Lipopeptide antibiotics disrupt interactions of undecaprenyl phosphate with UptA.

The peptidoglycan pathway represents one of the most successful antibacterial targets with the last critical step being the flipping of carrier lipid, undecaprenyl phosphate (C55-P), across the membrane to reenter the pathway. This translocation of C55-P is facilitated by DedA and DUF368 domain-containing family membrane proteins via unknown mechanisms. Here, we employ native mass spectrometry to investigate the interactions of UptA, a member of the DedA family of membrane protein from Bacillus subtilis, with C55-P, membrane phospholipids, and cell wall-targeting antibiotics. Our results show that UptA, expressed and purified in Escherichia coli, forms monomer-dimer equilibria, and binds to C55-P in a pH-dependent fashion. Specifically, we show that UptA interacts more favorably with C55-P over shorter-chain analogs and membrane phospholipids. Moreover, we demonstrate that lipopeptide antibiotics, amphomycin and aspartocin D, can directly inhibit UptA function by out-competing the substrate for the protein binding, in addition to their propensity to form complex with free C55-P. Overall, this study shows that UptA-mediated translocation of C55-P is potentially mediated by pH and anionic phospholipids and provides insights for future development of antibiotics targeting carrier lipid recycling.

Dietary flavonoids: Nano delivery and nanoparticles for cancer therapy.

Application of nanotechnologies to cancer therapy might increase solubility and/or bioavailability of bioactive compounds of natural or synthetic origin and offers other potential benefits in cancer therapy, including selective targeting. In the present review we aim to evaluate in vivo studies on the anticancer activity of nanoparticles (NPs) obtained from food-derived flavonoids. From a systematic search a total of 60 studies were identified. Most of the studies involved the flavanol epigallocatechin-3-O-gallate and the flavonol quercetin, in both delivery and co-delivery (with anti-cancer drugs) systems. Moreover, some studies investigated the effects of other flavonoids, such as anthocyanins aglycones anthocyanidins, flavanones, flavones and isoflavonoids. NPs inhibited tumor growth in both xenograft and chemical-induced animal models of cancerogenesis. Encapsulation improved bioavailability and/or reduced toxicity of both flavonoids and/or co-delivered drugs, such as doxorubicin, docetaxel, paclitaxel, honokiol and vincristine. Moreover, flavonoids have been successfully applied in molecular targeted nanosystems. Selectivity for cancer cells involves pH- and/or reactive oxygen species-mediated mechanisms. Furthermore, flavonoids are good candidates as drug delivery for anticancer drugs in green synthesis systems. In conclusion, although human studies are needed, NPs obtained from food-derived flavonoids have promising anticancer effects in vivo.

MmpL3 is the flippase for mycolic acids in mycobacteria.

The defining feature of the mycobacterial outer membrane (OM) is the presence of mycolic acids (MAs), which, in part, render the bilayer extremely hydrophobic and impermeable to external insults, including many antibiotics. Although the biosynthetic pathway of MAs is well studied, the mechanism(s) by which these lipids are transported across the cell envelope is(are) much less known. Mycobacterial membrane protein Large 3 (MmpL3), an essential inner membrane (IM) protein, is implicated in MA transport, but its exact function has not been elucidated. It is believed to be the cellular target of several antimycobacterial compounds; however, evidence for direct inhibition of MmpL3 activity is also lacking. Here, we establish that MmpL3 is the MA flippase at the IM of mycobacteria and is the molecular target of BM212, a 1,5-diarylpyrrole compound. We develop assays that selectively access mycolates on the surface of Mycobacterium smegmatis spheroplasts, allowing us to monitor flipping of MAs across the IM. Using these assays, we establish the mechanism of action of BM212 as a potent MmpL3 inhibitor, and use it as a molecular probe to demonstrate the requirement for functional MmpL3 in the transport of MAs across the IM. Finally, we show that BM212 binds MmpL3 directly and inhibits its activity. Our work provides fundamental insights into OM biogenesis and MA transport in mycobacteria. Furthermore, our assays serve as an important platform for accelerating the validation of small molecules that target MmpL3, and their development as future antituberculosis drugs.

Mycobacterial tryptophan biosynthesis: A promising target for tuberculosis drug development?

The biosynthetic pathways of amino acids are attractive targets for drug development against pathogens with an intracellular behavior like M. tuberculosis (Mtb). Indeed, while in the macrophages Mtb has restricted access to amino acids such as tryptophan (Trp). Auxotrophic Mtb strains, with mutations in the Trp biosynthetic pathway, showed reduced intracellular survival in cultured human and murine macrophages and failed to cause the disease in immunocompetent and immunocompromised mice. Herein we present recent efforts in the discovery of Trp biosynthesis inhibitors.

A novel antimycobacterial compound acts as an intracellular iron chelator.

Efficient iron acquisition is crucial for the pathogenesis of Mycobacterium tuberculosis. Mycobacterial iron uptake and metabolism are therefore attractive targets for antitubercular drug development. Resistance mutations against a novel pyrazolopyrimidinone compound (PZP) that is active against M. tuberculosis have been identified within the gene cluster encoding the ESX-3 type VII secretion system. ESX-3 is required for mycobacterial iron acquisition through the mycobactin siderophore pathway, which could indicate that PZP restricts mycobacterial growth by targeting ESX-3 and thus iron uptake. Surprisingly, we show that ESX-3 is not the cellular target of the compound. We demonstrate that PZP indeed targets iron metabolism; however, we found that instead of inhibiting uptake of iron, PZP acts as an iron chelator, and we present evidence that the compound restricts mycobacterial growth by chelating intrabacterial iron. Thus, we have unraveled the unexpected mechanism of a novel antimycobacterial compound.

Synthesis, biological evaluation and docking analysis of a new series of methylsulfonyl and sulfamoyl acetamides and ethyl acetates as potent COX-2 inhibitors.

We report herein the synthesis, biological evaluation and docking analysis of a new series of methylsulfonyl, sulfamoyl acetamides and ethyl acetates that selectively inhibit cyclooxygenase-2 (COX-2) isoform. Among the newly synthesized compounds, some of them were endowed with a good activity against COX-2 and a good selectivity COX-2/COX-1 in vitro as well as a desirable analgesic activity in vivo, proving that replacement of the ester moiety with an amide group gave access to more stable derivatives, characterized by a good COX-inhibition.

Enhancing the pharmacodynamic profile of a class of selective COX-2 inhibiting nitric oxide donors.

We report herein the development, synthesis, physicochemical and pharmacological characterization of a novel class of pharmacodynamic hybrids that selectively inhibit cyclooxygenase-2 (COX-2) isoform and present suitable nitric oxide releasing properties. The replacement of the ester moiety with the amide group gave access to in vivo more stable and active derivatives that highlighted outstanding pharmacological properties. In particular, the glycine derivative proved to be extremely active in suppressing hyperalgesia and edema.

Amino Acid Biosynthesis Inhibitors in Tuberculosis Drug Discovery.

According to the latest World Health Organization (WHO) report, an estimated 10.6 million people were diagnosed with tuberculosis (TB) in 2022, and 1.30 million died. A major concern is the emergence of multi-drug-resistant (MDR) and extensively drug-resistant (XDR) strains, fueled by the length of anti-TB treatment and HIV comorbidity. Innovative anti-TB agents acting with new modes of action are the only solution to counteract the spread of resistant infections. To escape starvation and survive inside macrophages, Mtb has evolved to become independent of the host by synthesizing its own amino acids. Therefore, targeting amino acid biosynthesis could subvert the ability of the mycobacterium to evade the host immune system, providing innovative avenues for drug discovery. The aim of this review is to give an overview of the most recent progress in the discovery of amino acid biosynthesis inhibitors. Among the hits discovered over the past five years, tryptophan (Trp) inhibitors stand out as the most advanced and have significantly contributed to demonstrating the feasibility of this approach for future TB drug discovery. Future efforts should be directed at prioritizing the chemical optimization of these hits to enrich the TB drug pipeline with high-quality leads.

Transmission-Blocking Strategies for Malaria Eradication: Recent Advances in Small-Molecule Drug Development.

Malaria drug research and development efforts have resurged in the last decade following the decelerating rate of mortality and malaria cases in endemic regions. The inefficiency of malaria interventions is largely driven by the spreading resistance of the Plasmodium falciparum parasite to current drug regimens and that of the malaria vector, the Anopheles mosquito, to insecticides. In response to the new eradication agenda, drugs that act by breaking the malaria transmission cycle (transmission-blocking drugs), which has been recognized as an important and additional target for intervention, are being developed. These drugs take advantage of the susceptibility of Plasmodium during population bottlenecks before transmission (gametocytes) and in the mosquito vector (gametes, zygotes, ookinetes, oocysts, sporozoites). To date, compounds targeting stage V gametocytes predominate in the chemical library of transmission-blocking drugs, and some of them have entered clinical trials. The targeting of Plasmodium mosquito stages has recently renewed interest in the development of innovative malaria control tools, which hold promise for the application of compounds effective at these stages. In this review, we highlight the major achievements and provide an update on the research of transmission-blocking drugs, with a particular focus on their chemical scaffolds, antiplasmodial activity, and transmission-blocking potential.

SAR Analysis of Novel Coxsackie virus A9 Capsid Binders.

Enterovirus infections are common in humans, yet there are no approved antiviral treatments. In this study we concentrated on inhibition of one of the Enterovirus B (EV-B), namely Coxsackievirus A9 (CVA9), using a combination of medicinal chemistry, virus inhibition assays, structure determination from cryogenic electron microscopy and molecular modeling, to determine the structure activity relationships for a promising class of novel N-phenylbenzylamines. Of the new 29 compounds synthesized, 10 had half maximal effective concentration (EC50) values between 0.64-10.46 µM, and of these, 7 had 50% cytotoxicity concentration (CC50) values higher than 200 µM. In addition, this new series of compounds showed promising physicochemical properties and act through capsid stabilization, preventing capsid expansion and subsequent release of the genome.

A class of pyrrole derivatives endowed with analgesic/anti-inflammatory activity.

We report the synthesis and bio-pharmacological evaluation of a class of pyrrole derivatives featuring a small appendage fragment (carbaldehyde, oxime, nitrile) on the central core. Compound 1c proved to be extremely effective in vivo, showing an interesting anti-nociceptic profile that is comparable to reference compounds already marketed, hence representing a great stimulus for a further improvement of this class of molecules.

Direct-Acting Antivirals and Host-Targeting Approaches against Enterovirus B Infections: Recent Advances.

Enterovirus B (EV-B)-related diseases, which can be life threatening in high-risk populations, have been recognized as a serious health problem, but their clinical treatment is largely supportive, and no selective antivirals are available on the market. As their clinical relevance has become more serious, efforts in the field of anti-EV-B inhibitors have greatly increased and many potential antivirals with very high selectivity indexes and promising in vitro activities have been discovered. The scope of this review encompasses recent advances in the discovery of new compounds with anti-viral activity against EV-B, as well as further progress in repurposing drugs to treat these infections. Current progress and future perspectives in drug discovery against EV-Bs are briefly discussed and existing gaps are spotlighted.

Antiviral Mechanisms of N-Phenyl Benzamides on Coxsackie Virus A9.

Enteroviruses are one of the most abundant groups of viruses infecting humans, and yet there are no approved antivirals against them. To find effective antiviral compounds against enterovirus B group viruses, an in-house chemical library was screened. The most effective compounds against Coxsackieviruses B3 (CVB3) and A9 (CVA9) were CL212 and CL213, two N-phenyl benzamides. Both compounds were more effective against CVA9 and CL213 gave a better EC50 value of 1 µM with high a specificity index of 140. Both drugs were most effective when incubated directly with viruses suggesting that they mainly bound to the virions. A real-time uncoating assay showed that the compounds stabilized the virions and radioactive sucrose gradient as well as TEM confirmed that the viruses stayed intact. A docking assay, taking into account larger areas around the 2-and 3-fold axes of CVA9 and CVB3, suggested that the hydrophobic pocket gives the strongest binding to CVA9 but revealed another binding site around the 3-fold axis which could contribute to the binding of the compounds. Together, our data support a direct antiviral mechanism against the virus capsid and suggest that the compounds bind to the hydrophobic pocket and 3-fold axis area resulting in the stabilization of the virion.

MmpL3 is the cellular target of the antitubercular pyrrole derivative BM212.

The 1,5-diarylpyrrole derivative BM212 was previously shown to be active against multidrug-resistant clinical isolates and Mycobacterium tuberculosis residing within macrophages as well as against Mycobacterium avium and other atypical mycobacteria. To determine its mechanism of action, we identified the cellular target. Spontaneous Mycobacterium smegmatis, Mycobacterium bovis BCG, and M. tuberculosis H37Rv mutants that were resistant to BM212 were isolated. By the screening of genomic libraries and by whole-genome sequencing, we found that all the characterized mutants showed mutations in the mmpL3 gene, allowing us to conclude that resistance to BM212 maps to the MmpL3 protein, a member of the MmpL (mycobacterial membrane protein, large) family. Susceptibility was unaffected by the efflux pump inhibitors reserpine, carbonylcyanide m-chlorophenylhydrazone, and verapamil. Uptake/efflux experiments with [(14)C]BM212 demonstrated that resistance is not driven by the efflux of BM212. Together, these data strongly suggest that the MmpL3 protein is the cellular target of BM212.

Malaria transmission blocking compounds: a patent review.

INTRODUCTION: Despite substantial progress in the field, malaria remains a global health issue and currently available control strategies are not sufficient to achieve eradication. Agents able to prevent transmission are likely to have a strong impact on malaria control and have been prioritized as a primary objective to reduce the number of secondary infections. Therefore, there is an increased interest in finding novel drugs targeting sexual stages of Plasmodium and innovative methods to target malaria transmission from host to vector, and vice versa. AREAS COVERED: This review covers innovative transmission-blocking inventions patented between 2015 and October 2021. The focus is on chemical interventions, which could be used as 'chemical vaccines' to prevent transmission (small molecules, carbohydrates, and polypeptides). EXPERT OPINION: Even though the development of novel strategies to block transmission still requires fundamental additional research and a deeper understanding of parasite sexual stages biology, the research in this field has significantly accelerated. Among innovative inventions patented over the last 6 years, the surface-delivery of antimalarial drugs to kill transmission-stages parasites in mosquitoes holds the highest promise for success in malaria control strategies, opening completely new scenarios in malaria transmission-blocking drug discovery.

Nano-Based Drug Delivery Systems of Potent MmpL3 Inhibitors for Tuberculosis Treatment.

Tuberculosis remains one of the world's deadliest infectious diseases, accounting for nearly 1.3 million deaths every year. Tuberculosis treatment is challenging because of the toxicity, decreased bioavailability at the target site of the conventional drugs and, most importantly, low adherence of patients; this leads to drug resistance. Here, we describe the development of suitable nanocarriers with specific physicochemical properties to efficiently deliver two potent antimycobacterial compounds. We prepared nanoemulsions and niosomes formulations and loaded them with two different MmpL3 inhibitors previously identified (NEs + BM635 and NIs + BM859). NEs + BM635 and NIs + BM859 were deeply characterized for their physicochemical properties and anti-mycobacterial activity. NEs + BM635 and NIs + BM859 showed good hydrodynamic diameter, ζ-Potential, PDI, drug-entrapment efficiency, polarity, and microviscosity and stability. Even though both formulations proved to perform well, only NIs + BM859 showed potent antimycobacterial activity against M. tuberculosis (MIC = 0.6 µM) compared to that of the free compound. This is most probably caused by the fact that BM635, being highly hydrophobic, encounters maximum hindrance in diffusion, whereas BM859, characterized by high solubility in aqueous medium (152 µM), diffuses more easily. The niosomal formulation described in this work may be a useful therapeutic tool for tuberculosis treatment, and further studies will follow to characterize the in vivo behavior of the formulation.

A Novel Class of Dual-Acting DCH-CORMs Counteracts Oxidative Stress-Induced Inflammation in Human Primary Tenocytes.

Carbon monoxide (CO) can prevent cell and tissue damage by restoring redox homeostasis and counteracting inflammation. CO-releasing molecules (CORMs) can release a controlled amount of CO to cells and are emerging as a safer therapeutic alternative to delivery of CO in vivo. Sustained oxidative stress and inflammation can cause chronic pain and disability in tendon-related diseases, whose therapeutic management is still a challenge. In this light, we developed three small subsets of 1,5-diarylpyrrole and pyrazole dicobalt(0)hexacarbonyl (DCH)-CORMs to assess their potential use in musculoskeletal diseases. A myoglobin-based spectrophotometric assay showed that these CORMs act as slow and efficient CO-releasers. Five selected compounds were then tested on human primary-derived tenocytes before and after hydrogen peroxide stimulation to assess their efficacy in restoring cell redox homeostasis and counteracting inflammation in terms of PGE2 secretion. The obtained results showed an improvement in tendon homeostasis and a cytoprotective effect, reflecting their activity as CO-releasers, and a reduction of PGE2 secretion. As these compounds contain structural fragments of COX-2 selective inhibitors, we hypothesized that such a composite mechanism of action results from the combination of CO-release and COX-2 inhibition and that these compounds might have a potential role as dual-acting therapeutic agents in tendon-derived diseases.

6-Fluorophenylbenzohydrazides inhibit Mycobacterium tuberculosis growth through alteration of tryptophan biosynthesis.

A major constraint in reducing tuberculosis epidemic is the emergence of strains resistant to one or more of clinically approved antibiotics, which emphasizes the need of novel drugs with novel targets. Genetic knockout strains of Mycobacterium tuberculosis (Mtb) have established that tryptophan (Trp) biosynthesis is essential for the bacterium to survive in vivo and cause disease in animal models. An anthranilate-like compound, 6-FABA, was previously shown to synergize with the host immune response to Mtb infection in vivo. Herein, we present a class of anthranilate-like compounds endowed with good antimycobacterial activity and low cytotoxicity. We show how replacing the carboxylic moiety with a hydrazide led to a significant improvement in both activity and cytotoxicity relative to the parent compound 6-FABA. Several new benzohydrazides (compounds 20-31, 33, 34, 36, 38 and 39) showed good activities against Mtb (0.625 ≤ MIC≤6.25 µM) and demonstrated no detectable cytotoxicity against Vero cell assay (CC50 ≥ 1360 µM). The target preliminary studies confirmed the hypothesis that this new class of compounds inhibits Trp biosynthesis. Taken together, these findings indicate that fluorophenylbenzohydrazides represent good candidates to be assessed for drug discovery.

Therapeutic potential for coxibs-nitric oxide releasing hybrids in cystic fibrosis.

This review discusses the rational for further studies of COX-2 inhibitors-NO releaser hybrids (NO-Coxibs) in the pharmacological treatment of the airway inflammation in Cystic Fibrosis (CF). Our research group developed several classes of NO-Coxibs for the pharmacological treatment of arthritis, and among them several compounds showed an outstanding in vivo efficacy and good pharmacokinetic properties. The good antiinflammatory properties displayed by these compounds during the previous screening could, by itself, suggest appropriate candidates for further testing in CF.

One-pot conversions of olefins to cyclic carbonates and secondary allylic and homoallylic amines to cyclic carbamates.

Sequential treatment of a 1,2-disubstituted olefin with m-CPBA, Br3CCO2H, and DBU results in the one-pot, stereospecific conversion of the olefin to the corresponding disubstituted cyclic carbonate (1,3-dioxolan-2-one). The reaction proceeds via an initial epoxidation followed by S(N)2-type epoxide ring opening by Br3CCO2H and subsequent base-promoted carbonate formation upon elimination of bromoform. When a solution of a secondary allylic or homoallylic amine and Br3CCO2H is sequentially treated with m-CPBA then DBU, the product of the reaction is a cyclic carbamate (1,3-oxazolidin-2-one or 1,3-oxazinan-2-one).