Publications by authors named "Fabio Zuccotto"

28 Publications

  • Page 1 of 1

Multiple unbiased approaches identify oxidosqualene cyclase as the molecular target of a promising anti-leishmanial.

Cell Chem Biol 2021 Feb 26. Epub 2021 Feb 26.

Division of Biological Chemistry and Drug Discovery, Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK. Electronic address:

Phenotypic screening identified a benzothiophene compound with activity against Leishmania donovani, the causative agent of visceral leishmaniasis. Using multiple orthogonal approaches, oxidosqualene cyclase (OSC), a key enzyme of sterol biosynthesis, was identified as the target of this racemic compound and its enantiomers. Whole genome sequencing and screening of a genome-wide overexpression library confirmed that OSC gene amplification is associated with resistance to compound 1. Introduction of an ectopic copy of the OSC gene into wild-type cells reduced susceptibility to these compounds confirming the role of this enzyme in resistance. Biochemical analyses demonstrated the accumulation of the substrate of OSC and depletion of its product in compound (S)-1-treated-promastigotes and cell-free membrane preparations, respectively. Thermal proteome profiling confirmed that compound (S)-1 binds directly to OSC. Finally, modeling and docking studies identified key interactions between compound (S)-1 and the LdOSC active site. Strategies to improve the potency for this promising anti-leishmanial are proposed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2021.02.008DOI Listing
February 2021

Spirocycle MmpL3 Inhibitors with Improved hERG and Cytotoxicity Profiles as Inhibitors of Growth.

ACS Omega 2021 Jan 13;6(3):2284-2311. Epub 2021 Jan 13.

TB Discovery Research, Infectious Disease Research Institute, 1616 Eastlake Avenue East, Suite 400, Seattle, Washington 98102, United States.

With the emergence of multi-drug-resistant strains of there is a pressing need for new oral drugs with novel mechanisms of action. A number of scaffolds with potent anti-tubercular activity have been identified from phenotypic screening that appear to target MmpL3. However, the scaffolds are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-à-go-go related gene (hERG) and IKr potassium current modulation. The spirocycle compound was reported to target MmpL3 and displayed promising efficacy in a murine model of acute tuberculosis (TB) infection. However, this highly lipophilic monobasic amine was cytotoxic and inhibited the hERG ion channel. Herein, the related spirocycles () are described, which were identified following phenotypic screening of the Eli Lilly corporate library against . The novel N-alkylated pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterion series, exemplified by lead , with decreased HepG2 cytotoxicity as well as limited hERG ion channel inhibition. Strains with mutations in MmpL3 were resistant to , and under replicating conditions, demonstrated bactericidal activity against . Unfortunately, compound had no efficacy in an acute model of TB infection; this was most likely due to the exposure remaining above the minimal inhibitory concentration for only a limited time.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsomega.0c05589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7841955PMC
January 2021

Instability of aquaglyceroporin (AQP) 2 contributes to drug resistance in Trypanosoma brucei.

PLoS Negl Trop Dis 2020 07 9;14(7):e0008458. Epub 2020 Jul 9.

School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.

Defining mode of action is vital for both developing new drugs and predicting potential resistance mechanisms. Sensitivity of African trypanosomes to pentamidine and melarsoprol is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a channel associated with water/glycerol transport. TbAQP2 is expressed at the flagellar pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; that TbAQP2 mediates pentamidine translocation across the plasma membrane or via binding to TbAQP2, with subsequent endocytosis and presumably transport across the endosomal/lysosomal membrane, but as trafficking and regulation of TbAQPs is uncharacterised this remains unresolved. We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated and is transported to the lysosome. Unexpectedly, mutation of potential ubiquitin conjugation sites, i.e. cytoplasmic-oriented lysine residues, reduced folding and tetramerization efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation, as observed in pentamidine-resistant parasites, also leads to impaired oligomerisation, mislocalisation and increased turnover. These data suggest that TbAQP2 stability is highly sensitive to mutation and that instability contributes towards the emergence of drug resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0008458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413563PMC
July 2020

The Q Site of Cytochrome is a Promiscuous Drug Target in and .

ACS Infect Dis 2020 03 30;6(3):515-528. Epub 2020 Jan 30.

Division of Biological Chemistry and Drug Discovery, Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.

Available treatments for Chagas' disease and visceral leishmaniasis are inadequate, and there is a pressing need for new therapeutics. Drug discovery efforts for both diseases principally rely upon phenotypic screening. However, the optimization of phenotypically active compounds is hindered by a lack of information regarding their molecular target(s). To combat this issue we initiate target deconvolution studies at an early stage. Here, we describe comprehensive genetic and biochemical studies to determine the targets of three unrelated phenotypically active compounds. All three structurally diverse compounds target the Q active-site of cytochrome , part of the cytochrome complex of the electron transport chain. Our studies go on to identify the Q site as a promiscuous drug target in and with a propensity to rapidly mutate. Strategies to rapidly identify compounds acting via this mechanism are discussed to ensure that drug discovery portfolios are not overwhelmed with inhibitors of a single target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsinfecdis.9b00426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076694PMC
March 2020

A platform for target prediction of phenotypic screening hit molecules.

J Mol Graph Model 2020 03 24;95:107485. Epub 2019 Oct 24.

Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Sir James Black Centre, DD1 5EH, United Kingdom. Electronic address:

Many drug discovery programmes, particularly for infectious diseases, are conducted phenotypically. Identifying the targets of phenotypic screening hits experimentally can be complex, time-consuming, and expensive. However, it would be valuable to know what the molecular target(s) is, as knowledge of the binding pose of the hit molecule in the binding site can facilitate the compound optimisation. Furthermore, knowing the target would allow de-prioritisation of less attractive chemical series or molecular targets. To generate target-hypotheses for phenotypic active compounds, an in silico platform was developed that utilises both ligand and protein-structure information to generate a ranked set of predicted molecular targets. As a result of the web-based workflow the user obtains a set of 3D structures of the predicted targets with the active molecule bound. The platform was exemplified using Mycobacterium tuberculosis, the causative organism of tuberculosis. In a test that we performed, the platform was able to predict the targets of 60% of compounds investigated, where there was some similarity to a ligand in the protein database.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmgm.2019.107485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983931PMC
March 2020

Identification of inhibitors of an unconventional Trypanosoma brucei kinetochore kinase.

PLoS One 2019 31;14(5):e0217828. Epub 2019 May 31.

Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United Kingdom.

The discovery of 20 unconventional kinetochore proteins in Trypanosoma brucei has opened a new and interesting area of evolutionary research to study a biological process previously thought to be highly conserved in all eukaryotes. In addition, the discovery of novel proteins involved in a critical cellular process provides an opportunity to exploit differences between kinetoplastid and human kinetochore proteins to develop therapeutics for diseases caused by kinetoplastid parasites. Consequently, we identified two of the unconventional kinetochore proteins as key targets (the highly related kinases KKT10 and KKT19). Recombinant T. brucei KKT19 (TbKKT19) protein was produced, a peptide substrate phosphorylated by TbKKT19 identified (KKLRRTLSVA), Michaelis constants for KKLRRTLSVA and ATP were determined (179 μM and 102 μM respectively) and a robust high-throughput compatible biochemical assay developed. This biochemical assay was validated pharmacologically with inhibition by staurosporine and hypothemycin (IC50 values of 288 nM and 65 nM respectively). Surprisingly, a subsequent high-throughput screen of a kinase-relevant compound library (6,624 compounds) yielded few hits (8 hits; final hit rate 0.12%). The low hit rate observed was unusual for a kinase target, particularly when screened against a compound library enriched with kinase hinge binding scaffolds. In an attempt to understand the low hit rate a TbKKT19 homology model, based on human cdc2-like kinase 1 (CLK1), was generated. Analysis of the TbKKT19 sequence and structure revealed no obvious features that could explain the low hit rates. Further work will therefore be necessary to explore this unique kinetochore kinase as well as to assess whether the few hits identified can be developed into tool molecules or new drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0217828PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544269PMC
January 2020

Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition.

Proc Natl Acad Sci U S A 2019 05 8;116(19):9318-9323. Epub 2019 Apr 8.

Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom;

Visceral leishmaniasis (VL), caused by the protozoan parasites and , is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant and isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the β5 subunit of the proteasome. High-resolution cryo-EM structures of apo and compound 8-bound 20S proteasome reveal a previously undiscovered inhibitor site that lies between the β4 and β5 proteasome subunits. This induced pocket exploits β4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1820175116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511062PMC
May 2019

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Proc Natl Acad Sci U S A 2019 04 20;116(14):7015-7020. Epub 2019 Mar 20.

Wellcome Centre for Anti-Infectives Research, Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, University of Dundee, DD1 5EH Dundee, United Kingdom;

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage and in cell-culture studies. Target deconvolution in has shown that cladosporin inhibits lysyl-tRNA synthetase (KRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both KRS1 and KRS (KRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between KRS1 and KRS. This series of compounds inhibit KRS and and in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for KRS1 and KRS vs. (human) KRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1814685116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452685PMC
April 2019

Pharmacological Validation of N-Myristoyltransferase as a Drug Target in Leishmania donovani.

ACS Infect Dis 2019 01 12;5(1):111-122. Epub 2018 Nov 12.

The Wellcome Trust Centre for Anti-Infectives Research, School of Life Sciences , University of Dundee , Dow Street , Dundee DD1 5EH , United Kingdom.

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and L. infantum, is responsible for ∼30 000 deaths annually. Available treatments are inadequate, and there is a pressing need for new therapeutics. N-Myristoyltransferase (NMT) remains one of the few genetically validated drug targets in these parasites. Here, we sought to pharmacologically validate this enzyme in Leishmania. A focused set of 1600 pyrazolyl sulfonamide compounds was screened against L. major NMT in a robust high-throughput biochemical assay. Several potent inhibitors were identified with marginal selectivity over the human enzyme. There was little correlation between the enzyme potency of these inhibitors and their cellular activity against L. donovani axenic amastigotes, and this discrepancy could be due to poor cellular uptake due to the basicity of these compounds. Thus, a series of analogues were synthesized with less basic centers. Although most of these compounds continued to suffer from relatively poor antileishmanial activity, our most potent inhibitor of LmNMT (DDD100097, K of 0.34 nM) showed modest activity against L. donovani intracellular amastigotes (EC of 2.4 μM) and maintained a modest therapeutic window over the human enzyme. Two unbiased approaches, namely, screening against our cosmid-based overexpression library and thermal proteome profiling (TPP), confirm that DDD100097 (compound 2) acts on-target within parasites. Oral dosing with compound 2 resulted in a 52% reduction in parasite burden in our mouse model of VL. Thus, NMT is now a pharmacologically validated target in Leishmania. The challenge in finding drug candidates remains to identify alternative strategies to address the drop-off in activity between enzyme inhibition and in vitro activity while maintaining sufficient selectivity over the human enzyme, both issues that continue to plague studies in this area.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsinfecdis.8b00226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6332449PMC
January 2019

Clinical and veterinary trypanocidal benzoxaboroles target CPSF3.

Proc Natl Acad Sci U S A 2018 09 5;115(38):9616-9621. Epub 2018 Sep 5.

The Wellcome Trust Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, DD1 5EH Dundee, United Kingdom;

African trypanosomes cause lethal and neglected tropical diseases, known as sleeping sickness in humans and nagana in animals. Current therapies are limited, but fortunately, promising therapies are in advanced clinical and veterinary development, including acoziborole (AN5568 or SCYX-7158) and AN11736, respectively. These benzoxaboroles will likely be key to the World Health Organization's target of disease control by 2030. Their mode of action was previously unknown. We have developed a high-coverage overexpression library and use it here to explore drug mode of action in Initially, an inhibitor with a known target was used to select for drug resistance and to test massive parallel library screening and genome-wide mapping; this effectively identified the known target and validated the approach. Subsequently, the overexpression screening approach was used to identify the target of the benzoxaboroles, Cleavage and Polyadenylation Specificity Factor 3 (CPSF3, Tb927.4.1340). We validated the CPSF3 endonuclease as the target, using independent overexpression strains. Knockdown provided genetic validation of CPSF3 as essential, and GFP tagging confirmed the expected nuclear localization. Molecular docking and CRISPR-Cas9-based editing demonstrated how acoziborole can specifically block the active site and mRNA processing by parasite, but not host CPSF3. Thus, our findings provide both genetic and chemical validation for CPSF3 as an important drug target in trypanosomes and reveal inhibition of mRNA maturation as the mode of action of the trypanocidal benzoxaboroles. Understanding the mechanism of action of benzoxaborole-based therapies can assist development of improved therapies, as well as the prediction and monitoring of resistance, if or when it arises.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1807915115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156652PMC
September 2018

Pharmacokinetics of β-Lactam Antibiotics: Clues from the Past To Help Discover Long-Acting Oral Drugs in the Future.

ACS Infect Dis 2018 10 10;4(10):1439-1447. Epub 2018 Sep 10.

Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, School of Life Sciences , University of Dundee , Dow Street , Dundee . DDI 5EH , U.K.

β-Lactams represent perhaps the most important class of antibiotics yet discovered. However, despite many years of active research, none of the currently approved drugs in this class combine oral activity with long duration of action. Recent developments suggest that new β-lactam antibiotics with such a profile would have utility in the treatment of tuberculosis. Consequently, the historical β-lactam pharmacokinetic data have been compiled and analyzed to identify possible directions and drug discovery strategies aimed toward new β-lactam antibiotics with this profile.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsinfecdis.8b00160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6189874PMC
October 2018

Cyclin-dependent kinase 12 is a drug target for visceral leishmaniasis.

Nature 2018 08 25;560(7717):192-197. Epub 2018 Jul 25.

Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, UK.

Visceral leishmaniasis causes considerable mortality and morbidity in many parts of the world. There is an urgent need for the development of new, effective treatments for this disease. Here we describe the development of an anti-leishmanial drug-like chemical series based on a pyrazolopyrimidine scaffold. The leading compound from this series (7, DDD853651/GSK3186899) is efficacious in a mouse model of visceral leishmaniasis, has suitable physicochemical, pharmacokinetic and toxicological properties for further development, and has been declared a preclinical candidate. Detailed mode-of-action studies indicate that compounds from this series act principally by inhibiting the parasite cdc-2-related kinase 12 (CRK12), thus defining a druggable target for visceral leishmaniasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-018-0356-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402543PMC
August 2018

On-Chip Screening of a Glycomimetic Library with C-Type Lectins Reveals Structural Features Responsible for Preferential Binding of Dectin-2 over DC-SIGN/R and Langerin.

Chemistry 2018 Sep 3;24(54):14448-14460. Epub 2018 Sep 3.

Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133, Milano, Italy.

A library of mannose- and fucose-based glycomimetics was synthesized and screened in a microarray format against a set of C-type lectin receptors (CLRs) that included DC-SIGN, DC-SIGNR, langerin, and dectin-2. Glycomimetic ligands able to interact with dectin-2 were identified for the first time. Comparative analysis of binding profiles allowed their selectivity against other CLRs to be probed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/chem.201802577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220942PMC
September 2018

Identification of Morpholino Thiophenes as Novel Mycobacterium tuberculosis Inhibitors, Targeting QcrB.

J Med Chem 2018 Aug 26;61(15):6592-6608. Epub 2018 Jul 26.

Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, College of Life Sciences , University of Dundee , Dundee DD1 5EH , United Kingdom.

With the emergence of multidrug-resistant strains of Mycobacterium tuberculosis there is a pressing need for new oral drugs with novel mechanisms of action. Herein, we describe the identification of a novel morpholino-thiophenes (MOT) series following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis strain H37Rv. The design, synthesis, and structure-activity relationships of a range of analogues around the confirmed actives are described. Optimized leads with potent whole cell activity against H37Rv, no cytotoxicity flags, and in vivo efficacy in an acute murine model of infection are described. Mode-of-action studies suggest that the novel scaffold targets QcrB, a subunit of the menaquinol cytochrome c oxidoreductase, part of the bc1-aa3-type cytochrome c oxidase complex that is responsible for driving oxygen-dependent respiration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b00172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6089501PMC
August 2018

Screening of a Novel Fragment Library with Functional Complexity against Mycobacterium tuberculosis InhA.

ChemMedChem 2018 04 19;13(7):672-677. Epub 2018 Feb 19.

Drug Discovery Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland, UK.

Our findings reported herein provide support for the benefits of including functional group complexity (FGC) within fragments when screening against protein targets such as Mycobacterium tuberculosis InhA. We show that InhA fragment actives with FGC maintained their binding pose during elaboration. Furthermore, weak fragment hits with functional group handles also allowed for facile fragment elaboration to afford novel and potent InhA inhibitors with good ligand efficiency metrics for optimization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cmdc.201700774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915743PMC
April 2018

Exhaustive sampling of the fragment space associated to a molecule leading to the generation of conserved fragments.

Chem Biol Drug Des 2018 03 12;91(3):655-667. Epub 2017 Dec 12.

Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, Scotland, UK.

The first step in hit optimization is the identification of the pharmacophore, which is normally achieved by deconstruction of the hit molecule to generate "deletion analogues." In silico fragmentation approaches often focus on the generation of small fragments that do not describe properly the fragment space associated to the deletion analogues. We present significant modifications to the molecular fragmentation programme molBLOCKS, which allows the exhaustive sampling of the fragment space associated with a molecule to generate all possible molecular fragments. This generates larger fragments, by combining the smallest fragments. Additionally, it has been modified to deal with the problem of changing pharmacophoric properties through fragmentation, by highlighting bond cuts. The modified molBLOCKS programme was used on a set of drug compounds, where it generated more unique fragments than standard fragmentation approaches by increasing the number of fragments derived per compound. This fragment set was found to be more diverse than those generated by standard fragmentation programmes and was relevant to drug discovery as it contains the key fragments representing the pharmacophoric elements associated with ligand recognition. The use of dummy atoms to highlight bond cuts further increases the information content of fragments by visualizing their previous bonding pattern.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/cbdd.13129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836963PMC
March 2018

Essential but Not Vulnerable: Indazole Sulfonamides Targeting Inosine Monophosphate Dehydrogenase as Potential Leads against Mycobacterium tuberculosis.

ACS Infect Dis 2017 01 17;3(1):18-33. Epub 2016 Oct 17.

Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health , Bethesda, Maryland 20892-3206, United States.

A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsinfecdis.6b00103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972394PMC
January 2017

Prediction of Drug Penetration in Tuberculosis Lesions.

ACS Infect Dis 2016 08 6;2(8):552-63. Epub 2016 Jul 6.

Public Health Research Institute Centre, New Jersey Medical School, Rutgers , 225 Warren Street, Newark, New Jersey 07103, United States.

The penetration of antibiotics in necrotic tuberculosis lesions is heterogeneous and drug-specific, but the factors underlying such differential partitioning are unknown. We hypothesized that drug binding to macromolecules in necrotic foci (or caseum) prevents passive drug diffusion through avascular caseum, a critical site of infection. Using a caseum binding assay and MALDI mass spectrometry imaging of tuberculosis drugs, we showed that binding to caseum inversely correlates with passive diffusion into the necrotic core. We developed a high-throughput assay relying on rapid equilibrium dialysis and a caseum surrogate designed to mimic the composition of native caseum. A set of 279 compounds was profiled in this assay to generate a large data set and explore the physicochemical drivers of free diffusion into caseum. Principle component analysis and modeling of the data set delivered an in silico signature predictive of caseum binding, combining 69 molecular descriptors. Among the major positive drivers of binding were high lipophilicity and poor solubility. Determinants of molecular shape such as the number of rings, particularly aromatic rings, number of sp(2) carbon counts, and volume-to-surface ratio negatively correlated with the free fraction, indicating that low-molecular-weight nonflat compounds are more likely to exhibit low caseum binding properties and diffuse effectively through caseum. To provide simple guidance in the property-based design of new compounds, a rule of thumb was derived whereby the sum of the hydrophobicity (clogP) and aromatic ring count is proportional to caseum binding. These tools can be used to ensure desirable lesion partitioning and guide the selection of optimal regimens against tuberculosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsinfecdis.6b00051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5028112PMC
August 2016

Discovery of 2-[1-(4,4-Difluorocyclohexyl)piperidin-4-yl]-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (NMS-P118): A Potent, Orally Available, and Highly Selective PARP-1 Inhibitor for Cancer Therapy.

J Med Chem 2015 Sep 26;58(17):6875-98. Epub 2015 Aug 26.

Oncology, Nerviano Medical Sciences Srl , Viale Pasteur 10, 20014 Nerviano, Milan, Italy.

The nuclear protein poly(ADP-ribose) polymerase-1 (PARP-1) has a well-established role in the signaling and repair of DNA and is a prominent target in oncology, as testified by the number of candidates in clinical testing that unselectively target both PARP-1 and its closest isoform PARP-2. The goal of our program was to find a PARP-1 selective inhibitor that would potentially mitigate toxicities arising from cross-inhibition of PARP-2. Thus, an HTS campaign on the proprietary Nerviano Medical Sciences (NMS) chemical collection, followed by SAR optimization, allowed us to discover 2-[1-(4,4-difluorocyclohexyl)piperidin-4-yl]-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (NMS-P118, 20by). NMS-P118 proved to be a potent, orally available, and highly selective PARP-1 inhibitor endowed with excellent ADME and pharmacokinetic profiles and high efficacy in vivo both as a single agent and in combination with Temozolomide in MDA-MB-436 and Capan-1 xenograft models, respectively. Cocrystal structures of 20by with both PARP-1 and PARP-2 catalytic domain proteins allowed rationalization of the observed selectivity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.5b00680DOI Listing
September 2015

A novel multiple-stage antimalarial agent that inhibits protein synthesis.

Nature 2015 06;522(7556):315-20

Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature14451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700930PMC
June 2015

Fragment-based hit discovery and structure-based optimization of aminotriazoloquinazolines as novel Hsp90 inhibitors.

Bioorg Med Chem 2014 Aug 14;22(15):4135-50. Epub 2014 Jun 14.

Oncology, Nerviano Medical Sciences, Viale Pasteur 10, 20014 Nerviano (MI), Italy. Electronic address:

In the last decade the heat shock protein 90 (Hsp90) has emerged as a major therapeutic target and many efforts have been dedicated to the discovery of Hsp90 inhibitors as new potent anticancer agents. Here we report the identification of a novel class of Hsp90 inhibitors by means of a biophysical FAXS-NMR based screening of a library of fragments. The use of X-ray structure information combined with modeling studies enabled the fragment evolution of the initial triazoloquinazoline hit to a class of compounds with nanomolar potency and drug-like properties suited for further lead optimization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2014.05.056DOI Listing
August 2014

Discovery of NMS-E973 as novel, selective and potent inhibitor of heat shock protein 90 (Hsp90).

Bioorg Med Chem 2013 Nov 19;21(22):7047-63. Epub 2013 Sep 19.

Nerviano Medical Sciences S.r.l., Oncology, Viale Pasteur 10, 20014 Nerviano, MI, Italy. Electronic address:

Novel small molecule inhibitors of heat shock protein 90 (Hsp90) were discovered with the help of a fragment based drug discovery approach (FBDD) and subsequent optimization with a combination of structure guided design, parallel synthesis and application of medicinal chemistry principles. These efforts led to the identification of compound 18 (NMS-E973), which displayed significant efficacy in a human ovarian A2780 xenograft tumor model, with a mechanism of action confirmed in vivo by typical modulation of known Hsp90 client proteins, and with a favorable pharmacokinetic and safety profile.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2013.09.018DOI Listing
November 2013

NMS-E973, a novel synthetic inhibitor of Hsp90 with activity against multiple models of drug resistance to targeted agents, including intracranial metastases.

Clin Cancer Res 2013 Jul 14;19(13):3520-32. Epub 2013 May 14.

Department of Biotechnology, Nerviano Medical Sciences Srl, Nerviano (MI), Italy.

Purpose: Recent developments of second generation Hsp90 inhibitors suggested a potential for development of this class of molecules also in tumors that have become resistant to molecular targeted agents. Disease progression is often due to brain metastases, sometimes related to insufficient drug concentrations within the brain. Our objective was to identify and characterize a novel inhibitor of Hsp90 able to cross the blood-brain barrier (BBB).

Experimental Design: Here is described a detailed biochemical and crystallographic characterization of NMS-E973. Mechanism-based anticancer activity was described in cell models, including models of resistance to kinase inhibitors. Pharmacokinetics properties were followed in plasma, tumor, liver, and brain. In vivo activity and pharmacodynamics, as well as the pharmacokinetic/pharmacodynamic relationships, were evaluated in xenografts, including an intracranially implanted melanoma model.

Results: NMS-E973, representative of a novel isoxazole-derived class of Hsp90 inhibitors, binds Hsp90α with subnanomolar affinity and high selectivity towards kinases, as well as other ATPases. It possesses potent antiproliferative activity against tumor cell lines and a favorable pharmacokinetic profile, with selective retention in tumor tissue and ability to cross the BBB. NMS-E973 induces tumor shrinkage in different human tumor xenografts, and is highly active in models of resistance to kinase inhibitors. Moreover, consistent with its brain penetration, NMS-E973 is active also in an intracranially implanted melanoma model.

Conclusions: Overall, the efficacy profile of NMS-E973 suggests a potential for development in different clinical settings, including tumors that have become resistant to molecular targeted agents, particularly in cases of tumors which reside beyond the BBB.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-12-3512DOI Listing
July 2013

Structure-based optimization of potent PDK1 inhibitors.

Bioorg Med Chem Lett 2010 Jul 8;20(14):4095-9. Epub 2010 Jun 8.

Nerviano Medical Sciences Srl, Viale Pasteur 10, 20014 Nerviano, Milano, Italy.

In this Letter is described the structure-based design of potent dihydro-pyrazoloquinazolines as PDK1 inhibitors. Starting from low potency HTS hits with the aid of X-ray crystallography and modeling, a medicinal chemistry activity was carried out to improve potency versus PDK1 and selectivity versus CDK2 protein kinase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2010.05.070DOI Listing
July 2010

Through the "gatekeeper door": exploiting the active kinase conformation.

J Med Chem 2010 Apr;53(7):2681-94

Department of Chemical Core Technologies, Nerviano Medical Sciences, Oncology, Viale Pasteur 10, Nerviano 20014, Italy.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm901443hDOI Listing
April 2010

2,4-Diaminopyrimidines as inhibitors of Leishmanial and Trypanosomal dihydrofolate reductase.

Bioorg Med Chem 2003 Nov;11(22):4693-711

Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, CF10 3XF Cardiff, UK.

This paper describes the synthesis of 4'-substituted and 3',4'-disubstituted 5-benzyl-2,4-diaminopyrimidines as selective inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Compounds were then assayed against the recombinant parasite and human enzymes. Some of the compounds showed good activity. They were also tested against the intact parasites using in vitro assays. Good activity was found against Trypanosoma cruzi, moderate activity against Trypanosoma brucei and Leishmania donovani. Molecular modeling was undertaken to explain the results. The leishmanial enzyme was found to have a more extensive lipophilic binding region in the active site than the human enzyme. Compounds which bound within the pocket showed the highest selectivity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2003.08.012DOI Listing
November 2003

Pharmacophore features distributions in different classes of compounds.

Authors:
Fabio Zuccotto

J Chem Inf Comput Sci 2003 Sep-Oct;43(5):1542-52

Inpharmatica Ltd, Molecular Design, 60 Charlotte Street, London, W1T 2NU, United Kingdom.

A pharmacophore analysis approach was used to investigate and compare different classes of compounds relevant to the drug discovery process (specifically, drug molecules, compounds in high throughput screening libraries, combinatorial chemistry building blocks and nondrug molecules). The distributions for a set of pharmacophore features including hydrogen bond acceptors, hydrogen bond donors, negatively ionizable centers, positively ionizable centers and hydrophobic points, were generated and examined. Significant differences were observed between the pharmacophore profiles obtained for the drug molecules and those obtained for the high-throughput screening compounds, which appear to be closely related to the nondrug pharmacophore distribution. It is suggested that the analysis of pharmacophore profiles could be used as an additional tool for the property-based optimization of compound selection and library design processes, thus improving the odds of success in lead discovery projects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ci034068kDOI Listing
July 2004