perturbed by our derivatives, thus validating the target.

Recent developments in ligands and chemical probes targeting solute carrier transporters.

Solute carrier (SLC) membrane transporters remain a largely unexploited target class, despite their central roles in cell identity and metabolism. This gap is reflected in the lack of high-quality chemical ligands or probes and in the small number of compounds that have progressed toward clinical development. In this review, we discuss recent advancements in SLC ligand discovery as well as new candidates that have been added to the investigational toolkit, with a particular focus on first-in-class ligands and the cognate discovery strategies. The availability of new probes expands the opportunity to elucidate the functions of SLCs and their relevance in physiology and explores any future potential of SLC druggability.

Design and Synthesis of Fluorescent Methylphenidate Analogues for a FRET-Based Assay of Synapsin III Binding.

We previously described synapsin III (Syn III) as a synaptic phosphoprotein that controls dopamine release in cooperation with α-synuclein (aSyn). Moreover, we found that in Parkinson's disease (PD), Syn III also participates in aSyn aggregation and toxicity. Our recent observations point to threo-methylphenidate (MPH), a monoamine re-uptake inhibitor that efficiently counteracts the freezing-gait characteristic of advanced PD, as a ligand for Syn III. We have designed and synthesised two different fluorescently labelled MPH derivatives, one with Rhodamine Red (RHOD) and one with 5-carboxytetramethylrhodamine (TAMRA), to be used for assessing MPH binding to Syn III by FRET. TAMRA-MPH exhibited the ideal characteristics to be used as a FRET acceptor, as it was able to enter into the SK-N-SH cells and could interact specifically with human green fluorescent protein (GFP)-tagged Syn III but not with GFP alone. Moreover, the uptake of TAMRA-MPH and co-localization with Syn III was also observed in primary mesencephalic neurons. These findings support that MPH is a Syn III ligand and that TAMRA-conjugated drug molecules might be valuable tools to study drug-ligand interactions by FRET or to detect Syn III in cytological and histological samples.

Benzamide Derivatives Targeting the Cell Division Protein FtsZ: Modifications of the Linker and the Benzodioxane Scaffold and Their Effects on Antimicrobial Activity.

Filamentous temperature-sensitive Z (FtsZ) is a prokaryotic protein with an essential role in the bacterial cell division process. It is widely conserved and expressed in both Gram-positive and Gram-negative strains. In the last decade, several research groups have pointed out molecules able to target FtsZ in Staphylococcus aureus, Bacillus subtilis and other Gram-positive strains, with sub-micromolar Minimum Inhibitory Concentrations (MICs). Conversely, no promising derivatives active on Gram-negatives have been found up to now. Here, we report our results on a class of benzamide compounds, which showed comparable inhibitory activities on both S. aureus and Escherichia coli FtsZ, even though they proved to be substrates of E. coli efflux pump AcrAB, thus affecting the antimicrobial activity. These surprising results confirmed how a single molecule can target both species while maintaining potent antimicrobial activity. A further computational study helped us decipher the structural features necessary for broad spectrum activity and assess the drug-like profile and the on-target activity of this family of compounds.

Alpha-synuclein/synapsin III pathological interplay boosts the motor response to methylphenidate.

Loss of dopaminergic nigrostriatal neurons and fibrillary α-synuclein (α-syn) aggregation in Lewy bodies (LB) characterize Parkinson's disease (PD). We recently found that Synapsin III (Syn III), a phosphoprotein regulating dopamine (DA) release with α-syn, is another key component of LB fibrils in the brain of PD patients and acts as a crucial mediator of α-syn aggregation and toxicity. Methylphenidate (MPH), a monoamine reuptake inhibitor (MRI) efficiently counteracting freezing of gait in advanced PD patients, can bind α-syn and controls α-syn-mediated DA overflow and presynaptic compartmentalization. Interestingly, MPH results also efficient for the treatment of attention deficits and hyperactivity disorder (ADHD), a neurodevelopmental psychiatric syndrome associated with Syn III and α-syn polymorphisms and constituting a risk factor for the development of LB disorders. Here, we studied α-syn/Syn III co-deposition and longitudinal changes of α-syn, Syn III and DA transporter (DAT) striatal levels in nigrostriatal neurons of a PD model, the human C-terminally truncated (1-120) α-syn transgenic (SYN120 tg) mouse, in comparison with C57BL/6J wild type (wt) and C57BL/6JOlaHsd α-syn null littermates. Then, we analyzed the locomotor response of these animals to an acute administration of MPH (d-threo) and other MRIs: cocaine, that we previously found to stimulate Syn III-reliant DA release in the absence of α-syn, or the selective DAT blocker GBR-12935, along aging. Finally, we assessed whether these drugs modulate α-syn/Syn III interaction by fluorescence resonance energy transfer (FRET) and performed in silico studies engendering a heuristic model of the α-syn conformations stabilized upon MPH binding. We found that only MPH was able to over-stimulate a Syn III-dependent/DAT-independent locomotor activity in the aged SYN120 tg mice showing α-syn/Syn III co-aggregates. MPH enhanced full length (fl) α-syn/Syn III and even more (1-120) α-syn/Syn III interaction in cells exhibiting α-syn/Syn III inclusions. Moreover, in silico studies confirmed that MPH may reduce α-syn fibrillation by stabilizing a protein conformation with increased lipid binding predisposition. Our observations indicate that the motor-stimulating effect of MPH can be positively fostered in the presence of α-syn/Syn III co-aggregation. This evidence holds significant implications for PD and ADHD therapeutic management.

Targeting Bacterial Cell Division: A Binding Site-Centered Approach to the Most Promising Inhibitors of the Essential Protein FtsZ.

Binary fission is the most common mode of bacterial cell division and is mediated by a multiprotein complex denominated the divisome. The constriction of the Z-ring splits the mother bacterial cell into two daughter cells of the same size. The Z-ring is formed by the polymerization of FtsZ, a bacterial protein homologue of eukaryotic tubulin, and it represents the first step of bacterial cytokinesis. The high grade of conservation of FtsZ in most prokaryotic organisms and its relevance in orchestrating the whole division system make this protein a fascinating target in antibiotic research. Indeed, FtsZ inhibition results in the complete blockage of the division system and, consequently, in a bacteriostatic or a bactericidal effect. Since many papers and reviews already discussed the physiology of FtsZ and its auxiliary proteins, as well as the molecular mechanisms in which they are involved, here, we focus on the discussion of the most compelling FtsZ inhibitors, classified by their main protein binding sites and following a medicinal chemistry approach.

Benzodioxane-Benzamides as Antibacterial Agents: Computational and SAR Studies to Evaluate the Influence of the 7-Substitution in FtsZ Interaction.

FtsZ is a crucial prokaryotic protein involved in bacterial cell replication. It recently arose as a promising target in the search for antimicrobial agents able to fight antimicrobial resistance. In this work, going on with our structure-activity relationship (SAR) study, we developed variously 7-substituted 1,4-benzodioxane compounds, linked to the 2,6-difluorobenzamide by a methylenoxy bridge. Compounds exhibit promising antibacterial activities not only against multidrug-resistant Staphylococcus aureus, but also on mutated Escherichia coli strains, thus enlarging their spectrum of action toward Gram-negative bacteria as well. Computational studies elucidated, through a validated FtsZ binding protocol, the structural features of new promising derivatives as FtsZ inhibitors.

How Reaction Conditions May Influence the Regioselectivity in the Synthesis of 2,3-Dihydro-1,4-benzoxathiine Derivatives.

The exploration of different reaction conditions aiming to obtain both 2,3-dihydro-1,4-benzoxathiine-2-yl derivatives and 2,3-dihydro-1,4-benzoxathiine-3-yl ones is reported. The treatment of 1,2-mercaptophenol with an organic base and a specific 2-bromo acrylate results in a solvent- and substrate-dependent exclusive solvation of O- and S-anions, thus managing the regioselectivity.

How do reaction conditions affect the enantiopure synthesis of 2-substituted-1,4-benzodioxane derivatives?

Several biologically active compounds structurally include the enantiopure 2-substituted-1,4-benzodioxane scaffold. The straightforward racemization that affects reactions involving most of the common chemical reactives is thus a crucial issue. The developing of a completely stereo-controlled synthetic route that does not affect the enantiomeric excess is consequently mandatory. It is also important to set up a reliable chiral HPLC method, able to follow the reaction, and to improve the synthetic performances. Here, we report the chiral investigation of two different synthons, we specifically evaluated the synthetic pathways that could be run in order to afford them, avoiding the racemization processes, which could normally occur in basic conditions. In addition, we developed peculiar chiral HPLC methods in order to resolve the enantiomers, define the enantiomeric excess, and fully characterize these compounds.

2,6-Difluorobenzamide Inhibitors of Bacterial Cell Division Protein FtsZ: Design, Synthesis, and Structure-Activity Relationships.

A wide variety of drug-resistant microorganisms are continuously emerging, restricting the therapeutic options for common bacterial infections. Antimicrobial agents that were originally potent are now no longer helpful, due to their weak or null activity toward these antibiotic-resistant bacteria. In addition, none of the recently approved antibiotics affect innovative targets, resulting in a need for novel drugs with innovative antibacterial mechanisms of action. The essential cell division protein filamentous temperature-sensitive Z (FtsZ) has emerged as a possible target, thanks to its ubiquitous expression and its homology to eukaryotic ß-tubulin. In the latest years, several compounds were shown to interact with this prokaryotic protein and selectively inhibit bacterial cell division. Recently, our research group developed interesting derivatives displaying good antibacterial activities against methicillin-resistant Staphylococcus aureus, as well as vancomycin-resistant Enterococcus faecalis and Mycobacterium tuberculosis. The aim of the present study was to summarize the structure-activity relationships of differently substituted heterocycles, linked by a methylenoxy bridge to the 2,6-difluorobenzamide, and to validate FtsZ as the real target of this class of antimicrobials.