Design, Synthesis, and Evaluation of An Anti-trypanosomal [1,2,4]Triazolo[1,5-a]pyrimidine Probe for Photoaffinity Labeling Studies.

Studies have shown that depending on the substitution pattern, microtubule (MT)-targeting 1,2,4-triazolo[1,5-a]pyrimidines (TPDs) can produce different cellular responses in mammalian cells that may be due to these compounds interacting with distinct binding sites within the MT structure. Selected TPDs are also potently bioactive against the causative agent of human African trypanosomiasis, Trypanosoma brucei, both in vitro and in vivo. So far, however, there has been no direct evidence of tubulin engagement by these TPDs in T. brucei. Therefore, to enable further investigation of anti-trypanosomal TPDs, a TPD derivative amenable to photoaffinity labeling (PAL) was designed, synthesized, and evaluated in PAL experiments using HEK293 cells and T. brucei. The data arising confirmed specific labeling of T. brucei tubulin. In addition, proteomic data revealed differences in the labeling profiles of tubulin between HEK293 and T. brucei, suggesting structural differences between the TPD binding site(s) in mammalian and trypanosomal tubulin.

Targeted Reversible Covalent Modification of a Noncatalytic Lysine of the Krev Interaction Trapped 1 Protein Enables Site-Directed Screening for Protein-Protein Interaction Inhibitors.

The covalent reversible modification of proteins is a validated strategy for the development of probes and candidate therapeutics. However, the covalent reversible targeting of noncatalytic lysines is particularly challenging. Herein, we characterize the 2-hydroxy-1-naphthaldehyde (HNA) fragment as a targeted covalent reversible ligand of a noncatalytic lysine (Lys720) of the Krev interaction trapped 1 (KRIT1) protein. We show that the interaction of HNA with KRIT1 is highly specific, results in prolonged residence time of >8 h, and inhibits the Heart of glass 1 (HEG1)-KRIT1 protein-protein interaction (PPI). Screening of HNA derivatives identified analogs exhibiting similar binding modes as the parent fragment but faster target engagement and stronger inhibition activity. These results demonstrate that HNA is an efficient site-directing fragment with promise in developing HEG1-KRIT1 PPI inhibitors. Further, the aldimine chemistry, when coupled with templating effects that promote proximity, can produce a long-lasting reversible covalent modification of noncatalytic lysines.

Structure-Activity Relationships, Tolerability and Efficacy of Microtubule-Active 1,2,4-Triazolo[1,5-a]pyrimidines as Potential Candidates to Treat Human African Trypanosomiasis.

Tubulin and microtubules (MTs) are potential protein targets to treat parasitic infections and our previous studies have shown that the triazolopyrimidine (TPD) class of MT-active compounds hold promise as antitrypanosomal agents. MT-targeting TPDs include structurally related but functionally diverse congeners that interact with mammalian tubulin at either one or two distinct interfacial binding sites; namely, the seventh and vinca sites, which are found within or between α,ß-tubulin heterodimers, respectively. Evaluation of the activity of 123 TPD congeners against cultured Trypanosoma brucei enabled a robust quantitative structure-activity relationship (QSAR) model and the prioritization of two congeners for in vivo pharmacokinetics (PK), tolerability and efficacy studies. Treatment of T. brucei-infected mice with tolerable doses of TPDs significantly decreased blood parasitemia within 24 h. Further, two once-weekly doses at 10 mg/kg of a candidate TPD significantly extended the survival of infected mice relative to infected animals treated with vehicle. Further optimization of dosing and/or the dosing schedule of these CNS-active TPDs may provide alternative treatments for human African trypanosomiasis.

PINK1-Dependent Mitophagy Inhibits Elevated Ubiquitin Phosphorylation Caused by Mitochondrial Damage.

Ubiquitin phosphorylation by the mitochondrial protein kinase PTEN-induced kinase 1 (PINK1), upon mitochondrial depolarization, is an important intermediate step in the recycling of damaged mitochondria via mitophagy. As mutations in PINK1 can cause early-onset Parkinson's disease (PD), there has been a growing interest in small-molecule activators of PINK1-mediated mitophagy as potential PD treatments. Herein, we show that N6-substituted adenosines, such as N6-(2-furanylmethyl)adenosine (known as kinetin riboside) and N6-benzyladenosine, activate PINK1 in HeLa cells and induce PINK1-dependent mitophagy in primary mouse fibroblasts. Interestingly, pre-treatment of HeLa cells and astrocytes with these compounds inhibited elevated ubiquitin phosphorylation that is induced by established mitochondrial depolarizing agents, carbonyl cyanide m-chlorophenyl-hydrazine and niclosamide. Together, this highlights N6-substituted adenosines as progenitor PINK1 activators that could potentially be developed, in the future, as treatments for aged and sporadic PD patients who have elevated phosphorylated ubiquitin levels in the brain.

Biocatalytic and Chemo-Enzymatic Synthesis of Quinolines and 2-Quinolones by Monoamine Oxidase (MAO-N) and Horseradish Peroxidase (HRP) Biocatalysts.

The oxidative aromatization of aliphatic N-heterocycles is a fundamental organic transformation for the preparation of a diverse array of heteroaromatic compounds. Despite many attempts to improve the efficiency and practicality of this transformation, most synthetic methodologies still require toxic and expensive reagents as well as harsh conditions. Herein, we describe two enzymatic strategies for the oxidation of 1,2,3,4-tetrahydroquinolines (THQs) and N-cyclopropyl-N-alkylanilines into quinolines and 2-quinolones, respectively. Whole cells and purified monoamine oxidase (MAO-N) enzymes were used to effectively catalyze the biotransformation of THQs into the corresponding aromatic quinoline derivatives, while N-cyclopropyl-N-alkylanilines were converted into 2-quinolone compounds through a horseradish peroxidase (HRP)-catalyzed annulation/aromatization reaction followed by Fe-mediated oxidation.

Structure-Activity Relationships, Tolerability and Efficacy of Microtubule-Active 1,2,4-Triazolo[1,5- a ]pyrimidines as Potential Candidates to Treat Human African Trypanosomiasis.

Tubulin and microtubules (MTs) are potential protein targets to treat parasitic infections and our previous studies have shown that the triazolopyrimidine (TPD) class of MT- active compounds hold promise as antitrypanosomal agents. MT-targeting TPDs include structurally related but functionally diverse congeners that interact with mammalian tubulin at either one or two distinct interfacial binding sites; namely, the seventh and vinca sites, which are found within or between α,ß-tubulin heterodimers, respectively. Evaluation of the activity of 123 TPD congeners against cultured Trypanosoma brucei enabled a robust quantitative structure-activity relationship (QSAR) model and the prioritization of two congeners for in vivo pharmacokinetics (PK), tolerability and efficacy studies. Treatment of T. brucei -infected mice with tolerable doses of TPDs 3 and 4 significantly decreased blood parasitemia within 24 h. Further, two once-weekly doses of 4 at 10 mg/kg significantly extended the survival of infected mice relative to infected animals treated with vehicle. Further optimization of dosing and/or the dosing schedule of these CNS-active TPDs may provide alternative treatments for human African trypanosomiasis.

Microtubule-Stabilizing 1,2,4-Triazolo[1,5-a]pyrimidines as Candidate Therapeutics for Neurodegenerative Disease: Matched Molecular Pair Analyses and Computational Studies Reveal New Structure-Activity Insights.

Microtubule (MT)-stabilizing 1,2,4-triazolo[1,5-a]pyrimidines (TPDs) hold promise as candidate therapeutics for Alzheimer's disease (AD) and other neurodegenerative conditions. However, depending on the choice of substituents around the TPD core, these compounds can elicit markedly different cellular phenotypes that likely arise from the interaction of TPD congeners with either one or two spatially distinct binding sites within tubulin heterodimers (i.e., the seventh site and the vinca site). In the present study, we report the design, synthesis, and evaluation of a series of new TPD congeners, as well as matched molecular pair analyses and computational studies, that further elucidate the structure-activity relationships of MT-active TPDs. These studies led to the identification of novel MT-normalizing TPD candidates that exhibit favorable ADME-PK, including brain penetration and oral bioavailability, as well as brain pharmacodynamic activity.

Rational design of novel nucleoside analogues reveals potent antiviral agents for EV71.

Different viruses belonging to distinct viral families, such as enterovirus 71, rely on the host methyltransferase METTL3 for the completion of fundamental cytoplasmic stages of their life cycle. Modulation of the activity of this enzyme could therefore provide a broad-spectrum approach to interfere with viral infections caused by viruses that depend on its activity for the completion of their viral cycle. With the aim to identify antiviral therapeutics with this effect, a series of new nucleoside analogues was rationally designed to act as inhibitors of human METTL3, as a novel approach to interfere with a range of viral infections. Guided by molecular docking studies on the SAM binding pocket of the enzyme, 24 compounds were prepared following multiple-step synthetic protocols, and evaluated for their ability to interfere with the replication of different viruses in cell-based systems, and to directly inhibit the activity of METTL3. While different molecules displayed moderate inhibition of the human methyltransferase in vitro, multiple novel, potent and selective inhibitors of enterovirus 71 were identified.

Geneticin shows selective antiviral activity against SARS-CoV-2 by interfering with programmed -1 ribosomal frameshifting.

SARS-CoV-2 is currently causing an unprecedented pandemic. While vaccines are massively deployed, we still lack effective large-scale antiviral therapies. In the quest for antivirals targeting conserved structures, we focused on molecules able to bind viral RNA secondary structures. Aminoglycosides are a class of antibiotics known to interact with the ribosomal RNA of both prokaryotes and eukaryotes and have previously been shown to exert antiviral activities by interacting with viral RNA. Here we show that the aminoglycoside geneticin is endowed with antiviral activity against all tested variants of SARS-CoV-2, in different cell lines and in a respiratory tissue model at non-toxic concentrations. The mechanism of action is an early inhibition of RNA replication and protein expression related to a decrease in the efficiency of the -1 programmed ribosomal frameshift (PRF) signal of SARS-CoV-2. Using in silico modeling, we have identified a potential binding site of geneticin in the pseudoknot of frameshift RNA motif. Moreover, we have selected, through virtual screening, additional RNA binding compounds, interacting with the same site with increased potency.

A Composite System Based upon Hydroxypropyl Cyclodextrins and Soft Hydrogel Contact Lenses for the Delivery of Therapeutic Doses of Econazole to the Cornea, In Vitro.

Fungal keratitis, a disease in which the cornea becomes inflamed due to an invasive fungal infection, remains difficult to treat due in part to limited choices of available treatments. Topical eye drops are first-line treatment, but can be ineffective as low levels of drug reach the target site due to precorneal losses and the impenetrability of the cornea. The aim of this study was to determine the corneal delivery of econazole using a novel topical enhancement approach using a composite delivery system based upon cyclodextrins and soft hydrogel contact lenses. Excess econazole nitrate was added to hydroxypropyl-α-cyclodextrin (HP-α-CD) and hydroxypropyl-ß-cyclodextrin (HP-ß-CD) solutions, and the solubility determined using HPLC. Proprietary soft hydrogel contact lenses were then impregnated with saturated solutions and applied to freshly enucleated porcine eyeballs. Econazole nitrate 'eye drops' at the same concentrations served as the control. After 6 h, the corneas were excised and drug-extracted, prior to quantification using HPLC. Molecular dynamic simulations were performed to examine econazole−HP-ß-CD inclusion complexation and dissociation. The minimum inhibitory concentration (MIC) of econazole was determined against four fungal species associated with keratitis, and these data were then related to the amount of drug delivered to the cornea, using an average corneal volume of 0.19 mL. The solubility of econazole increased greatly in the presence of HP-ß-CD and more so with HP-α-CD (p < 0.001), with ratios >> 2. Hydrogel contact lenses delivered ×2.8 more drug across the corneas in comparison to eye drops alone, and ×5 more drug delivered to the cornea when cyclodextrin was present. Molecular graphics demonstrated dynamic econazole release, which would create transient enhanced drug concentration at the cornea surface. The solution-only drops achieved the least satisfactory result, producing sub-MIC levels with factors of ×0.81 for both Fusarium semitectum and Fusarium solani and ×0.40 for both Scolecobasidium tshawytschae and Bipolaris hawaiiensis. All other treatments delivered econazole at > MIC for all four fungal species. The efficacies of the delivery platforms evaluated were ranked: HP-α-CD contact lens > HP-ß-CD contact lens > contact lens = HP-α-CD drops > HP-ß-CD drops > solution-only drops. In summary, the results in this study have demonstrated that a composite drug delivery system based upon econazole−HP-ß-CD inclusion complexes loaded into contact lenses can achieve significantly greater corneal drug delivery with the potential for improved clinical responses.

Geneticin shows selective antiviral activity against SARS-CoV-2 by interfering with programmed -1 ribosomal frameshifting.

SARS-CoV-2 is currently causing an unprecedented pandemic. While vaccines are massively deployed, we still lack effective large-scale antiviral therapies. In the quest for antivirals targeting conserved structures, we focused on molecules able to bind viral RNA secondary structures. Aminoglycosides are a class of antibiotics known to interact with the ribosomal RNA of both prokaryotes and eukaryotes and have previously been shown to exert antiviral activities by interacting with viral RNA. Here we show that the aminoglycoside geneticin is endowed with antiviral activity against all tested variants of SARS-CoV-2, in different cell lines and in a respiratory tissue model at non-toxic concentrations. The mechanism of action is an early inhibition of RNA replication and protein expression related to a decrease in the efficiency of the -1 programmed ribosomal frameshift (PRF) signal of SARS-CoV-2. Using in silico modelling, we have identified a potential binding site of geneticin in the pseudoknot of frameshift RNA motif. Moreover, we have selected, through virtual screening, additional RNA binding compounds, interacting with the same site with increased potency.

Identification of SARS-CoV-2 inhibitors targeting Mpro and PLpro using in-cell-protease assay.

SARS-CoV-2 proteases Mpro and PLpro are promising targets for antiviral drug development. In this study, we present an antiviral screening strategy involving a novel in-cell protease assay, antiviral and biochemical activity assessments, as well as structural determinations for rapid identification of protease inhibitors with low cytotoxicity. We identified eight compounds with anti-SARS-CoV-2 activity from a library of 64 repurposed drugs and modeled at protease active sites by in silico docking. We demonstrate that Sitagliptin and Daclatasvir inhibit PLpro, and MG-101, Lycorine HCl, and Nelfinavir mesylate inhibit Mpro of SARS-CoV-2. The X-ray crystal structure of Mpro in complex with MG-101 shows a covalent bond formation between the inhibitor and the active site Cys145 residue indicating its mechanism of inhibition is by blocking the substrate binding at the active site. Thus, we provide methods for rapid and effective screening and development of inhibitors for blocking virus polyprotein processing as SARS-CoV-2 antivirals. Additionally, we show that the combined inhibition of Mpro and PLpro is more effective in inhibiting SARS-CoV-2 and the delta variant.

A Time-Kill Assay Study on the Synergistic Bactericidal Activity of Pomegranate Rind Extract and Zn (II) against Methicillin-Resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa.

There is a need for new antimicrobial systems due to increased global resistance to current antimicrobials. Pomegranate rind extract (PRE) and Zn (II) ions both possess a level of antimicrobial activity and work has previously shown that PRE/Zn (II) in combination possesses synergistic activity against Herpes simplex virus and Micrococcus luteus. Here, we determined whether such synergistic activity extended to other, more pathogenic, bacteria. Reference strains of methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa were cultured and subjected to challenge by PRE, Zn (II), or PRE + Zn (II), in time-kill assays. Data were obtained independently by two researchers using different PRE preparations. Statistically significant synergistic activity for PRE + Zn (II) was shown for all four bacterial strains tested compared to untreated controls, although the extent of efficacy and timescales varied. Zn (II) exerted activity and at 1 h, it was not possible to distinguish with PRE + Zn (II) combination treatment in all cases. PRE alone showed low activity against all four bacteria. Reproducible synergistic bactericidal activity involving PRE and Zn (II) has been confirmed. Potential mechanisms are discussed. The development of a therapeutic system that possesses demonstrable antimicrobial activity is supported which lends itself particularly to topical delivery applications, for example MRSA infections.

Structure-Activity Relationship Studies on Novel Antiviral Agents for Norovirus Infections.

Human norovirus is the leading cause of acute gastroenteritis worldwide, affecting every year 685 million people. Norovirus outbreaks are associated with very significant economic losses, with an estimated societal cost of 60 billion USD per year. Despite this, no therapeutic options or vaccines are currently available to treat or prevent this infection. An antiviral therapy that can be used as treatment and as a prophylactic measure in the case of outbreaks is urgently needed. We previously described the computer-aided design and synthesis of novel small-molecule agents able to inhibit the replication of human norovirus in cell-based systems. These compounds are non-nucleoside inhibitors of the viral polymerase and are characterized by a terminal para-substituted phenyl group connected to a central phenyl ring by an amide-thioamide linker, and a terminal thiophene ring. Here we describe new modifications of these scaffolds focused on exploring the role of the substituent at the para position of the terminal phenyl ring and on removing the thioamide portion of the amide-thioamide linker, to further explore structure-activity relationships (SARs) and improve antiviral properties. According to three to four-step synthetic routes, we prepared thirty novel compounds, which were then evaluated against the replication of both murine (MNV) and human (HuNoV) norovirus in cells. Derivatives in which the terminal phenyl group has been replaced by an unsubstituted benzoxazole or indole, and the thioamide component of the amide-thioamide linker has been removed, showed promising results in inhibiting HuNoV replication at low micromolar concentrations. Particularly, compound 28 was found to have an EC50 against HuNoV of 0.9 µM. Although the most active novel derivatives were also associated with an increased cytotoxicity in the human cell line, these compounds represent a very promising starting point for the development of new analogues with reduced cytotoxicity and improved selectivity indexes. In addition, the experimental biological data have been used to create an initial 3D quantitative structure-activity relationship model, which could be used to guide the future design of novel potential anti-norovirus agents.

Ligand-based rational design, synthesis and evaluation of novel potential chemical chaperones for opsin.

Inherited blinding diseases retinitis pigmentosa (RP) and a subset of Leber's congenital amaurosis (LCA) are caused by the misfolding and mistrafficking of rhodopsin molecules, which aggregate and accumulate in the endoplasmic reticulum (ER), leading to photoreceptor cell death. One potential therapeutic strategy to prevent the loss of photoreceptors in these conditions is to identify opsin-binding compounds that act as chemical chaperones for opsin, aiding its proper folding and trafficking to the outer cell membrane. Aiming to identify novel compounds with such effect, a rational ligand-based approach was applied to the structure of the visual pigment chromophore, 11-cis-retinal, and its locked analogue 11-cis-6mr-retinal. Following molecular docking studies on the main chromophore binding site of rhodopsin, 49 novel compounds were synthesized according to optimized one-to seven-step synthetic routes. These agents were evaluated for their ability to compete for the chromophore binding site of opsin, and their capacity to increase the trafficking of the P23H opsin mutant from the ER to the cell membrane. Different new molecules displayed an effect in at least one assay, acting either as chemical chaperones or as stabilizers of the 9-cis-retinal-rhodopsin complex. These compounds could provide the basis to develop novel therapeutics for RP and LCA.

Structure property relationships of N-acylsulfonamides and related bioisosteres.

The N-acylsulfonamide functional group is a feature of the pharmacophore of several biologically active molecules, including marketed drugs. Although this acidic moiety presents multiple points of attachments that could be exploited to introduce structural diversification, depending on the circumstances, the replacement of the functional group itself with a suitable surrogate, or bioisostere, may be desirable. A number of N-acylsulfonamide bioisosteres have been developed over the years that provide opportunities to modulate both structure and physicochemical properties of this important structural motif. To enable an assessment of the relative impact on physicochemical properties that these replacements may have compared to the N-acylsulfonamide group, we conducted a structure-property relationship study based on matched molecular pairs, in which the N-acylsulfonamide moiety of common template reference structures is replaced with a series of bioisosteres. The data presented, which include an assessment of relative changes in acidity, permeability, lipophilicity and intrinsic solubility, provides a basis for informed decisions when deploying N-acylsulfonamides, or surrogates thereof, in analog design.

Sulfonated cryogel scaffolds for focal delivery in ex-vivo brain tissue cultures.

The human brain has unique features that are difficult to study in animal models, including the mechanisms underlying neurodevelopmental and psychiatric disorders. Despite recent advances in human primary brain tissue culture systems, the use of these models to elucidate cellular disease mechanisms remains limited. A major reason for this is the lack of tools available to precisely manipulate a specific area of the tissue in a reproducible manner. Here we report an easy-to-use tool for site-specific manipulation of human brain tissue in culture. We show that line-shaped cryogel scaffolds synthesized with precise microscale dimensions allow the targeted delivery of a reagent to a specific region of human brain tissue in culture. 3-sulfopropyl acrylate (SPA) was incorporated into the cryogel network to yield a negative surface charge for the reversible binding of molecular cargo. The fluorescent dyes BODIPY and DiI were used as model cargos to show that placement of dye loaded scaffolds onto brain tissue in culture resulted in controlled delivery without a burst release, and labelling of specific regions without tissue damage. We further show that cryogels can deliver tetrodotoxin to tissue, inhibiting neuronal function in a reversible manner. The robust nature and precise dimensions of the cryogel resulted in a user-friendly and reproducible tool to manipulate primary human tissue cultures. These easy-to-use cryogels offer an innovate approach for more complex manipulations of ex-vivo tissue.

Evaluation of the Structure-Activity Relationship of Microtubule-Targeting 1,2,4-Triazolo[1,5-a]pyrimidines Identifies New Candidates for Neurodegenerative Tauopathies.

Studies in tau and Aß plaque transgenic mouse models demonstrated that brain-penetrant microtubule (MT)-stabilizing compounds, including the 1,2,4-triazolo[1,5-a]pyrimidines, hold promise as candidate treatments for Alzheimer's disease and related neurodegenerative tauopathies. Triazolopyrimidines have already been investigated as anticancer agents; however, the antimitotic activity of these compounds does not always correlate with stabilization of MTs in cells. Indeed, previous studies from our laboratories identified a critical role for the fragment linked at C6 in determining whether triazolopyrimidines promote MT stabilization or, conversely, disrupt MT integrity in cells. To further elucidate the structure-activity relationship (SAR) and to identify potentially improved MT-stabilizing candidates for neurodegenerative disease, a comprehensive set of 68 triazolopyrimidine congeners bearing structural modifications at C6 and/or C7 was designed, synthesized, and evaluated. These studies expand upon prior understanding of triazolopyrimidine SAR and enabled the identification of novel analogues that, relative to the existing lead, exhibit improved physicochemical properties, MT-stabilizing activity, and pharmacokinetics.

Discovery of Novel 2-Aniline-1,4-naphthoquinones as Potential New Drug Treatment for Leber's Hereditary Optic Neuropathy (LHON).

Leber's hereditary optic neuropathy (LHON) is a rare genetic mitochondrial disease and the primary cause of chronic visual impairment for at least 1 in 10 000 individuals in the U.K. Treatment options remain limited, with only a few drug candidates and therapeutic approaches, either approved or in development. Recently, idebenone has been investigated as drug therapy in the treatment of LHON, although evidence for the efficacy of idebenone is limited in the literature. NAD(P)H:quinone oxidoreductase 1 (NQO1) and mitochondrial complex III were identified as the major enzymes involved in idebenone activity. Based on this mode of action, computer-aided techniques and structure-activity relationship (SAR) optimization studies led to the discovery of a series naphthoquinone-related small molecules, with comparable adenosine 5'-triphosphate (ATP) rescue activity to idebenone. Among these, three compounds showed activity in the nanomolar range and one, 2-((4-fluoro-3-(trifluoromethyl)phenyl)amino)-3-(methylthio)naphthalene-1,3-dione (1), demonstrated significantly higher potency ex vivo, and significantly lower cytotoxicity, than idebenone.