Gabriele Costantino

Target Flexibility: An Emerging Consideration in Drug Discovery and Design†

Department of General and Inorganic Chemistry, UniVersity of Parma, Via G.P. Usberti 17/A 43100, Parma, Italy, National Institute for Biosystems and Biostructures, Rome, Italy, Department of Medicinal Chemistry and Institute for Structural Biology & Drug DiscoVery, Virginia Commonwealth UniVersity, Richmond, Virginia 23298-0540, Department of Pharmaceutics, UniVersity of Parma, Via GP Usberti 27/A, 43100 Parma, Italy, High Performance Systems, CINECA Supercomputing Centre, Casalecchio di Reno, Bologna, Italy, Dulbecco Telethon Institute, Department of Chemistry, UniVersity of Modena and Reggio Emilia, Via Campi 183, 41100 Modena, Italy, Department of Mathematics and Natural Sciences, Pharmaceutical Institute, Christian-Albrechts-UniVersity, Gutenbergstrasse 76, 24118 Kiel, Germany, Departments of Biochemistry & Molecular Biology, Computer Science & Engineering, and Physics & Astronomy, Michigan State UniVersity, East Lansing, Michigan 48824-1319, Department of Molecular Biology, MB-5, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037-1000, Molecular Modeling and Bioinformatics Unit, Institute of Biomedical Research, Scientific Park of Barcelona, Department of Biochemistry and Molecular Biology, UniVersity of Barcelona, Josep Samitier 1-5, Barcelona 08028, Spain, Department of Experimental Medicine, UniVersity of Parma, Via Volturno, 39, 43100, Parma, Italy, Department of Chemical, Food, Pharmaceutical and Pharmacological Sciences, UniVersity of Piemonte Orientale “Amedeo AVogadro”, Via BoVio 6, 28100 NoVara, Italy, Institute of Pharmacy and Food Chemistry, UniVersity of Wurzburg, Am Hubland, D-97074 Wurzburg, Germany

Membrane-Sensitive Conformational States of Helix 8 in the Metabotropic Glu2 Receptor, a Class C GPCR

The recent elucidation of the X-ray structure of several class A GPCRs clearly indicates that the amphipathic helix 8 (H8) is a conserved structural domain in most crystallized GPCRs. Very little is known about the presence and the possible role of an analogous H8 domain in the distantly related class C GPCRs. In this study, we investigated the structural properties for the H8 domain of the mGluR2 receptor, a class C GPCR, by applying extended molecular dynamics simulations. Our study indicates that the amphipathic H8 adopts membrane-sensitive conformational states, which depend on the membrane composition. Cholesterol-rich membranes stabilize the helical structure of H8 whereas cholesterol-depleted membranes induce a disruption of H8. The observed link between membrane cholesterol levels and H8 conformational states suggests that H8 behaves as a sensor of cholesterol concentration.

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

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.

Design and synthesis of trans-2-substituted-cyclopropane-1-carboxylic acids as the first non-natural small molecule inhibitors of O-acetylserine sulfhydrylase

O-Acetylserine sulfhydrylase (isoform A, OASS-A) is a pyridoxal-5′-phosphate-dependent enzyme responsible for cysteine biosynthesis in many pathological microorganisms. It is proposed that inhibition of OASS-A could represent a novel strategy to overcome bacterial resistance to antibiotics. A class of 2-substituted-cyclopropane-1-carboxylic acids was synthesized, based on structural determinants grasped by analyzing a group of synthetic pentapeptides known to efficiently bind OASS-A from Haemophilus influenzae (HiOASS-A). The cyclopropane derivatives were submitted to a binding affinity assay with HiOASS-A and three of them, with Kdiss in the low micromolar range, showed higher affinity than the most active synthetic pentapeptide. Thus, in this communication we report the first example of potent non-natural small molecule inhibitors of HiOASS-A. In addition, a molecular modelling study suggested a possible inhibition mechanism, through which the new cyclopropane ligands block HiOASS-A. Noteworthily, the novel, small-sized, non-peptidic inhibitors retain the structural motifs of the bulky peptides, which are relevant for the enzyme inhibition.

Discovery and characterization of novel potent PARP-1 inhibitors endowed with neuroprotective properties: From TIQ-A to HYDAMTIQ

Activation of poly(ADP-ribose) polymerase (PARP) is an important factor in controlling cell survival or death. As a consequence, therapeutic interventions with PARP-1 inhibitors are sought in different pathological conditions such as cancer, cardiovascular and inflammatory diseases, as well as brain ischemia. In the first part of this work, as a continuation of our efforts in the field, we report the design, synthesis and biological appraisal of novel potent PARP-1 inhibitors. A crystallization experiment is carried out to ascertain the mode of binding to PARP-1 of the most potent compound, namely 2-((dimethylamino)methyl)-9-hydroxythieno[2,3-c]isoquinolin-5(4H)-one (HYDAMTIQ), whilst molecular modeling studies are performed to infer the role of water molecules in ligand binding. In the second part of the work, we discuss the results of HYDAMTIQ in models of brain ischemia as well as its preliminary physicochemical and pharmacokinetic characterization. Collectively, the data obtained qualify HYDAMTIQ as a novel lead candidate for advancement to clinical settings of brain ischemia.

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

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 MIC90 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.

Synthesis of atypical bile acids for use as investigative tools for the genetic defect of 3β-hydroxy-Δ5-C27-steroid oxidoreductase deficiency

Deficiency of 3β-hydroxy-Δ(5)-C27-steroid oxidoreductase (HSD3B7), an enzyme catalyzing the second step in the pathway for bile acid synthesis, leads to a complete lack of the primary bile acids, cholic and chenodeoxycholic acids, and the accumulation of 3β,7α-dihydroxy- and 3β,7α,12α-trihydroxy-Δ(5)-cholenoic acids. Patients affected by this autosomal recessive genetic defect develop cholestatic liver disease that is clinically responsive to primary bile acid therapy. Reference standards of these compounds are needed to facilitate diagnosis and to accurately quantify biochemical responses to therapy. Described are a novel synthesis of atypical bile acids that characterize the HSD3B7 deficiency and their effect on bile acid-activated nuclear receptors, target genes and cytochromes involved in bile acid homeostasis and detoxification. The failure of 3β-hydroxy-Δ(5)-cholenoic acids to function as FXR, PXR and CAR agonists and to exert hepatoprotective actions explains the mechanism for progressive cholestatic liver disease in patients with HSD3B7 deficiency.

Modulation of bacterial metabolism by the microenvironment controls MAIT cell stimulation

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.

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

Abstract 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.

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

Abstract 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.