Publications by authors named "Marco Pieroni"

47 Publications

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

ACS Infect Dis 2021 02 29;7(2):281-292. Epub 2021 Jan 29.

P4T Group, Department of Food and Drugs, University of Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy.

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 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, , serovar Typhimurium, , , methicillin-resistant , and . 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.
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http://dx.doi.org/10.1021/acsinfecdis.0c00378DOI Listing
February 2021

Aspergillus fumigatus tryptophan metabolic route differently affects host immunity.

Cell Rep 2021 Jan;34(4):108673

Department of Medicine and Surgery, University of Perugia, 06132 Perugia, Italy.

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.
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http://dx.doi.org/10.1016/j.celrep.2020.108673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7844877PMC
January 2021

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

Authors:
Marco Pieroni

Expert Opin Ther Pat 2020 Dec 22;30(12):901-905. Epub 2020 Sep 22.

P4T Group, University of Parma , Parma, Italy.

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 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.
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http://dx.doi.org/10.1080/13543776.2020.1822328DOI Listing
December 2020

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

ACS Med Chem Lett 2020 Jul 8;11(7):1435-1441. Epub 2020 Jun 8.

P4T group and Food and Drug Department, University of Parma, 43124 Parma, Italy.

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.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357219PMC
July 2020

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

ACS Med Chem Lett 2020 May 13;11(5):790-797. Epub 2020 Feb 13.

P4T group and Laboratory of Biochemistry and Molecular Biology, Department of Food and Drug, University of Parma, 43124 Parma, Italy.

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 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 IC <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.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236226PMC
May 2020

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

Biochem J 2019 12;476(24):3751-3768

Department of Experimental Medicine, University of Perugia, Perugia, Italy.

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.
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http://dx.doi.org/10.1042/BCJ20190507DOI Listing
December 2019

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

Pharmaceutics 2019 May 1;11(5). Epub 2019 May 1.

Food and Drug Department, University of Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy.

Tuberculosis resistant cases have been estimated to grow every year. Besides , 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.
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http://dx.doi.org/10.3390/pharmaceutics11050203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6571635PMC
May 2019

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

Front Mol Biosci 2018 28;5:104. Epub 2018 Nov 28.

Department of Experimental Medicine, University of Perugia, Perugia, Italy.

The rise in the frequency of nosocomial infections is becoming a major problem for public health, in particular in immunocompromised patients. 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 L-tryptophan regulates 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 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.
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http://dx.doi.org/10.3389/fmolb.2018.00104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279937PMC
November 2018

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

J Enzyme Inhib Med Chem 2019 Dec;34(1):31-43

a P4T group, Department of Food and Drug, University of Parma, Parma, Italy.

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.
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http://dx.doi.org/10.1080/14756366.2018.1518959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217552PMC
December 2019

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

J Enzyme Inhib Med Chem 2018 Dec;33(1):1537-1544

a Department of Food and Drugs , University of Parma , Parma , Italy.

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 β-CAs and provides a proof of concept of microbial CAs suitability as an antimicrobial drug target.
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http://dx.doi.org/10.1080/14756366.2018.1516652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6179086PMC
December 2018

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

Future Microbiol 2018 09 27;13:1383-1402. Epub 2018 Sep 27.

Global Health & Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Lisboa, Portugal.

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.
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http://dx.doi.org/10.2217/fmb-2018-0110DOI Listing
September 2018

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

J Enzyme Inhib Med Chem 2018 Dec;33(1):1444-1452

b Laboratory of Biochemistry and Molecular Biology, Department of Food and Drug , University of Parma , Parma , Italy.

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.
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http://dx.doi.org/10.1080/14756366.2018.1512596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6147075PMC
December 2018

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

Front Microbiol 2018 3;9:1367. Epub 2018 Jul 3.

P4T Group, Department of Food and Drug, University of Parma, Parma, Italy.

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 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 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.
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http://dx.doi.org/10.3389/fmicb.2018.01367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6037898PMC
July 2018

Modulation of bacterial metabolism by the microenvironment controls MAIT cell stimulation.

Mucosal Immunol 2018 07 9;11(4):1060-1070. Epub 2018 May 9.

Experimental Immunology, Department of Biomedicine, University and University Hospital Basel, 4031, Basel, Switzerland.

Mucosal-associated invariant T (MAIT) cells are abundant innate-like T lymphocytes in mucosal tissues and recognize a variety of riboflavin-related metabolites produced by the microbial flora. Relevant issues are whether MAIT cells are heterogeneous in the colon, and whether the local environment influences microbial metabolism thereby shaping MAIT cell phenotypes and responses. We found discrete MAIT cell populations in human colon, characterized by the diverse expression of transcription factors, cytokines and surface markers, indicative of activated and precisely controlled lymphocyte populations. Similar phenotypes were rare among circulating MAIT cells and appeared when circulating MAIT cells were stimulated with the synthetic antigens 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil, and 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil. Furthermore, bacteria grown in colon-resembling conditions with low oxygen tension and harvested at stationary growth phase, potently activated human MAIT cells. The increased activation correlated with accumulation of the above antigenic metabolites as indicated by mass spectrometry. Thus, the colon environment contributes to mucosal immunity by directly affecting bacterial metabolism, and indirectly controlling the stimulation and differentiation of MAIT cells.
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http://dx.doi.org/10.1038/s41385-018-0020-9DOI Listing
July 2018

Efflux Activity Differentially Modulates the Levels of Isoniazid and Rifampicin Resistance among Multidrug Resistant and Monoresistant Mycobacterium tuberculosis Strains.

Antibiotics (Basel) 2018 Mar 3;7(1). Epub 2018 Mar 3.

Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade NOVA de Lisboa, UNL, Lisboa 1349-008, Portugal.

With the growing body of knowledge on the contribution of efflux activity to drug resistance, increased attention has been given to the use of efflux inhibitors as adjuvants of tuberculosis therapy. Here, we investigated how efflux activity modulates the levels of efflux between monoresistant and multi- and extensively drug resistant (M/XDR) clinical isolates. The strains were characterized by antibiotic susceptibility testing in the presence/absence of efflux inhibitors, molecular typing, and genetic analysis of drug-resistance-associated genes. Efflux activity was quantified by real-time fluorometry. The results demonstrated that all the clinical strains, susceptible or resistant, presented a faster, rapid, and non-specific efflux-mediated short-term response to drugs. The synergism assays demonstrated that the efflux inhibitors were more effective in reducing the resistance levels in the M/XDR strains than in the monoresistant strains. This indicated that M/XDR strains presented a more prolonged response to drugs mediated by efflux compared to the monoresistant strains, but both maintain it as a long-term stress response. This work shows that efflux activity modulates the levels of drug resistance between monoresistant and M/XDR clinical strains, allowing the bacteria to survive in the presence of noxious compounds.
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http://dx.doi.org/10.3390/antibiotics7010018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5872129PMC
March 2018

Integration of Enhanced Sampling Methods with Saturation Transfer Difference Experiments to Identify Protein Druggable Pockets.

J Chem Inf Model 2018 03 6;58(3):710-723. Epub 2018 Mar 6.

Food and Drug Department , P4T group , Parco Area Delle Scienze 27/A - 43124 , Parma , Italy.

Saturation transfer difference (STD) is an NMR technique conventionally applied in drug discovery to identify ligand moieties relevant for binding to protein cavities. This is important to direct medicinal chemistry efforts in small-molecule optimization processes. However, STD does not provide any structural details about the ligand-target complex under investigation. Herein, we report the application of a new integrated approach, which combines enhanced sampling methods with STD experiments, for the characterization of ligand-target complexes that are instrumental for drug design purposes. As an example, we have studied the interaction between StOASS-A, a potential antibacterial target, and an inhibitor previously reported. This approach allowed us to consider the ligand-target complex from a dynamic point of view, revealing the presence of an accessory subpocket which can be exploited to design novel StOASS-A inhibitors. As a proof of concept, a small library of derivatives was designed and evaluated in vitro, displaying the expected activity.
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http://dx.doi.org/10.1021/acs.jcim.7b00733DOI Listing
March 2018

Substituted N-Phenyl-5-(2-(phenylamino)thiazol-4-yl)isoxazole-3-carboxamides Are Valuable Antitubercular Candidates that Evade Innate Efflux Machinery.

J Med Chem 2017 08 10;60(16):7108-7122. Epub 2017 Aug 10.

Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL , Rua da Junqueira, 100, 1349-008 Lisbon, Portugal.

Tuberculosis remains one of the deadliest infectious diseases in the world, and the increased number of multidrug-resistant and extremely drug-resistant strains is a significant reason for concern. This makes the discovery of novel antitubercular agents a cogent priority. We have previously addressed this need by reporting a series of substituted 2-aminothiazoles capable to inhibit the growth of actively replicating, nonreplicating persistent, and resistant Mycobacterium tuberculosis strains. Clues from the structure-activity relationships lining up the antitubercular activity were exploited for the rational design of improved analogues. Two compounds, namely N-phenyl-5-(2-(p-tolylamino)thiazol-4-yl)isoxazole-3-carboxamide 7a and N-(pyridin-2-yl)-5-(2-(p-tolylamino)thiazol-4-yl)isoxazole-3-carboxamide 8a, were found to show high inhibitory activity toward susceptible M. tuberculosis strains, with an MIC of 0.125-0.25 μg/mL (0.33-0.66 μM) and 0.06-0.125 μg/mL (0.16-0.32 μM), respectively. Moreover, they maintained good activity also toward resistant strains, and they were selective over other bacterial species and eukaryotic cells, metabolically stable, and apparently not susceptible to the action of efflux pumps.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00793DOI Listing
August 2017

Accepting the Invitation to Open Innovation in Malaria Drug Discovery: Synthesis, Biological Evaluation, and Investigation on the Structure-Activity Relationships of Benzo[b]thiophene-2-carboxamides as Antimalarial Agents.

J Med Chem 2017 03 14;60(5):1959-1970. Epub 2017 Feb 14.

Centro Interdipartimentale Misure (CIM) 'G. Casnati', University of Parma , Parco Area delle Scienze 23/A, 43124 Parma, Italy.

Malaria eradication is a global health priority, but current therapies are not always suitable for providing a radical cure. Artemisinin has paved the way for the current malaria treatment, the so-called Artemisinin-based Combination Therapy (ACT). However, with the detection of resistance to ACT, innovative compounds active against multiple parasite species and at multiple life stages are needed. GlaxoSmithKline has recently disclosed the results of a phenotypic screening of an internal library, publishing a collection of 400 antimalarial chemotypes, termed the "Malaria Box". After analysis of the data set, we have carried out a medicinal chemistry campaign in order to define the structure-activity relationships for one of the released compounds, which embodies a benzothiophene-2-carboxamide core. Thirty-five compounds were prepared, and a description of the structural features responsible for the in vitro activity against different strains of P. falciparum, the toxicity, and the metabolic stability is herein reported.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01685DOI Listing
March 2017

Discovery of Multitarget Agents Active as Broad-Spectrum Antivirals and Correctors of Cystic Fibrosis Transmembrane Conductance Regulator for Associated Pulmonary Diseases.

J Med Chem 2017 02 10;60(4):1400-1416. Epub 2017 Feb 10.

P4T Group, Dipartimento di Scienze degli Alimenti e del Farmaco, Università degli Studi di Parma , Viale delle Scienze, 27/A, 43124 Parma, Italy.

Enteroviruses (EVs) are among the most frequent infectious agents in humans worldwide and represent the leading cause of upper respiratory tract infections. No drugs for the treatment of EV infections are currently available. Recent studies have also linked EV infection with pulmonary exacerbations, especially in cystic fibrosis (CF) patients, and the importance of this link is probably underestimated. The aim of this work was to develop a new class of multitarget agents active both as broad-spectrum antivirals and as correctors of the F508del-cystic fibrosis transmembrane conductance regulator (CFTR) folding defect responsible for >90% of CF cases. We report herein the discovery of the first small molecules able to simultaneously act as correctors of the F508del-CFTR folding defect and as broad-spectrum antivirals against a panel of EVs representative of all major species.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01521DOI Listing
February 2017

An Experimental Model for the Rapid Screening of Compounds with Potential Use Against Mycobacteria.

Assay Drug Dev Technol 2016 11;14(9):524-534

1 Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa , UNL, Lisbon, Portugal .

Infections caused by Mycobacterium tuberculosis and other mycobacteria are major challenges for global public health. Particularly worrisome are infections caused by multidrug-resistant bacteria, which are increasingly difficult to treat because of the loss of efficacy of the current antibacterial agents, a problem that continues to escalate worldwide. There has been a limited interest and investment on the development of new antibacterial agents in the past decades. This has led to the current situation, in which there is an urgent demand for innovative therapeutic alternatives to fight infections caused by multidrug-resistant pathogens, such as multidrug-resistant tuberculosis. The identification of compounds that can act as adjuvants in antimycobacterial therapeutic regimens is an appealing strategy to restore the efficacy lost by some of the antibiotics currently used and shorten the duration of the therapeutic regimen. In this work, by setting Mycobacterium smegmatis as a model organism, we have developed a methodological strategy to identify, in a fast and simple approach, compounds with antimycobacterial activity or with potential adjuvant properties, by either inhibition of efflux or other unrelated mechanisms. Such an approach may increase the rate of identification of promising molecules, to be further explored in pathogenic models for their potential use either as antimicrobials or as adjuvants, in combination with available therapeutic regimens for the treatment of mycobacterial infections. This method allowed us to identify a new molecule that shows promising activity as an efflux inhibitor in M. smegmatis.
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http://dx.doi.org/10.1089/adt.2016.752DOI Listing
November 2016

Cyclopropane-1,2-dicarboxylic acids as new tools for the biophysical investigation of O-acetylserine sulfhydrylases by fluorimetric methods and saturation transfer difference (STD) NMR.

J Enzyme Inhib Med Chem 2016 31;31(sup4):78-87. Epub 2016 Aug 31.

a Department of Pharmacy , and.

Cysteine is a building block for many biomolecules that are crucial for living organisms. O-Acetylserine sulfhydrylase (OASS), present in bacteria and plants but absent in mammals, catalyzes the last step of cysteine biosynthesis. This enzyme has been deeply investigated because, beside the biosynthesis of cysteine, it exerts a series of "moonlighting" activities in bacteria. We have previously reported a series of molecules capable of inhibiting Salmonella typhimurium (S. typhymurium) OASS isoforms at nanomolar concentrations, using a combination of computational and spectroscopic approaches. The cyclopropane-1,2-dicarboxylic acids presented herein provide further insights into the binding mode of small molecules to OASS enzymes. Saturation transfer difference NMR (STD-NMR) was used to characterize the molecule/enzyme interactions for both OASS-A and B. Most of the compounds induce a several fold increase in fluorescence emission of the pyridoxal 5'-phosphate (PLP) coenzyme upon binding to either OASS-A or OASS-B, making these compounds excellent tools for the development of competition-binding experiments.
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http://dx.doi.org/10.1080/14756366.2016.1218486DOI Listing
February 2017

Discovery of New Potential Anti-Infective Compounds Based on Carbonic Anhydrase Inhibitors by Rational Target-Focused Repurposing Approaches.

ChemMedChem 2016 09 15;11(17):1904-14. Epub 2016 Jun 15.

Università degli Studi di Parma, Dipartimento di Farmacia, P4T group, Parco Area delle Scienze, Via G.P. Usberti 27A, 43121, Parma, Italy.

In academia, compound recycling represents an alternative drug discovery strategy to identify new pharmaceutical targets from a library of chemical compounds available in house. Herein we report the application of a rational target-based drug-repurposing approach to find diverse applications for our in-house collection of compounds. The carbonic anhydrase (CA, EC 4.2.1.1) metalloenzyme superfamily was identified as a potential target of our compounds. The combination of a thoroughly validated docking screening protocol, together with in vitro assays against various CA families and isoforms, allowed us to identify two unprecedented chemotypes as CA inhibitors. The identified compounds have the capacity to preferentially bind pathogenic (bacterial/protozoan) CAs over human isoforms and represent excellent hits for further optimization in hit-to-lead campaigns.
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http://dx.doi.org/10.1002/cmdc.201600180DOI Listing
September 2016

A combined ligand- and structure-based approach for the identification of rilmenidine-derived compounds which synergize the antitumor effects of doxorubicin.

Bioorg Med Chem 2016 07 24;24(14):3174-83. Epub 2016 May 24.

Center for Multidisciplinary Research, Institute of Nuclear Sciences Vinca, University of Belgrade, POB 522, Mihaila Petrovica Alasa 14, 11001 Belgrade, Serbia. Electronic address:

The clonidine-like central antihypertensive agent rilmenidine, which has high affinity for I1-type imidazoline receptors (I1-IR) was recently found to have cytotoxic effects on cultured cancer cell lines. However, due to its pharmacological effects resulting also from α2-adrenoceptor activation, rilmenidine cannot be considered a suitable anticancer drug candidate. Here, we report the identification of novel rilmenidine-derived compounds with anticancer potential and devoid of α2-adrenoceptor effects by means of ligand- and structure-based drug design approaches. Starting from a large virtual library, eleven compounds were selected, synthesized and submitted to biological evaluation. The most active compound 5 exhibited a cytotoxic profile similar to that of rilmenidine, but without appreciable affinity to α2-adrenoceptors. In addition, compound 5 significantly enhanced the apoptotic response to doxorubicin, and may thus represent an important tool for the development of better adjuvant chemotherapeutic strategies for doxorubicin-insensitive cancers.
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http://dx.doi.org/10.1016/j.bmc.2016.05.043DOI Listing
July 2016

Rational Design, Synthesis, and Preliminary Structure-Activity Relationships of α-Substituted-2-Phenylcyclopropane Carboxylic Acids as Inhibitors of Salmonella typhimurium O-Acetylserine Sulfhydrylase.

J Med Chem 2016 Mar 2;59(6):2567-78. Epub 2016 Mar 2.

National Institute of Biostructures and Biosystems , Viale delle Medaglie d'Oro 305, 00136 Rome, Italy.

Cysteine is a building block for several biomolecules that are crucial for living organisms. The last step of cysteine biosynthesis is catalyzed by O-acetylserine sulfydrylase (OASS), a highly conserved pyridoxal 5'-phosphate (PLP)-dependent enzyme, present in different isoforms in bacteria, plants, and nematodes, but absent in mammals. Beside the biosynthesis of cysteine, OASS exerts a series of "moonlighting" activities in bacteria, such as transcriptional regulation, contact-dependent growth inhibition, swarming motility, and induction of antibiotic resistance. Therefore, the discovery of molecules capable of inhibiting OASS would be a valuable tool to unravel how this protein affects the physiology of unicellular organisms. As a continuation of our efforts toward the synthesis of OASS inhibitors, in this work we have used a combination of computational and spectroscopic approaches to rationally design, synthesize, and test a series of substituted 2-phenylcyclopropane carboxylic acids that bind to the two S. typhymurium OASS isoforms at nanomolar concentrations.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01775DOI Listing
March 2016

Mutation of Rv2887, a marR-like gene, confers Mycobacterium tuberculosis resistance to an imidazopyridine-based agent.

Antimicrob Agents Chemother 2015 Nov 24;59(11):6873-81. Epub 2015 Aug 24.

Center for Tuberculosis Research, Department of Medicine, Johns Hopkins University, Baltimore, Maryland, USA

Drug resistance is a major problem in Mycobacterium tuberculosis control, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity against M. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independent M. tuberculosis mutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations in Rv2887 were common to all three MP-III-71-resistant mutants, and we confirmed the role of Rv2887 as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified in Escherichia coli to negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation of Rv2887 abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations of Rv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance of M. tuberculosis Rv2887 mutants may involve efflux pump upregulation and also drug methylation.
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http://dx.doi.org/10.1128/AAC.01341-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604386PMC
November 2015

Rational Design and Synthesis of Thioridazine Analogues as Enhancers of the Antituberculosis Therapy.

J Med Chem 2015 Aug 3;58(15):5842-53. Epub 2015 Aug 3.

‡Grupo de Micobactérias, Unidade de Microbiologia Médica, Global Health and Tropical Medicine (GHTM), Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT, UNL), Rua da Junqueira, 100, 1349-008 Lisbon, Portugal.

Tuberculosis, caused by Mycobacterium tuberculosis, is still one of the leading infectious diseases globally. Therefore, novel approaches are needed to face this disease. Efflux pumps are known to contribute to the emergence of M. tuberculosis drug resistance. Thioridazine has shown good anti-TB properties both in vitro and in vivo, likely due to its capacity to inhibit efflux mechanisms. Here we report the design and synthesis of a number of putative efflux inhibitors inspired by the structure of thioridazine. Compounds were evaluated for their in vitro and ex vivo activity against M. tuberculosis H37Rv. Compared to the parent molecule, some of the compounds synthesized showed higher efflux inhibitory capacity, less cytotoxicity, and a remarkable synergistic effect with anti-TB drugs both in vitro and in human macrophages, demonstrating their potential to be used as coadjuvants for the treatment of tuberculosis.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00428DOI Listing
August 2015

Cyclopropane derivatives as potential human serine racemase inhibitors: unveiling novel insights into a difficult target.

J Enzyme Inhib Med Chem 2016 Aug 2;31(4):645-52. Epub 2015 Jul 2.

a Dipartimento di Farmacia , Università degli studi di Parma , Parma , Italy .

d-Serine is the co-agonist of NMDA receptors and binds to the so-called glycine site. d-Serine is synthesized by human serine racemase (SR). Over activation of NMDA receptors is involved in many neurodegenerative diseases and, therefore, the inhibition of SR might represent a novel strategy for the treatment of these pathologies. SR is a very difficult target, with only few compounds so far identified exhibiting weak inhibitory activity. This study was aimed at the identification of novel SR inhibitor by mimicking malonic acid, the best-known SR inhibitor, with a cyclopropane scaffold. We developed, synthesized, and tested a series of cyclopropane dicarboxylic acid derivatives, complementing the synthetic effort with molecular docking. We identified few compounds that bind SR in high micromolar range with a lack of significant correlation between experimental and predicted binding affinities. The thorough analysis of the results can be exploited for the development of more potent SR inhibitors.
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http://dx.doi.org/10.3109/14756366.2015.1057720DOI Listing
August 2016

Discovery of antitubercular 2,4-diphenyl-1H-imidazoles from chemical library repositioning and rational design.

Eur J Med Chem 2015 Jul 5;100:44-9. Epub 2015 Jun 5.

Dipartimento di Farmacia, University of Parma, Parco Area delle Scienze 27/A, Parma, 43124, Italy. Electronic address:

TB, caused by Mycobacterium tuberculosis, is one of the deadliest infections worldwide. The co-infection with HIV and the emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains have further increased the burden for this disease. In the attempt to respond to the constant need of novel therapeutic options, we herein report the discovery of 2,4-diphenyl-1H-imidazoles as effective antitubercular agents, with MIC in the low micromolar range against actively replicating and persistent M. tuberculosis strains. The good activity, along with the lack of toxicity and the feasible synthesis, underscore their value as novel scaffolds for the development of new anti-TB drugs.
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http://dx.doi.org/10.1016/j.ejmech.2015.05.048DOI Listing
July 2015

Further insights into the SAR of α-substituted cyclopropylamine derivatives as inhibitors of histone demethylase KDM1A.

Eur J Med Chem 2015 Mar 7;92:377-86. Epub 2015 Jan 7.

Dipartimento Farmaceutico, University of Parma, Parco Area delle Scienze 27/A, Parma 43124, Italy. Electronic address:

Epigenetics alterations including histone methylation and acetylation, and DNA methylation, are thought to play important roles in the onset and progression of cancer in numerous tumour cell lines. Lysine-specific demethylase 1 (LSD1 or KDM1A) is highly expressed in different cancer types and inhibiting KDM1A activity seems to have high therapeutic potential in cancer treatment. In the recent years, several inhibitors of KDM1A have been prepared and disclosed. The majority of these derivatives were designed based on the structure of tranylcypromine, as the cyclopropane core is responsible for the covalent interaction between the inhibitor and the catalytic domain of KDM proteins. In this study, we have further extended the SAR regarding compounds 1a-e, which were recently found to inhibit KDM1A with good activity. The decoration of the phenyl ring at the β-position of the cyclopropane ring with small functional groups, mostly halogenated, and in particular at the meta position, led to a significant improvement of the inhibitory activity against KDM1A, as exemplified by compound 44a, which has a potency in the low nanomolar range (31 nM).
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http://dx.doi.org/10.1016/j.ejmech.2014.12.032DOI Listing
March 2015

Inhibitors of the sulfur assimilation pathway in bacterial pathogens as enhancers of antibiotic therapy.

Curr Med Chem 2015 ;22(2):187-213

Dipartimento di Farmacia, Universita di Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italy.

The rising emergence of antibiotic resistance urges the search for new strategies to defeat microorganisms that lead to persistent infections of the host. Tolerant to antibiotics, slowly replicating bacteria often cause latent and persistent infections that are the most challenging for pharmacological treatment. Persistence inside the host requires an extensive re-programming of the pathogen metabolic functions, due to the extremely hostile environment they face. Therefore, targeting key metabolic functions could result in better antibiotic treatments, shortened latency periods, and increased susceptibility to traditional antibiotics. Bacteria, differently from mammals, assimilate inorganic sulfur into cysteine, the precursor of a number of key metabolites including reducing agents, cofactors and membrane components. Inhibition of cysteine biosynthesis was proven to interfere heavily with the ability of pathogens to fight oxidative stress, to infect the host and to establish long-term infections. This review has the purpose of i) briefly summarizing the key structural and functional properties of transporters and enzymes involved in sulfur assimilation, ii) presenting biological evidence that supports the exploitation of this pathway for the identification of potential targets and, iii) highlighting intense efforts and advancements in the search of promising candidates for the development of novel compounds that enhance antibiotics therapy.
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http://dx.doi.org/10.2174/0929867321666141112122553DOI Listing
July 2015