A Fluorescence-Based Assay for Screening β-Lactams Targeting the Mycobacterium tuberculosis Transpeptidase LdtMt2

Mycobacterium tuberculosis l,d -transpeptidases (Ldts), which are involved in cell-wall biosynthesis, have emerged as promising targets for the treatment of tuberculosis. However, an efficient method for testing inhibition of these enzymes is not currently available. We present a fluorescence-based assay for Ldt_Mt2, which is suitable for high-throughput screening. Two fluorogenic probes were identified that release a fluorophore upon reaction with Ldt_Mt2, thus making it possible to assess the availability of the catalytic site in the presence of inhibitors. The assay was applied to a panel of β-lactam antibiotics and related inhibitors; the results validate observations that the (carba)penem subclass of β-lactams are more potent Ldt inhibitors than other β-lactam classes, though unexpected variations in potency were observed. The method will enable systematic structure–activity relationship studies on Ldts, thereby facilitating the identification of new antibiotics active against M. tuberculosis.     

Thiazolopyrimidine Inhibitors of 2‐Methylerythritol 2,4‐Cyclodiphosphate Synthase (IspF) from Mycobacterium tuberculosis and Plasmodium falciparum

A library of 40 000 compounds was screened for inhibitors of 2‐methylerythritol 2,4‐cyclodiphosphate synthase (IspF) protein from Arabidopsis thaliana using a photometric assay. A thiazolopyrimidine derivative resulting from the high‐throughput screen was found to inhibit the IspF proteins of Mycobacterium tuberculosis, Plasmodium falciparum, and A. thaliana with IC₅₀ values in the micromolar range. Synthetic efforts afforded derivatives that inhibit IspF protein from M. tuberculosis and P. falciparum with IC₅₀ values in the low micromolar range. Several compounds act as weak inhibitors in the P. falciparum red blood cell assay.     

Discovery of Plasmepsin Inhibitors by Fragment‐Based Docking and Consensus Scoring

Plasmepsins (PMs) are essential proteases of the plasmodia parasites and are therefore promising targets for developing drugs against malaria. We have discovered six inhibitors of PM II by high‐throughput fragment‐based docking of a diversity set of ～40 000 molecules, and consensus scoring with force field energy functions. Using the common scaffold of the three most active inhibitors (IC₅₀=2–5 μM ), another seven inhibitors were identified by substructure search. Furthermore, these 13 inhibitors belong to at least three different classes of compounds. The in silico approach was very effective since a total of 13 active compounds were discovered by testing only 59 molecules in an enzymatic assay. This hit rate is about one to two orders of magnitude higher than those reported for medium‐ and high‐throughput screening techniques in vitro. Interestingly, one of the inhibitors identified by docking was halofantrine, an antimalarial drug of unknown mechanism. Explicit water molecular dynamics simulations were used to discriminate between two putative binding modes of halofantrine in PM II.     

Integrated Target-Based and Phenotypic Screening Approaches for the Identification of Anti-Tubercular Agents That Bind to the Mycobacterial Adenylating Enzyme MbtA

Iron is essential for the pathogenicity and virulence of Mycobacterium tuberculosis, which synthesises salicyl-capped siderophores (mycobactins) to acquire this element from the host. MbtA is the adenylating enzyme that catalyses the initial reaction of mycobactin biosynthesis and is solely expressed by mycobacteria. A 3200-member library comprised of lead-like, structurally diverse compounds was screened against M. tuberculosis for whole-cell inhibitory activity. A set of 846 compounds that inhibited the tubercle bacilli growth were then tested for their ability to bind to MbtA using a fluorescence-based thermal shift assay and NMR-based Water-LOGSY and saturation transfer difference (STD) experiments. We identified an attractive hit molecule, 5-hydroxyindol-3-ethylamino-(2-nitro-4-trifluoromethyl)benzene ( 5 ), that bound with high affinity to MbtA and produced a MIC₉₀ value of 13 μm . The ligand was docked into the MbtA crystal structure and displayed an excellent fit within the MbtA active pocket, adopting a binding mode different from that of the established MbtA inhibitor Sal-AMS.     

N‐Phenylbenzamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore

Persistent opening of the mitochondrial permeability transition pore (PTP), an inner membrane channel, leads to mitochondrial dysfunction and renders the PTP a therapeutic target for a host of life‐threatening diseases. Herein, we report our effort toward identifying small‐molecule inhibitors of this target through structure–activity relationship optimization studies, which led to the identification of several potent analogues around the N‐phenylbenzamide compound series identified by high‐throughput screening. In particular, compound 4 (3‐(benzyloxy)‐5‐chloro‐N‐(4‐(piperidin‐1‐ylmethyl)phenyl)benzamide) displayed noteworthy inhibitory activity in the mitochondrial swelling assay (EC₅₀=280 nm ), poor‐to‐very‐good physicochemical as well as in vitro pharmacokinetic properties, and conferred very high calcium retention capacity to mitochondria. From the data, we believe compound 4 in this series represents a promising lead for the development of PTP inhibitors of pharmacological relevance.     

Nitroarenes as Antitubercular Agents: Stereoelectronic Modulation to Mitigate Mutagenicity

Nitroarenes are less preferred in drug discovery due to their potential to be mutagenic. However, several nitroarenes were shown to be promising antitubercular agents with specific modes of action, namely, nitroimidazoles and benzothiazinones. The nitro group in these compounds is activated through different mechanisms, both enzymatic and non‐enzymatic, in mycobacteria prior to binding to the target of interest. From a whole‐cell screening program, we identified a novel lead nitrobenzothiazole (BT) series that acts by inhibition of decaprenylphosphoryl‐β‐d ‐ribose 2′‐epimerase (DprE1) of Mycobacterium tuberculosis (Mtb). The lead was found to be mutagenic to start with. Our efforts to mitigate mutagenicity resulted in the identification of 6‐methyl‐7‐nitro‐5‐(trifluoromethyl)‐1,3‐benzothiazoles (cBTs), a novel class of antitubercular agents that are non‐mutagenic and exhibit an improved safety profile. The methyl group ortho to the nitro group decreases the electron affinity of the series, and is hence responsible for the non‐mutagenic nature of these compounds. Additionally, the co‐crystal structure of cBT in complex with Mtb DprE1 established the mode of binding. This investigation led to a new non‐mutagenic antitubercular agent and demonstrates that the mutagenic nature of nitroarenes can be solved by modulation of stereoelectronic properties.     

2‐Substituted 6‐(Het)aryl‐7‐deazapurine Ribonucleosides: Synthesis,Inhibition of Adenosine Kinases,and Antimycobacterial Activity

A series of 6‐(hetero)aryl‐ or 6‐methyl‐7‐deazapurine ribonucleosides bearing a substituent at position 2 (Cl, F, NH₂, or CH₃) were prepared by cross‐coupling reactions at position 6 and functional group transformations at position 2. Cytostatic, antiviral, and antimicrobial activity assays were performed. The title compounds were observed to be potent and selective inhibitors of Mycobacterium tuberculosis adenosine kinase (ADK), but not human ADK; moreover, they were found to be non‐cytotoxic. The antimycobacterial activities against M. tuberculosis, however, were only moderate. The reason for this could be due to either poor uptake through the cell wall or to parallel biosynthesis of adenosine monophosphate by the salvage pathway.     

Targeting tumour proliferation with a small-molecule inhibitor of AICAR transformylase homodimerization

Aminoimidazole carboxamide ribonucleotide transformylase/ inosine monophosphate cyclohydrolase (ATIC) is a bifunctional homodimeric enzyme that catalyzes the last two steps of de novo purine biosynthesis. Homodimerization of ATIC, a protein–protein interaction with an interface of over 5000 Ų, is required for its aminoimidazole carboxamide ribonucleotide (AICAR) transformylase activity, with the active sites forming at the interface of the interacting proteins. Here, we report the development of a small‐molecule inhibitor of AICAR transformylase that functions by preventing the homodimerization of ATIC. The compound is derived from a previously reported cyclic hexapeptide inhibitor of AICAR transformylase (with a K_i of 17 μM ), identified by high‐throughput screening. The active motif of the cyclic peptide is identified as an arginine‐tyrosine dipeptide, a capped analogue of which inhibits AICAR transformylase with a K_i value of 84 μM . A library of nonnatural analogues of this dipeptide was designed, synthesized, and assayed. The most potent compound inhibits AICAR transformylase with a K_i value of 685 nM , a 25‐fold improvement in activity from the parent cyclic peptide. The potential for this AICAR transformylase inhibitor in cancer therapy was assessed by studying its effect on the proliferation of a model breast cancer cell line. Using a nonradioactive proliferation assay and live cell imaging, a dose‐dependent reduction in cell numbers and cell division rates was observed in cells treated with our ATIC dimerization inhibitor.     

Synthesis of Long-Chain β-Lactones and Their Antibacterial Activities against Pathogenic Mycobacteria

In the quest for new antibacterial agents, a series of novel long- and medium-chain mono- and disubstituted β-lactones was developed. Their activity against three pathogenic mycobacteria—M. abscessus, M. marinum, and M. tuberculosis—was assessed by the resazurin microtiter assay (REMA). Among the 16 β-lactones synthesized, only 3-hexadecyloxetan-2-one (VM005) exhibited promising activity against M. abscessus, whereas most of the β-lactones showed interesting activities against M. marinum, similar to that of the classical antibiotic, isoniazid. Regarding M. tuberculosis, six compounds were found to be active against this mycobacterium, with β-lactone VM008 [trans-(Z)-3-(hexadec-7-en-1-yl)-4-propyloxetan-2-one] being the best growth inhibitor. The promising antibacterial activities of the best compounds in this series suggest that these molecules may serve as leads for the development of much more efficient antimycobacterial agents.     

Triazolophthalazines: Easily Accessible Compounds with Potent Antitubercular Activity

Tuberculosis (TB) remains one of the major causes of death worldwide, in particular because of the emergence of multidrug‐resistant TB. Herein we explored the potential of an alternative class of molecules as anti‐TB agents. Thus, a series of novel 3‐substituted triazolophthalazines was quickly and easily prepared from commercial hydralazine hydrochloride as starting material and were further evaluated for their antimycobacterial activities and cytotoxicities. Four of the synthesized compounds were found to effectively inhibit the Mycobacterium tuberculosis (M.tb) H₃₇Rv strain with minimum inhibitory concentration (MIC) values <10 μg mL^?1, whereas no compounds displayed cytotoxicity against HCT116 human cell lines (IC₅₀>100 μm ). More remarkably, the most potent compounds proved to be active to a similar extent against various multidrug‐resistant M.tb strains, thus uncovering a mode of action distinct from that of standard antitubercular agents. Overall, their ease of preparation, combined with their attractive antimycobacterial activities, make such triazolophthalazine‐based derivatives promising leads for further development.     

A high‐throughput assay for screening modifiers of calcium oxalate crystallization

Controlling crystal habit using growth modifiers provides novel avenues for tailoring properties of crystalline materials. Here, we report on the design of a high‐throughput screening assay for rapid identification of growth modifiers using calcium oxalate monohydrate crystallization as a model system. We conducted a systematic study of assay parameters (sample volume, shaking, and temperature) on crystallization kinetics. Crystallization half‐time (t_1/2), defined as the time at which crystallization is 50% complete, was obtained from the logistic fit of kinetic data and used as a measure of growth modifier potency. A test library of 13 peptides composed of aspartic acid and alanine residues was screened to determine their growth promotion or inhibition potentials. Leads identified from this study are in good agreement with ion‐selective electrode measurements and a single time point measurement of free calcium ion concentration is an excellent end‐point for evaluating modifier potency. © 2016 American Institute of Chemical Engineers AIChE J, 62: 3538–3546, 2016     

Reversed‐Phase Liquid Chromatography–Quadrupole‐Time‐of‐Flight Mass Spectrometry for High‐Throughput Molecular Profiling of Sea Cucumber Cerebrosides

Usually, the chemical structures of cerebrosides in sea creatures are more complicated than those from terrestrial plants and animals. Very little is known about the method for high‐throughput molecular profiling of cerebrosides in sea cucumbers. In this study, cerebrosides from four species of edible sea cucumbers, specifically, Apostichopus japonicas, Thelenota ananas, Acaudina molpadioides and Bohadschia marmorata, were rapidly identified using reversed‐phase liquid chromatography–quadrupole‐time‐of‐flight mass spectrometry (RPLC‐QToF‐MS). [M + H]⁺ in positive electrospray ionization (ESI) mode were used to obtain the product ion spectra. The cerebroside molecules were selected according to the neutral loss fragments of 180 Da and then identified according to pairs of specific products of long‐chain bases (LCB) and their precursor ions. A typical predominant LCB was 2‐amino‐1,3‐dihydroxy‐4‐heptadecene (d17:1), which was acylated to form saturated and monounsaturated non‐hydroxy and monohydroxy fatty acids with 17–25 carbon atoms. Simultaneously, the occurrence of 2‐hydroxy‐tricosenoic acid (C23:1h) was characteristic of sea cucumber cerebrosides, whereas this molecule was rarely discovered in plants, mammals, or fungi. The profiles of LCB and fatty acids (FA) distribution might be related to the genera of sea cucumber. These data will be useful for identification of cerebrosides using RPLC‐QToF‐MS.     

Identification and Optimization of the First Highly Selective GLUT1 Inhibitor BAY‐876

Despite the long‐known fact that the facilitative glucose transporter GLUT1 is one of the key players safeguarding the increase in glucose consumption of many tumor entities even under conditions of normal oxygen supply (known as the Warburg effect), only few endeavors have been undertaken to find a GLUT1‐selective small‐molecule inhibitor. Because other transporters of the GLUT1 family are involved in crucial processes, these transporters should not be addressed by such an inhibitor. A high‐throughput screen against a library of ～3 million compounds was performed to find a small molecule with this challenging potency and selectivity profile. The N‐(1H‐pyrazol‐4‐yl)quinoline‐4‐carboxamides were identified as an excellent starting point for further compound optimization. After extensive structure–activity relationship explorations, single‐digit nanomolar inhibitors with a selectivity factor of >100 against GLUT2, GLUT3, and GLUT4 were obtained. The most promising compound, BAY‐876 [N⁴‐[1‐(4‐cyanobenzyl)‐5‐methyl‐3‐(trifluoromethyl)‐1H‐pyrazol‐4‐yl]‐7‐fluoroquinoline‐2,4‐dicarboxamide], showed good metabolic stability in vitro and high oral bioavailability in vivo.     

Identification of the first known inhibitors of O-acetylpeptidoglycan esterase: a potential new antibacterial target

The O‐acetylation of peptidoglycan (PG) is now known to occur in 53 species, including numerous human pathogens such as, Staphylococcus aureus, Bacillus anthracis, species of Enterococcus, Campylobacter jejuni, Helicobacter pylori, Neisseria gonorrhoeae and N. meningitidis. This modification, which occurs at the C‐6 hydroxyl of N‐acetylmuramoyl residues within PG, serves to regulate autolytic activity during PG metabolism and contributes to pathogenesis and persistence within a host. O‐Acetylpeptidoglycan esterase (Ape) was recently discovered as an enzyme responsible for the removal of O‐acetyl groups from PG, thus permitting the continued maintenance and metabolism of the sacculus. Recombinant Ape1 from N. gonorrhoeae was purified to apparent homogeneity and optimal storage conditions for the enzyme were determined. Using 4‐methylumbelliferyl acetate as substrate, a fluorogenic assay amenable for the high‐throughput screening for potential inhibitors was developed and Ape1 was screened against a subset of compounds of the Canadian Compound Collection. The overall Z′ score for the screen was 0.62, indicative of a well‐suited assay with a sufficient signal window, and the threshold was set at 65 %. After eliminating a number of false‐positives, seven compounds were identified as true inhibitors of Ape1, the first to be identified for this class of enzyme. Dose–response curves were generated leading to the identification of five of these compounds with IC₅₀ values ranging between 0.3 and 23 μM . Of these, purpurin was selected for further analysis and it was found to inhibit the growth of both Gram‐positive and Gram‐negative bacteria that produce both O‐acetylated PG and Ape.     

Discovery and Biophysical Evaluation of First Low Nanomolar Hits Targeting CYP125 of M. tuberculosis

Tuberculosis, which is predominantly caused by Mycobacterium tuberculosis (Mtb), is still the most lethal bacterial infection with 1.5 million casualties in 2014. Moreover, the fact that the appearance of resistant mutants and long‐term treatment are coupled with economic problems in developing countries hampers an efficient therapy. Interference with the essential cholesterol metabolism of Mtb could be a promising novel strategy to fight Mtb infections. CYP125, a P450 enzyme in Mtb, has been shown to play an important role in this metabolic pathway. For this reason, we used a combined screening approach involving surface plasmon resonance spectroscopy and a heme coordination assay to identify new CYP125 binders by employing a focused P450‐inhibitor library. We identified the first hits with high affinity and favorable ligand efficiencies. Furthermore, frontrunner compounds also showed selectivity toward CYP121 specific to Mtb and required for its survival. To date, these are the first compounds targeting CYP125 with low nanomolar affinity.     

3‐Substituted Indazoles as Configurationally Locked 4EGI‐1 Mimetics and Inhibitors of the eIF4E/eIF4G Interaction

4EGI‐1, the prototypic inhibitor of eIF4E/eIF4G interaction, was identified in a high‐throughput screening of small‐molecule libraries with the aid of a fluorescence polarization assay that measures inhibition of binding of an eIF4G‐derived peptide to recombinant eIF4E. As such, the molecular probe 4EGI‐1 has potential for the study of molecular mechanisms involved in human disorders characterized by loss of physiological restraints on translation initiation. A hit‐to‐lead optimization campaign was carried out to overcome the configurational instability in 4EGI‐1, which stems from the E‐to‐Z isomerization of the hydrazone function. We identified compound 1 a , in which the labile hydrazone was incorporated into a rigid indazole scaffold, as a promising rigidified 4EGI‐1 mimetic lead. In a structure–activity relationship study directed towards probing the structural latitude of this new chemotype as an inhibitor of eIF4E/eIF4G interaction and translation initiation we identified 1 d , an indazole‐based 4EGI‐1 mimetic, as a new and improved lead inhibitor of eIF4E/eIF4G interaction and a promising molecular probe candidate for elucidation of the role of cap‐dependent translation initiation in a host of pathophysiological states.     

α‐Keto Phenylamides as P1′‐Extended Proteasome Inhibitors

The major challenge for proteasome inhibitor design lies in achieving high selectivity for, and activity against, the target, which requires specific interactions with the active site. Novel ligands aim to overcome off‐target‐related side effects such as peripheral neuropathy, which is frequently observed in cancer patients treated with the FDA‐approved proteasome inhibitors bortezomib ( 1 ) or carfilzomib ( 2 ). A systematic comparison of electrophilic headgroups recently identified the class of α‐keto amides as promising for next generation drug development. On the basis of crystallographic knowledge, we were able to develop a structure–activity relationship (SAR)‐based approach for rational ligand design using an electronic parameter (Hammett’s σ) and in silico molecular modeling. This resulted in the tripeptidic α‐keto phenylamide BSc4999 [(S)‐3‐(benzyloxycarbonyl‐(S)‐leucyl‐(S)‐leucylamino)‐5‐methyl‐2‐oxo‐N‐(2,4‐dimethylphenyl)hexanamide, 6 a ], a highly potent (IC₅₀=38 nM ), cell‐permeable, and slowly reversible covalent inhibitor which targets both the primed and non‐primed sites of the proteasome’s substrate binding channel as a special criterion for selectivity. The improved inhibition potency and selectivity of this new α‐keto phenylamide makes it a promising candidate for targeting a wider range of tumor subtypes than commercially available proteasome inhibitors and presents a new candidate for future studies.     

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.     

Materials genomics‐guided ab initio screening of MOFs with open copper sites for acetylene storage

Discovering high‐performance metal‐organic frameworks (MOFs) with open metal sites has become an increasingly hot research topic in the field of safe storage and transportation of acetylene. Following the concept of Materials Genomics proposed recently, a database of 502 experimental MOFs was built by searching the structures deposited in the CSD with the dicopper paddle‐wheel node Cu₂(COO)₄ as the characteristic materials gene. On the basis of the developed ab initio force field, a high‐throughput computational screening was conducted to examine the property‐performance relationships of MOFs containing Cu‐OMS for C₂H₂ storage at ambient conditions. The optimal ranges of the structural and energetic features for the design of such MOFs were suggested. From our computational screening, three potentially promising MOFs were identified which exhibit a performance outperforming those MOFs reported experimentally so far with record high gravimetric C₂H₂ uptakes, both in the total and deliverable adsorption capacities. © 2017 American Institute of Chemical Engineers AIChE J, 64: 1389–1398, 2018