Expanding the knowledge around antitubercular 5-(2-aminothiazol-4-yl)isoxazole-3-carboxamides: Hit-to-lead optimization and release of a novel antitubercular chemotype via scaffold derivatization.

Tuberculosis is one of the deadliest infectious diseases in the world, and the increased number of multidrug-resistant and extensively drug-resistant strains is a reason for concern. We have previously reported a series of substituted 5-(2-aminothiazol-4-yl)isoxazole-3-carboxamides with growth inhibitory activity against Mycobacterium tuberculosis strains and low propensity to be substrate of efflux pumps. Encouraged by these preliminary results, we have undertaken a medicinal chemistry campaign to determine the metabolic fate of these compounds and to delineate a reliable body of Structure-Activity Relationships. Keeping intact the (thiazol-4-yl)isoxazole-3-carboxamide core, as it is deemed to be the pharmacophore of the molecule, we have extensively explored the structural modifications able to confer good activity and avoid rapid clearance. Also, a small set of analogues based on isostere manipulation of the 2-aminothiazole were prepared and tested, with the aim to disclose novel antitubercular chemotypes. These studies, combined, were instrumental in designing improved compounds such as 42g and 42l, escaping metabolic degradation by human liver microsomes and, at the same time, maintaining good antitubercular activity against both drug-susceptible and drug-resistant strains.

Identification of Human Alanine-Glyoxylate Aminotransferase Ligands as Pharmacological Chaperones for Variants Associated with Primary Hyperoxaluria Type 1.

Primary hyperoxaluria type I (PH1) is a rare kidney disease due to the deficit of alanine:glyoxylate aminotransferase (AGT), a pyridoxal-5'-phosphate-dependent enzyme responsible for liver glyoxylate detoxification, which in turn prevents oxalate formation and precipitation as kidney stones. Many PH1-associated missense mutations cause AGT misfolding. Therefore, the use of pharmacological chaperones (PCs), small molecules that promote correct folding, represents a useful therapeutic option. To identify ligands acting as PCs for AGT, we first performed a small screening of commercially available compounds. We tested each molecule by a dual approach aimed at defining the inhibition potency on purified proteins and the chaperone activity in cells expressing a misfolded variant associated with PH1. We then performed a chemical optimization campaign and tested the resulting synthetic molecules using the same approach. Overall, the results allowed us to identify a promising hit compound for AGT and draw conclusions about the requirements for optimal PC activity.

Inhibitors of O-Acetylserine Sulfhydrylase with a Cyclopropane-Carboxylic Acid Scaffold Are Effective Colistin Adjuvants in Gram Negative Bacteria.

Antibacterial adjuvants are of great significance, since they allow one to downscale the therapeutic dose of conventional antibiotics and reduce the insurgence of antibacterial resistance. Herein, we report that O-acetylserine sulfhydrylase (OASS) inhibitors could be used as colistin adjuvants to treat infections caused by critical pathogens spreading worldwide, Escherichia coli, Salmonella enterica serovar Typhimurium, and Klebsiella pneumoniae. Starting from a hit compound endowed with a nanomolar dissociation constant, we have rationally designed and synthesized a series of derivatives to be tested against S. Typhimurium OASS isoenzymes, StOASS-A and StOASS-B. All acidic derivatives have shown good activities in the nanomolar range against both OASS isoforms in vitro. Minimal Inhibitory Concentrations (MICs) were then evaluated, as well as compounds' toxicity. The compounds endowed with good activity in vitro and low cytotoxicity have been challenged as a potential colistin adjuvant against pathogenic bacteria in vitro and the fractional inhibitory concentration (FIC) index has been calculated to define additive or synergistic effects. Finally, the target engagement inside the S. Typhimurium cells was confirmed by using a mutant strain in which the OASS enzymes were inactivated. Our results provide a robust proof of principle supporting OASS as a potential nonessential antibacterial target to develop a new class of adjuvants.

Crystal structure of Aspergillus fumigatus AroH, an aromatic amino acid aminotransferase.

Aspergillus fumigatus is a saprophytic ubiquitous fungus whose spores can trigger reactions such as allergic bronchopulmonary aspergillosis or the fatal invasive pulmonary aspergillosis. To survive in the lungs, the fungus must adapt to a hypoxic and nutritionally restrictive environment, exploiting the limited availability of aromatic amino acids (AAAs) in the best possible way, as mammals do not synthesize them. A key enzyme for AAAs catabolism in A. fumigatus is AroH, a pyridoxal 5'-phosphate-dependent aromatic aminotransferase. AroH was recently shown to display a broad substrate specificity, accepting L-kynurenine and α-aminoadipate as amino donors besides AAAs. Given its pivotal role in the adaptability of the fungus to nutrient conditions, AroH represents a potential target for the development of innovative therapies against A. fumigatus-related diseases. We have solved the crystal structure of Af-AroH at 2.4 Å resolution and gained new insight into the dynamics of the enzyme's active site, which appears to be crucial for the design of inhibitors. The conformational plasticity of the active site pocket is probably linked to the wide substrate specificity of AroH.

Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

Discovery of Substituted (2-Aminooxazol-4-yl)Isoxazole-3-carboxylic Acids as Inhibitors of Bacterial Serine Acetyltransferase in the Quest for Novel Potential Antibacterial Adjuvants.

Many bacteria and actinomycetales use L-cysteine biosynthesis to increase their tolerance to antibacterial treatment and establish a long-lasting infection. In turn, this might lead to the onset of antimicrobial resistance that currently represents one of the most menacing threats to public health worldwide. The biosynthetic machinery required to synthesise L-cysteine is absent in mammals; therefore, its exploitation as a drug target is particularly promising. In this article, we report a series of inhibitors of Salmonella thyphimurium serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of L-cysteine biosynthesis. The development of such inhibitors started with the virtual screening of an in-house library of compounds that led to the selection of seven structurally unrelated hit derivatives. A set of molecules structurally related to hit compound 5, coming either from the original library or from medicinal chemistry efforts, were tested to determine a preliminary structure-activity relationship and, especially, to improve the inhibitory potency of the derivatives, that was indeed ameliorated by several folds compared to hit compound 5 Despite these progresses, at this stage, the most promising compound failed to interfere with bacterial growth when tested on a Gram-negative model organism, anticipating the need for further research efforts.

Aspergillus fumigatus tryptophan metabolic route differently affects host immunity.

Indoleamine 2,3-dioxygenases (IDOs) degrade l-tryptophan to kynurenines and drive the de novo synthesis of nicotinamide adenine dinucleotide. Unsurprisingly, various invertebrates, vertebrates, and even fungi produce IDO. In mammals, IDO1 also serves as a homeostatic regulator, modulating immune response to infection via local tryptophan deprivation, active catabolite production, and non-enzymatic cell signaling. Whether fungal Idos have pleiotropic functions that impact on host-fungal physiology is unclear. Here, we show that Aspergillus fumigatus possesses three ido genes that are expressed under conditions of hypoxia or tryptophan abundance. Loss of these genes results in increased fungal pathogenicity and inflammation in a mouse model of aspergillosis, driven by an alternative tryptophan degradation pathway to indole derivatives and the host aryl hydrocarbon receptor. Fungal tryptophan metabolic pathways thus cooperate with the host xenobiotic response to shape host-microbe interactions in local tissue microenvironments.

Investigational Studies on a Hit Compound Cyclopropane-Carboxylic Acid Derivative Targeting O-Acetylserine Sulfhydrylase as a Colistin Adjuvant.

Antibacterial adjuvants are of great significance, since they allow the therapeutic dose of conventional antibiotics to be lowered and reduce the insurgence of antibiotic resistance. Herein, we report that an O-acetylserine sulfhydrylase (OASS) inhibitor can be used as a colistin adjuvant to treat infections caused by Gram-positive and Gram-negative pathogens. A compound that binds OASS with a nM dissociation constant was tested as an adjuvant of colistin against six critical pathogens responsible for infections spreading worldwide, Escherichia coli, Salmonella enterica serovar Typhimurium, Klebisiella pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Staphylococcus pseudintermedius. The compound showed promising synergistic or additive activities against all of them. Knockout experiments confirmed the intracellular target engagement supporting the proposed mechanism of action. Moreover, compound toxicity was evaluated by means of its hemolytic activity against sheep defibrinated blood cells, showing a good safety profile. The 3D structure of the compound in complex with OASS was determined at 1.2 Å resolution by macromolecular crystallography, providing for the first time structural insights about the nature of the interaction between the enzyme and this class of competitive inhibitors. Our results provide a robust proof of principle supporting OASS as a potential nonessential antibacterial target to develop a new class of adjuvants and the structural basis for further structure-activity relationship studies.

Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

Nitric oxide-releasing cyclodextrins as biodegradable antibacterial scaffolds: a patent evaluation of US2019343869(A1).

INTRODUCTION: Antimicrobial resistance is one of the major scourges for health care worldwide; therefore, novel investigational approaches are needed to potentiate and preserve the current antibacterial arsenal. Cyclodextrins are known to improve the formulability of several different therapeutic agents. When functionalized with nitric oxide (NO) releasing groups, and suitably loaded with an antibacterial or antitumoral agents, they can exert additive activity, especially toward certain bacterial strains and cell cancer lines. AREAS COVERED: US2019343869 describes NO-releasing cyclodextrins, a method for their synthesis, a composition that is based on them, and their application as anticancer or antibacterial agents, especially toward planktonic P. aeruginosa and the biofilm resulting from infection. Anticancer activity is measured against A549 cells. The amount of NO released is in the range of 0.5 µmol to 2.5 µmol per milligram of functionalized cyclodextrin with a half-life for NO release in a range of between about 0.7-4.2 hours. EXPERT OPINION: The results support the use of NO-releasing cyclodextrins as a matrix for the delivery of antibacterial and anticancer drugs in a suitable formulation. However, antibacterial activity seems to be weak, and more focused studies are needed.

2-Aminooxazole as a Novel Privileged Scaffold in Antitubercular Medicinal Chemistry.

To obtain effective eradication of numerous infectious diseases such as tuberculosis, it is important to supply the medicinal chemistry arsenal with novel chemical agents. Isosterism and bioisosterism are widely known concepts in the field of early drug discovery, and in several cases, rational isosteric replacements have contributed to improved efficacy and physicochemical characteristics throughout the hit-to-lead optimization process. However, sometimes the synthesis of isosteres might not be as straightforward as that of the parent compounds, and therefore, novel synthetic strategies must be elaborated. In this regard, we herein report the evaluation of a series of N-substituted 4-phenyl-2-aminooxazoles that, despite being isosteres of a widely used nucleus such as the 2-aminothiazole, have been only seldom explored. After elaboration of a convenient synthetic strategy, a small set of 2-aminothiazoles and their 2-aminooxazole counterparts were compared with regard to antitubercular activity and physicochemical characteristics.

Inhibition of Nonessential Bacterial Targets: Discovery of a Novel Serine O-Acetyltransferase Inhibitor.

In ϒ-proteobacteria and Actinomycetales, cysteine biosynthetic enzymes are indispensable during persistence and become dispensable during growth or acute infection. The biosynthetic machinery required to convert inorganic sulfur into cysteine is absent in mammals; therefore, it is a suitable drug target. We searched for inhibitors of Salmonella serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of l-cysteine biosynthesis. The virtual screening of three ChemDiv focused libraries containing 91 243 compounds was performed to identify potential SAT inhibitors. Scaffold similarity and the analysis of the overall physicochemical properties allowed the selection of 73 compounds that were purchased and evaluated on the recombinant enzyme. Six compounds displaying an IC50 <100 µM were identified via an indirect assay using Ellman's reagent and then tested on a Gram-negative model organism, with one of them being able to interfere with bacterial growth via SAT inhibition.

Cycloserine enantiomers are reversible inhibitors of human alanine:glyoxylate aminotransferase: implications for Primary Hyperoxaluria type 1.

Peroxisomal alanine:glyoxylate aminotransferase (AGT) is responsible for glyoxylate detoxification in human liver and utilizes pyridoxal 5'-phosphate (PLP) as coenzyme. The deficit of AGT leads to Primary Hyperoxaluria Type I (PH1), a rare disease characterized by calcium oxalate stones deposition in the urinary tract as a consequence of glyoxylate accumulation. Most missense mutations cause AGT misfolding, as in the case of the G41R, which induces aggregation and proteolytic degradation. We have investigated the interaction of wild-type AGT and the pathogenic G41R variant with d-cycloserine (DCS, commercialized as Seromycin), a natural product used as a second-line treatment of multidrug-resistant tuberculosis, and its synthetic enantiomer l-cycloserine (LCS). In contrast with evidences previously reported on other PLP-enzymes, both ligands are AGT reversible inhibitors showing inhibition constants in the micromolar range. While LCS undergoes half-transamination generating a ketimine intermediate and behaves as a classical competitive inhibitor, DCS displays a time-dependent binding mainly generating an oxime intermediate. Using a mammalian cellular model, we found that DCS, but not LCS, is able to promote the correct folding of the G41R variant, as revealed by its increased specific activity and expression as a soluble protein. This effect also translates into an increased glyoxylate detoxification ability of cells expressing the variant upon treatment with DCS. Overall, our findings establish that DCS could play a role as pharmacological chaperone, thus suggesting a new line of intervention against PH1 based on a drug repositioning approach. To a widest extent, this strategy could be applied to other disease-causing mutations leading to AGT misfolding.

Sodium Hyaluronate Nanocomposite Respirable Microparticles to Tackle Antibiotic Resistance with Potential Application in Treatment of Mycobacterial Pulmonary Infections.

Tuberculosis resistant cases have been estimated to grow every year. Besides Mycobacterium tuberculosis, other mycobacterial species are responsible for an increasing number of difficult-to-treat infections. To increase efficacy of pulmonary treatment of mycobacterial infections an inhalable antibiotic powder targeting infected alveolar macrophages (AMs) and including an efflux pump inhibitor was developed. Low molecular weight sodium hyaluronate sub-micron particles were efficiently loaded with rifampicin, isoniazid and verapamil, and transformed in highly respirable microparticles (mean volume diameter: 1 µm) by spray drying. These particles were able to regenerate their original size upon contact with aqueous environment with mechanical stirring or sonication. The in vitro drugs release profile from the powder was characterized by a slow release rate, favorable to maintain a high drug level inside AMs. In vitro antimicrobial activity and ex vivo macrophage infection assays employing susceptible and drug resistant strains were carried out. No significant differences were observed when the powder, which did not compromise the AMs viability after a five-day exposure, was compared to the same formulation without verapamil. However, both preparations achieved more than 80% reduction in bacterial viability irrespective of the drug resistance profile. This approach can be considered appropriate to treat mycobacterial respiratory infections, regardless the level of drug resistance.

Refining the structure-activity relationships of 2-phenylcyclopropane carboxylic acids as inhibitors of O-acetylserine sulfhydrylase isoforms.

The lack of efficacy of current antibacterials to treat multidrug resistant bacteria poses a life-threatening alarm. In order to develop enhancers of the antibacterial activity, we carried out a medicinal chemistry campaign aiming to develop inhibitors of enzymes that synthesise cysteine and belong to the reductive sulphur assimilation pathway, absent in mammals. Previous studies have provided a novel series of inhibitors for O-acetylsulfhydrylase - a key enzyme involved in cysteine biosynthesis. Despite displaying nanomolar affinity, the most active representative of the series was not able to interfere with bacterial growth, likely due to poor permeability. Therefore, we rationally modified the structure of the hit compound with the aim of promoting their passage through the outer cell membrane porins. The new series was evaluated on the recombinant enzyme from Salmonella enterica serovar Typhimurium, with several compounds able to keep nanomolar binding affinity despite the extent of chemical manipulation.

Biochemical Characterization of Aspergillus fumigatus AroH, a Putative Aromatic Amino Acid Aminotransferase.

The rise in the frequency of nosocomial infections is becoming a major problem for public health, in particular in immunocompromised patients. Aspergillus fumigatus is an opportunistic fungus normally present in the environment directly responsible for lethal invasive infections. Recent results suggest that the metabolic pathways related to amino acid metabolism can regulate the fungus-host interaction and that an important role is played by enzymes involved in the catabolism of L-tryptophan. In particular, in A. fumigatus L-tryptophan regulates Aro genes. Among them, AroH encodes a putative pyridoxal 5'-phosphate-dependent aminotransferase. Here we analyzed the biochemical features of recombinant purified AroH by spectroscopic and kinetic analyses corroborated by in silico studies. We found that the protein is dimeric and tightly binds the coenzyme forming a deprotonated internal aldimine in equilibrium with a protonated ketoenamine form. By setting up a new rapid assay method, we measured the kinetic parameters for the overall transamination of substrates and we demonstrated that AroH behaves as an aromatic amino acid aminotransferase, but also accepts L-kynurenine and α-aminoadipate as amino donors. Interestingly, computational approaches showed that the predicted overall fold and active site topology of the protein are similar to those of its yeast ortholog, albeit with some differences in the regions at the entrance of the active site, which could possibly influence substrate specificity. Should targeting fungal metabolic adaptation be of therapeutic value, the results of the present study may pave the way to the design of specific AroH modulators as potential novel agents at the host/fungus interface.

Discovering a new class of antifungal agents that selectively inhibits microbial carbonic anhydrases.

Infections caused by pathogens resistant to the available antimicrobial treatments represent nowadays a threat to global public health. Recently, it has been demonstrated that carbonic anhydrases (CAs) are essential for the growth of many pathogens and their inhibition leads to growth defects. Principal drawbacks in using CA inhibitors (CAIs) as antimicrobial agents are the side effects due to the lack of selectivity toward human CA isoforms. Herein we report a new class of CAIs, which preferentially interacts with microbial CA active sites over the human ones. The mechanism of action of these inhibitors was investigated against an important fungal pathogen, Cryptococcus neoformans, revealing that they are also able to inhibit CA in microbial cells growing in vitro. At our best knowledge, this is the first report on newly designed synthetic compounds selectively targeting ß-CAs and provides a proof of concept of microbial CAs suitability as an antimicrobial drug target.

Discovery of novel fragments inhibiting O-acetylserine sulphhydrylase by combining scaffold hopping and ligand-based drug design.

Several bacteria rely on the reductive sulphur assimilation pathway, absent in mammals, to synthesise cysteine. Reduction of virulence and decrease in antibiotic resistance have already been associated with mutations on the genes that codify cysteine biosynthetic enzymes. Therefore, inhibition of cysteine biosynthesis has emerged as a promising strategy to find new potential agents for the treatment of bacterial infection. Following our previous efforts to explore OASS inhibition and to expand and diversify our library, a scaffold hopping approach was carried out, with the aim of identifying a novel fragment for further development. This novel chemical tool, endowed with favourable pharmacological characteristics, was successfully developed, and a preliminary Structure-Activity Relationship investigation was carried out.

Adjuvant therapies against tuberculosis: discovery of a 2-aminothiazole targeting Mycobacterium tuberculosis energetics.

AIM: To evaluate the activity of the 2-aminothiazole UPAR-174 following an unexplored approach: targeting Mycobacterium tuberculosis with lipophilic compounds that present antituberculosis and efflux inhibitory activity. METHODS: Antituberculosis activity was assessed against replicating, nonreplicating and intracellular bacilli. Its capacity to inhibit active efflux was determined. ATP quantification and membrane potential analysis were performed. Intracellular activity was studied on human-monocyte-derived macrophages. RESULTS: UPAR-174 is an efflux inhibitor active against replicating, nonreplicating and intracellular M. tuberculosis. It dissipates the membrane potential and causes ATP depletion. CONCLUSION: Targeting M. tuberculosis with lipophilic efflux inhibitors, exploring their dual activity - dissipation of the proton motive force and efflux inhibition - represents an attractive strategy to fight against drug-resistant tuberculosis.

Challenging the Drug-Likeness Dogma for New Drug Discovery in Tuberculosis.

The emergence of multi- and extensively drug resistant tuberculosis worldwide poses a great threat to human health and highlight the need to discover and develop new, effective and inexpensive antituberculosis agents. High-throughput screening assays against well-validated drug targets and structure based drug design have been employed to discover new lead compounds. However, the great majority fail to demonstrate any antimycobacterial activity when tested against Mycobacterium tuberculosis in whole-cell screening assays. This is mainly due to some of the intrinsic properties of the bacilli, such as the extremely low permeability of its cell wall, slow growth, drug resistance, drug tolerance, and persistence. In this sense, understanding the pathways involved in M. tuberculosis drug tolerance, persistence, and pathogenesis, may reveal new approaches for drug development. Moreover, the need for compounds presenting a novel mode of action is of utmost importance due to the emergence of resistance not only to the currently used antituberculosis agents, but also to those in the pipeline. Cheminformatics studies have shown that drugs endowed with antituberculosis activity have the peculiarity of being more lipophilic than many other antibacterials, likely because this leads to improved cell penetration through the extremely waxy mycobacterial cell wall. Moreover, the interaction of the lipophilic moiety with the membrane alters its stability and functional integrity due to the disruption of the proton motive force, resulting in cell death. When a ligand-based medicinal chemistry campaign is ongoing, it is always difficult to predict whether a chemical modification or a functional group would be suitable for improving the activity. Nevertheless, in the "instruction manual" of medicinal chemists, certain functional groups or certain physicochemical characteristics (i.e., high lipophilicity) are considered red flags to look out for in order to safeguard drug-likeness and avoid attritions in the drug discovery process. In this review, we describe how antituberculosis compounds challenge established rules such as the Lipinski's "rule of five" and how medicinal chemistry for antituberculosis compounds must be thought beyond such dogmatic schemes.