Fragment‐Based Phenotypic Lead Discovery: Cell‐Based Assay to Target Leishmaniasis

A rapid and practical approach for the discovery of new chemical matter for targeting pathogens and diseases is described. Fragment‐based phenotypic lead discovery (FPLD) combines aspects of traditional fragment‐based lead discovery (FBLD), which involves the screening of small‐molecule fragment libraries to target specific proteins, with phenotypic lead discovery (PLD), which typically involves the screening of drug‐like compounds in cell‐based assays. To enable FPLD, a diverse library of fragments was first designed, assembled, and curated. This library of soluble, low‐molecular‐weight compounds was then pooled to expedite screening. Axenic cultures of Leishmania promastigotes were screened, and single hits were then tested for leishmanicidal activity against intracellular amastigote forms in infected murine bone‐marrow‐derived macrophages without evidence of toxicity toward mammalian cells. These studies demonstrate that FPLD can be a rapid and effective means to discover hits that can serve as leads for further medicinal chemistry purposes or as tool compounds for identifying known or novel targets.     

Fragment‐Based Lead Discovery: Screening and Optimizing Fragments for Thermolysin Inhibition

Fragment‐based drug discovery has gained a foothold in today's lead identification processes. We present the application of in silico fragment‐based screening for the discovery of novel lead compounds for the metalloendoproteinase thermolysin. We have chosen thermolysin to validate our screening approach as it is a well‐studied enzyme and serves as a model system for other proteases. A protein‐targeted virtual library was designed and screening was carried out using the program AutoDock. Two fragment hits could be identified. For one of them, the crystal structure in complex with thermolysin is presented. This compound was selected for structure‐based optimization of binding affinity and improvement of ligand efficiency, while concomitantly keeping the fragment‐like properties of the initial hit. Redesigning the zinc coordination group revealed a novel class of fragments possessing K_i values as low as 128 μM , thus they provide a good starting point for further hit evolution in a tailored lead design.     

Monitoring Binding of HIV‐1 Capsid Assembly Inhibitors Using 19F Ligand‐and 15N Protein‐Based NMR and X‐ray Crystallography: Early Hit Validation of a Benzodiazepine Series

The emergence of resistance to existing classes of antiretroviral drugs underlines the need to find novel human immunodeficiency virus (HIV)‐1 targets for drug discovery. The viral capsid protein (CA) represents one such potential target. Recently, a series of benzodiazepine inhibitors was identified via high‐throughput screening using an in vitro capsid assembly assay (CAA). Here, we demonstrate how a combination of NMR and X‐ray co‐crystallography allowed for the rapid characterization of the early hits from this inhibitor series. Ligand‐based ¹⁹F NMR was used to confirm inhibitor binding specificity and reversibility as well as to identify the N‐terminal domain of the capsid (CA_NTD) as its molecular target. Protein‐based NMR (¹H and ¹⁵N chemical shift perturbation analysis) identified key residues within the CA_NTD involved in inhibitor binding, while X‐ray co‐crystallography confirmed the inhibitor binding site and its binding mode. Based on these results, two conformationally restricted cyclic inhibitors were designed to further validate the possible binding modes. These studies were crucial to early hit confirmation and subsequent lead optimization.     

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

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.     

Discovery of Mono‐ and Disubstituted 1H‐Pyrazolo[3,4]pyrimidines and 9H‐Purines as Catalytic Inhibitors of Human DNA Topoisomerase IIα

Human DNA topoisomerase IIα (htIIα) is a validated target for the development of anticancer agents. Based on structural data regarding the binding mode of AMP‐PNP (5′‐adenylyl‐β,γ‐imidodiphosphate) to htIIα, we designed a two‐stage virtual screening campaign that combines structure‐based pharmacophores and molecular docking. In the first stage, we identified several monosubstituted 9H‐purine compounds and a novel class of 1H‐pyrazolo[3,4]pyrimidines as inhibitors of htIIα. In the second stage, disubstituted analogues with improved cellular activities were discovered. Compounds from both classes were shown to inhibit htIIα‐mediated DNA decatenation, and surface plasmon resonance (SPR) experiments confirmed binding of these two compounds on the htIIα ATPase domain. Proposed complexes and interaction patterns between both compounds and htIIα were further analyzed in molecular dynamics simulations. Two compounds identified in the second stage showed promising anticancer activities in hepatocellular carcinoma (HepG2) and breast cancer (MCF‐7) cell lines. The discovered compounds are suitable starting points for further hit‐to‐lead development in anticancer drug discovery.     

Biofragments: An Approach towards Predicting Protein Function Using Biologically Related Fragments and its Application to Mycobacterium tuberculosis CYP126

We present a novel fragment‐based approach that tackles some of the challenges for chemical biology of predicting protein function. The general approach, which we have termed biofragments, comprises two key stages. First, a biologically relevant fragment library (biofragment library) can be designed and constructed from known sets of substrate‐like ligands for a protein class of interest. Second, the library can be screened for binding to a novel putative ligand‐binding protein from the same or similar class, and the characterization of hits provides insight into the basis of ligand recognition, selectivity, and function at the substrate level. As a proof‐of‐concept, we applied the biofragments approach to the functionally uncharacterized Mycobacterium tuberculosis (Mtb) cytochrome P450 isoform, CYP126. This led to the development of a tailored CYP biofragment library with notable 3D characteristics and a significantly higher screening hit rate (14 %) than standard drug‐like fragment libraries screened previously against Mtb CYP121 and 125 (4 % and 1 %, respectively). Biofragment hits were identified that make both substrate‐like type‐I and inhibitor‐like type‐II interactions with CYP126. A chemical‐fingerprint‐based substrate model was built from the hits and used to search a virtual TB metabolome, which led to the discovery that CYP126 has a strong preference for the recognition of aromatics and substrate‐like type‐I binding of chlorophenol moieties within the active site near the heme. Future catalytic analyses will be focused on assessing CYP126 for potential substrate oxidative dehalogenation.     

Development of Fragment‐Based n‐FABS NMR Screening Applied to the Membrane Enzyme FAAH

Despite the recognized importance of membrane proteins as pharmaceutical targets, the reliable identification of fragment hits that are able to bind these proteins is still a major challenge. Among different ¹⁹F NMR spectroscopic methods, n‐fluorine atoms for biochemical screening (n‐FABS) is a highly sensitive technique that has been used efficiently for fragment screening, but its application for membrane enzymes has not been reported yet. Herein, we present the first successful application of n‐FABS to the discovery of novel fragment hits, targeting the membrane‐bound enzyme fatty acid amide hydrolase (FAAH), using a library of fluorinated fragments generated based on the different local environment of fluorine concept. The use of the recombinant fusion protein MBP‐FAAH and the design of compound 11 as a suitable novel fluorinated substrate analogue allowed n‐FABS screening to be efficiently performed using a very small amount of enzyme. Notably, we have identified 19 novel fragment hits that inhibit FAAH with a median effective concentration (IC₅₀) in the low mM –μM range. To the best of our knowledge, these results represent the first application of a ¹⁹F NMR fragment‐based functional assay to a membrane protein.     

The Discovery and Development of the N‐Substituted trans‐3,4‐Dimethyl‐4‐(3′‐hydroxyphenyl)piperidine Class of Pure Opioid Receptor Antagonists

N‐Substituted trans‐3,4‐dimethyl‐4‐(3‐hydroxyphenyl)piperidines are a class of pure opioid receptor antagonists with a novel pharmacophore. This opioid receptor antagonist pharmacophore was used as a lead structure to design and develop several interesting and useful opioid receptor antagonists. In this review we describe: 1) early SAR studies that led to the discovery of LY255582 and analogues that are nonselective opioid receptor antagonists developed for the treatment of obesity; 2) the discovery and commercialization of LY246736 (alvimopan; ENTEREG®), a peripherally selective opioid receptor antagonist that accelerates the time to upper and lower GI recovery following surgeries that include partial bowel resection with primary anastomosis; and 3) the discovery and development of the potent and selective κ opioid receptor antagonist JDTic and analogues as potential pharmacotherapies for treating depression, anxiety, and substance abuse (nicotine, alcohol, and cocaine). In addition, the use of JDTic for obtaining the X‐ray structure of the human κ opioid receptor is discussed.     

Preparation and characterization of mPEG‐g‐α‐zein biohybrid micelles as a nano‐carrier

A new kind of block copolymer micelles methoxy polyethylene glycol (mPEG) grafted α‐zein protein (mPEG‐g‐α‐zein) was synthesized. The chemical composition of mPEG‐g‐α‐zein was identified with the help of FT‐IR and ¹H‐NMR. The biohybrid polymer can self‐assemble into spherical core–shell nanoparticles in aqueous solution. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to investigate the self‐assembled morphology of mPEG‐g‐α‐zein. Dynamic light scattering (DLS) results showed that the particle size of mPEG‐g‐α‐zein was about 90 nm. Moreover, the nanoparticles had a very low critical micelle concentration value with only 0.02 mg/mL. Then, the anticancer drug curcumin (CUR) was encapsulated into the biohybrid polymer micelles. The in vitro drug release profile showed a zero‐order release of CUR up to 12 h at 37°C. Cell viability studies revealed that the mPEG‐g‐α‐zein polymer exhibited low cytotoxicity for HepG2 cells (human hepatoma cells). Consequently, the mPEG‐g‐α‐zein micelles can be used as a potential nano‐carrier to encapsulate hydrophobic drugs and nutrients. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42555.     

Structure‐Guided Discovery of Antitubercular Agents That Target the Gyrase ATPase Domain

In this study we explored the pharmaceutically underexploited ATPase domain of DNA gyrase (GyrB) as a potential platform for developing novel agents that target Mycobacterium tuberculosis. In this effort a combination of ligand‐ and structure‐based pharmacophore modeling was used to identify structurally diverse small‐molecule inhibitors of the mycobacterial GyrB domain based on the crystal structure of the enzyme with a pyrrolamide inhibitor (PDB ID: 4BAE ). Pharmacophore modeling and subsequent in vitro screening resulted in an initial hit compound 5 [(E)‐5‐(5‐(2‐(1H‐benzo[d]imidazol‐2‐yl)‐2‐cyanovinyl)furan‐2‐yl)isophthalic acid; IC₅₀=4.6±0.1 μm ], which was subsequently tailored through a combination of molecular modeling and synthetic chemistry to yield the optimized lead compound 24 [(E)‐3‐(5‐(2‐cyano‐2‐(5‐methyl‐1H‐benzo[d]imidazol‐2‐yl)vinyl)thiophen‐2‐yl)benzoic acid; IC₅₀=0.3±0.2 μm ], which was found to display considerable in vitro efficacy against the purified GyrB enzyme and potency against the H₃₇Rv strain of M. tuberculosis. Structural handles were also identified that will provide a suitable foundation for further optimization of these potent analogues.     

Ergoline‐Derived Inverse Agonists of the Human H3 Receptor for the Treatment of Narcolepsy

Ergoline derivative (6aR,9R)‐4‐(2‐(dimethylamino)ethyl)‐N‐phenyl‐9‐(pyrrolidine‐1‐carbonyl)‐6,6a,8,9‐tetrahydroindolo[4,3‐fg]quinoline‐7(4H)‐carboxamide ( 1 ), a CXCR3 antagonist, also inhibits human histamine H3 receptors (H3R) and represents a structurally novel H3R inverse agonist chemotype. It displays favorable pharmacokinetic and in vitro safety profiles, and served as a lead compound in a program to explore ergoline derivatives as potential drug candidates for the treatment of narcolepsy. A key objective of this work was to enhance the safety and efficacy profiles of 1 , while minimizing its duration of action to mitigate the episodes of insomnia documented with previously reported clinical candidates during the night following administration. Modifications to the ergoline core at positions 1, 6 and 8 were systematically investigated, and derivative 23 (1‐((4aR,8R,9aR)‐8‐(hydroxymethyl)‐1‐(2‐((R)‐2‐methylpyrrolidin‐1‐yl)ethyl)‐4,4a,7,8,9,9a‐hexahydroindolo[1,14‐fg]quinolin‐6(1H)‐yl)ethanone) was identified as a promising lead compound. Derivative 23 has a desirable pharmacokinetic profile and demonstrated efficacy by enhancing brain concentrations of tele‐methylhistamine, a major histamine metabolite. This validates the potential of the ergoline scaffold to serve as a template for the development of H3R inverse agonists.     

Phage Display on the Anti‐infective Target 1‐Deoxy‐d‐xylulose‐5‐phosphate Synthase Leads to an Acceptor–Substrate Competitive Peptidic Inhibitor

Enzymes of the 2‐C‐methyl‐d ‐erythritol‐4‐phosphate pathway for the biosynthesis of isoprenoid precursors are validated drug targets. By performing phage display on 1‐deoxy‐d ‐xylulose‐5‐phosphate synthase (DXS), which catalyzes the first step of this pathway, we discovered several peptide hits and recognized false‐positive hits. The enriched peptide binder P12 emerged as a substrate (d ‐glyceraldehyde‐3‐phosphate)‐competitive inhibitor of Deinococcus radiodurans DXS. The results indicate possible overlap of the cofactor‐ and acceptor‐substrate‐binding pockets and provide inspiration for the design of inhibitors of DXS with a unique and novel mechanism of inhibition.     

Synthesis and characterization of 2‐hydroxyethylmethacrylate/2‐(3‐indol‐yl)ethylmethacrylamide‐based novel hydrogels as drug carrier with in vitro antibacterial properties

In this study, a new cationic monomer 2‐(3‐indol‐yl)ethylmethacrylamide (IEMA) derived from tryptamine was synthesized in a single step and characterized by Fourier transform infrared (FTIR), ¹H‐NMR, and ¹³C‐NMR. Then, one‐step preparation of novel poly[2‐hydroxyethylmethacrylate‐c‐2‐(3‐indol‐yl)ethylmethacrylamide], or p(HEMA‐c‐IEMA), copolymeric hydrogels has been performed successfully with IEMA and 2‐hydroxyethylmethacrylate (HEMA) as monomers using free radical aqueous polymerization. The hydrogels were characterized with scanning electron microscopy, FTIR, elemental analysis, thermogravimetric analysis, and texture profile analysis instruments. p(HEMA‐c‐IEMA) hydrogels were used for swelling, diffusion, drug release, and antibacterial activity studies. The drug‐release behavior of the hydrogels was determined as a function of time at 37 °C in pH 1.2 and 7.2. The swelling and drug‐release studies showed that an increased IEMA amount caused a higher increase in swelling and drug‐release values. Additionally, zero‐order, first‐order, and Higuchi equation kinetic models were applied to the drug‐release data, and the data fit well in the Higuchi model, and the Peppas power‐law model was applied to the release mechanism. Finally, the antibacterial activities of the hydrogels were screened against Gram‐positive bacteria (Bacillus cereus and Staphylococcus aureus) and Gram‐negative bacteria (Escherichia coli and Salmonella typhimurium). © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 45550.     

Preliminary Evaluation of Artemisinin–Cholesterol Conjugates as Potential Drugs for the Treatment of Intractable Forms of Malaria and Tuberculosis

To evaluate the feasibility of developing drugs that may be active against both malaria and tuberculosis (TB) by using in part putative cholesterol transporters in the causative pathogens and through enhancement of passive diffusion in granulomatous TB, artemisinin–cholesterol conjugates were synthesized by connecting the component molecules through various linkers. The compounds were screened in vitro against Plasmodium falciparum (Pf) and Mycobacterium tuberculosis (Mtb). Antimalarial activities (IC₅₀) against Pf drug‐sensitive NF54, and drug‐resistant K1 and W2 strains ranged from 0.03–2.6, 0.03–1.9, and 0.02–1.7 μm . Although the compounds are less active than the precursor artemisinin derivatives, the cholesterol moiety renders the compounds relatively insoluble in the culture medium, and variation in solubilities among the different compounds may reflect in the range of efficacies observed. Activities against Mtb H37Rv were assessed using a standardized colony‐forming unit (CFU) assay after 24 h pretreatment of cultures with each of the compounds. Percentage inhibition ranged from 3–38 % and 18–52 % at 10 and 80 μm , respectively. Thus, in contrast to the comparator drug artemether, the conjugates display enhanced activities. The immediate aims include the preparation of conjugates with enhanced aqueous solubilities, assays against malaria and TB in vivo, and for TB, assays using an infected macrophage model and assessment of granuloma influx.     

Discovery of Natural Products Possessing Selective Eukaryotic Readthrough Activity: 3‐epi‐Deoxynegamycin and Its Leucine Adduct

Herein we report the first discovery of natural readthrough products that do not display antimicrobial activity. Two natural negamycins, 3‐epi‐deoxynegamycin and its leucine adduct, isolated 37 years ago, were found to be potent readthrough agents against nonsense mutations of eukaryotes, but not prokaryotes, without displaying antimicrobial activity. These results suggest that the compounds are valuable leads for the development of readthrough drugs against nonsense‐mediated genetic diseases without the potential for contributing to the emergence of drug‐resistant bacteria.     

Identification of a Small‐Molecule Inhibitor of HIV‐1 Assembly that Targets the Phosphatidylinositol (4,5)‐bisphosphate Binding Site of the HIV‐1 Matrix Protein

The development of drug resistance remains a critical problem for current HIV‐1 antiviral therapies, creating a need for new inhibitors of HIV‐1 replication. We previously reported on a novel anti‐HIV‐1 compound, N²‐(phenoxyacetyl)‐N‐[4‐(1‐piperidinylcarbonyl)benzyl]glycinamide ( 14 ), that binds to the highly conserved phosphatidylinositol (4,5)‐bisphosphate (PI(4,5)P₂) binding pocket of the HIV‐1 matrix (MA) protein. In this study, we re‐evaluate the hits from the virtual screen used to identify compound 14 and test them directly in an HIV‐1 replication assay using primary human peripheral blood mononuclear cells. This study resulted in the identification of three new compounds with antiviral activity; 2‐(4‐{[3‐(4‐fluorophenyl)‐1,2,4‐oxadiazol‐5‐yl]methyl})‐1‐piperazinyl)‐N‐(4‐methylphenyl)acetamide ( 7 ), 3‐(2‐ethoxyphenyl)‐5‐[[4‐(4‐nitrophenyl)piperazin‐1‐yl]methyl]‐1,2,4‐oxadiazole ( 17 ), and N‐[4‐ethoxy‐3‐(1‐piperidinylsulfonyl)phenyl]‐2‐(imidazo[2,1‐b][1,3]thiazol‐6‐yl)acetamide ( 18 ), with compound 7 being the most potent of these hits. Mechanistic studies on 7 demonstrated that it directly interacts with and functions through HIV‐1 MA. In accordance with our drug target, compound 7 competes with PI(4,5)P₂ for MA binding and, as a result, diminishes the production of new virus. Mutation of residues within the PI(4,5)P₂ binding site of MA decreased the antiviral effect of compound 7 . Additionally, compound 7 displays a broadly neutralizing anti‐HIV activity, with IC₅₀ values of 7.5–15.6 μM for the group M isolates tested. Taken together, these results point towards a novel chemical probe that can be used to more closely study the biological role of MA and could, through further optimization, lead to a new class of anti‐HIV‐1 therapeutics.     

Encapsulation of diclofenac sodium with acidic copolymer hydrogels based on PEG/poly(N‐isopropylacrylamide‐co‐2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid) semi‐interpenetrating network using in situ loading technique

A pH‐ and temperature‐responsive semi‐interpenetrating copolymer PEG6000/poly(NIPA‐co‐AMPS) (PEG/AMPS‐co‐NIPA SIPN), for short PEG SIPN, was made by ammonium persulfate‐initiated suspension copolymerization of N‐isopropylacrylamide, 2‐acrylamido‐2‐methylpropanesulphonic acid, and N,N′‐methylene‐bis‐acrylamide (MBAA; crosslinker) in the presence of PEG6000. The PEG SIPN copolymer matrices containing nanostructures made in the high‐temperature copolymerization resulted in channels for PEG and facile migration of drugs. In drug encapsulation or drug‐loading process, one can easily ignore or pay less attention to the interaction between a drug and its encapsulation materials; however, the ignored interactions may induce problems in drug properties or the release behavior in use. Sodium diclofenac (DFNa) precipitates as the carboxylic acid form in an acidic environment, and it is challenging to encapsulate sodium diclofenac in such an acidic matrix without precipitation of the sparingly soluble acid form of DFNa on the surface of the polymer substrate. To avoid bulky precipitation in drug loading, an in situ loading technique was developed for producing gel spheres with DFNa uniformly distributed in the polymer matrix. The technique is based on fast polymerization of spherical droplets of a pregel solution in which the drug is dissolved. Diffusion‐loading prodrugs were made in comparison with in situ loading prodrugs in thermal, release kinetics, and release behavior. Drug release profiles (in pH 7.4 phosphate buffer) show that the new drug loading technique gives controlled release during a period of about 7 days at 37°C. By contrast, gel spheres loaded with sodium diclofenac using the conventional diffusion technique produced almost total release of the drug within about 24 h. The thermal stability of sodium diclofenac, the PEG/AMPS‐co‐NIPA SIPN, and the prodrugs made with the SIPN and sodium diclofenac was studied. A near zero‐order release kinetics was found in the in vitro release of sodium diclofenac with in situ loading PEG SIPN prodrug. We have, for the first time, studied sodium diclofenac release behavior from the PEG SIPN hydrogel systems. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Ribosomal Synthesis of Natural‐Product‐Like Bicyclic Peptides in Escherichia coli

Methods to access natural‐product‐like macrocyclic peptides can disclose new opportunities for the exploration of this important structural class for chemical biology and drug discovery applications. Here, the scope and mechanism of a novel strategy for directing the biosynthesis of thioether‐bridged bicyclic peptides in bacterial cells was investigated. This method entails split intein‐catalyzed head‐to‐tail cyclization of a ribosomally produced precursor peptide, combined with inter‐side‐chain crosslinking through a genetically encoded cysteine‐reactive amino acid. This strategy could be successfully applied to achieve formation of structurally diverse bicyclic peptides with high efficiency and selectivity in Escherichia coli. Insights into the sequence of reactions underlying the peptide bicyclization process were gained from time‐course experiments. Finally, the potential utility of this methodology toward the discovery of macrocyclic peptides with enhanced functional properties was demonstrated through the isolation of a bicyclic peptide with sub‐micromolar affinity for streptavidin.     

Enantiopure Indolizinoindolones with in vitro Activity against Blood‐ and Liver‐Stage Malaria Parasites

Malaria continues to be a major cause of morbidity and mortality to this day, and resistance to drugs like chloroquine has led to an urgent need to discover novel chemical entities aimed at new targets. Here, we report the discovery of a novel class of potential antimalarial compounds containing an indolizinoindolone scaffold. These novel enantiopure indolizinoindolones were synthesized, in good‐to‐excellent yields and excellent diastereoselectivities, by cyclocondensation reaction of (S)‐ or (R)‐tryptophanol and 2‐acyl benzoic acids, followed by intramolecular α‐amidoalkylation. Interestingly, we were able to synthesize for the first time 7,13b‐cis indolizinoindolones in a two‐step route. The novel compounds showed promising activity against erythrocytic stages of the human malaria parasite, Plasmodium falciparum, and liver stages of the rodent parasite Plasmodium berghei. In particular, an (S)‐tryptophanol‐derived isoindolinone was identified as a promising starting scaffold to search for novel antimalarials, combining excellent activity against both stages of the parasite′s life cycle with low cytotoxicity and excellent metabolic and chemical stability in vitro.