Meeting Proceedings from ICBS 2022 - Uncovering Solutions for Diseases.

ICBS 2022 was a refreshing multi-day event where it was justified that the advancement of chemical biology did not halt due to the pandemic, but in contrast, amazing findings were discovered within the restrictions of the SARS-CoV-2 pandemic. All aspects of this annual gathering reinforced that interconnecting the branches of chemical biology through collaboration, the sharing of ideas and knowledge, and networking are enabling the discovery and diversification of applications that will arm scientists of this world in "uncovering solutions for diseases."

Inhibition of Streptococcus pneumoniae growth by masarimycin.

Despite renewed interest, development of chemical biology methods to study peptidoglycan metabolism has lagged in comparison to the glycobiology field in general. To address this, a panel of diamides were screened against the Gram-positive bacterium Streptococcus pneumoniae to identify inhibitors of bacterial growth. The screen identified the diamide masarimycin as a bacteriostatic inhibitor of S. pneumoniae growth with an MIC of 8 µM. The diamide inhibited detergent-induced autolysis in a concentration-dependent manner, indicating perturbation of peptidoglycan degradation as the mode-of-action. Cell based screening of masarimycin against a panel of autolysin mutants, identified a higher MIC against a ΔlytB strain lacking an endo-N-acetylglucosaminidase involved in cell division. Subsequent biochemical and phenotypic analyses suggested that the higher MIC was due to an indirect interaction with LytB. Further analysis of changes to the cell surface in masarimycin treated cells identified the overexpression of several moonlighting proteins, including elongation factor Tu which is implicated in regulating cell shape. Checkerboard assays using masarimycin in concert with additional antibiotics identified an antagonistic relationship with the cell wall targeting antibiotic fosfomycin, which further supports a cell wall mode-of-action.

Development of a Bioluminescent High-Throughput Screening Assay for Nicotinamide Mononucleotide Adenylyltransferase (NMNAT).

Nicotinamide mononucleotide adenylyltransferase (NMNAT; EC 2.7.7.1) catalyzes the reversible production of NAD+ from NMN+ and ATP and is a potential drug target for cancer and neurodegenerative diseases. A sensitive bioluminescent assay format suitable to high-throughput screening (HTS) and mechanistic follow-up has not been reported and is of value to identify new modulators of NMNATs. To this end, we report the development of a bioluminescent assay using Photinus pyralis ATP-dependent luciferase and luciferin for NMNAT1 in a 384-well plate format. We also report a mechanistic follow-up paradigm using this format to determine time dependence and competition with substrates. The assay and follow-up paradigm were used to screen 912 compounds from the National Cancer Institute (NCI) Mechanistic Diversity Set II and the Approved Oncology Set VI against NMNAT1. Twenty inhibitors with greater than 35% inhibition at 20 µM were identified. The follow-up studies showed that seven actives were time-dependent inhibitors of NMNAT1. 2,3-Dibromo-1,4-naphthoquinone was the most potent, time-dependent inhibitor with IC50 values of 0.76 and 0.26 µM for inhibition of the forward and reverse reactions of the enzyme, respectively, and was shown to be NMN and ATP competitive. The bioluminescent NMNAT assay and mechanistic-follow-up will be of use to identify new modulators of NAD biosynthesis.

Trophic upgrading and mobilization of wax esters in microzooplankton.

Heterotrophic protists play pivotal roles in aquatic ecosystems by transferring matter and energy, including lipids, from primary producers to higher trophic predators. Using Oxyrrhis marina as a model organism, changes to the non-saponifiable protist lipids were investigated under satiation and starvation conditions. During active feeding on the alga Cryptomonas sp., the O. marina hexane soluble non-saponifiable fraction lipid profile reflected its food source with the observed presence of long chain mono-unsaturated fatty alcohols up to C25:1. Evidence of trophic upgrading in O. marina was observed with long chain mono-unsaturated fatty alcohol accumulation of up to C35:1. To the best of our knowledge, this is the first evidence that heterotrophic dinoflagellates are capable of producing ester derived alcohols and that dinoflagellates like O. marina are capable of synthesizing fatty alcohols up to C35. Additionally, we show evidence of trophic upgrading of lipids. During a 20-day resource deprivation, the lipid profile remained constant. During starvation, the mobilization of wax esters as energy stores was observed with long chain fatty alcohols mobilized first. Changes in lipid class profile and utilization of wax esters in O. marina provides insight into the types of lipids available for energy demand, the transfer of lipids through the base of marine food webs, and the catabolic response induced by resource deprivation.

Microbial Sterolomics as a Chemical Biology Tool.

Metabolomics has become a powerful tool in chemical biology. Profiling the human sterolome has resulted in the discovery of noncanonical sterols, including oxysterols and meiosis-activating sterols. They are important to immune responses and development, and have been reviewed extensively. The triterpenoid metabolite fusidic acid has developed clinical relevance, and many steroidal metabolites from microbial sources possess varying bioactivities. Beyond the prospect of pharmacognostical agents, the profiling of minor metabolites can provide insight into an organism's biosynthesis and phylogeny, as well as inform drug discovery about infectious diseases. This review aims to highlight recent discoveries from detailed sterolomic profiling in microorganisms and their phylogenic and pharmacological implications.

A Four-Point Screening Method for Assessing Molecular Mechanism of Action (MMOA) Identifies Tideglusib as a Time-Dependent Inhibitor of Trypanosoma brucei GSK3ß.

BACKGROUND: New therapeutics are needed for neglected tropical diseases including Human African trypanosomiasis (HAT), a progressive and fatal disease caused by the protozoan parasites Trypanosoma brucei gambiense and T. b. rhodesiense. There is a need for simple, efficient, cost effective methods to identify new molecules with unique molecular mechanisms of action (MMOAs). The mechanistic features of a binding mode, such as competition with endogenous substrates and time-dependence can affect the observed inhibitory IC50, and differentiate molecules and their therapeutic usefulness. Simple screening methods to determine time-dependence and competition can be used to differentiate compounds with different MMOAs in order to identify new therapeutic opportunities. METHODOLOGY/PRINCIPAL FINDINGS: In this work we report a four point screening methodology to evaluate the time-dependence and competition for inhibition of GSK3ß protein kinase isolated from T. brucei. Using this method, we identified tideglusib as a time-dependent inhibitor whose mechanism of action is time-dependent, ATP competitive upon initial binding, which transitions to ATP non-competitive with time. The enzyme activity was not recovered following 100-fold dilution of the buffer consistent with an irreversible mechanism of action. This is in contrast to the T. brucei GSK3ß inhibitor GW8510, whose inhibition was competitive with ATP, not time-dependent at all measured time points and reversible in dilution experiments. The activity of tideglusib against T. brucei parasites was confirmed by inhibition of parasite proliferation (GI50 of 2.3 µM). CONCLUSIONS/SIGNIFICANCE: Altogether this work demonstrates a straightforward method for determining molecular mechanisms of action and its application for mechanistic differentiation of two potent TbGSK3ß inhibitors. The four point MMOA method identified tideglusib as a mechanistically differentiated TbGSK3ß inhibitor. Tideglusib was shown to inhibit parasite growth in this work, and has been reported to be well tolerated in one year of dosing in human clinical studies. Consequently, further supportive studies on the potential therapeutic usefulness of tideglusib for HAT are justified.

Enzyme Activity Assays for Protein Kinases: Strategies to Identify Active Substrates.

Protein kinases are an important class of enzymes and drug targets. New opportunities to discover medicines for neglected diseases can be leveraged by the extensive kinase tools and knowledge created in targeting human kinases. A valuable tool for kinase drug discovery is an enzyme assay that measures catalytic function. The functional assay can be used to identify inhibitors, estimate affinity, characterize molecular mechanisms of action (MMOAs) and evaluate selectivity. However, establishing an enzyme assay for a new kinases requires identification of a suitable substrate. Identification of a new kinase's endogenous physiologic substrate and function can be extremely costly and time consuming. Fortunately, most kinases are promiscuous and will catalyze the phosphotransfer from ATP to alternative substrates with differing degrees of catalytic efficiency. In this manuscript we review strategies and successes in the identification of alternative substrates for kinases from organisms responsible for many of the neglected tropical diseases (NTDs) towards the goal of informing strategies to identify substrates for new kinases. Approaches for establishing a functional kinase assay include measuring auto-activation and use of generic substrates and peptides. The most commonly used generic substrates are casein, myelin basic protein, and histone. Sequence homology modeling can provide insights into the potential substrates and the requirement for activation. Empirical approaches that can identify substrates include screening of lysates (which may also help identify native substrates) and use of peptide arrays. All of these approaches have been used with a varying degree of success to identify alternative substrates.

Fluorinated Sterols Are Suicide Inhibitors of Ergosterol Biosynthesis and Growth in Trypanosoma brucei.

Trypanosoma brucei, the causal agent for sleeping sickness, depends on ergosterol for growth. Here, we describe the effects of a mechanism-based inhibitor, 26-fluorolanosterol (26FL), which converts in vivo to a fluorinated substrate of the sterol C24-methyltransferase essential for sterol methylation and function of ergosterol, and missing from the human host. 26FL showed potent inhibition of ergosterol biosynthesis and growth of procyclic and bloodstream forms while having no effect on cholesterol biosynthesis or growth of human epithelial kidney cells. During exposure of cloned TbSMT to 26-fluorocholesta-5,7,24-trienol, the enzyme is gradually killed as a consequence of the covalent binding of the intermediate C25 cation to the active site (kcat/kinact = 0.26 min(-1)/0.24 min(-1); partition ratio of 1.08), whereas 26FL is non-productively bound. These results demonstrate that poisoning of ergosterol biosynthesis by a 26-fluorinated Δ(24)-sterol is a promising strategy for developing a new treatment for trypanosomiasis.

The Role of Binding Kinetics in GPCR Drug Discovery.

Binding kinetics are the rates of association and dissociation of a drug-protein complex and are important molecular descriptors for the optimization of drug binding to G-protein coupled receptors (GPCRs). There are now many examples of binding kinetics in GPCR drug discovery. In this report, the first principles and examples of binding kinetics in GPCR drug discovery are reviewed. Addressed are the influence of binding kinetics on the translation of binding to the therapeutic window in the context of the equilibrium state of the system and molecular mechanisms of slow binding including induced fit, displacement of water, rebinding and heterovalency.

Identification of natural RORγ ligands that regulate the development of lymphoid cells.

Mice deficient in the nuclear hormone receptor RORγt have defective development of thymocytes, lymphoid organs, Th17 cells, and type 3 innate lymphoid cells. RORγt binds to oxysterols derived from cholesterol catabolism, but it is not clear whether these are its natural ligands. Here, we show that sterol lipids are necessary and sufficient to drive RORγt-dependent transcription. We combined overexpression, RNAi, and genetic deletion of metabolic enzymes to study RORγ-dependent transcription. Our results are consistent with the RORγt ligand(s) being a cholesterol biosynthetic intermediate (CBI) downstream of lanosterol and upstream of zymosterol. Analysis of lipids bound to RORγ identified molecules with molecular weights consistent with CBIs. Furthermore, CBIs stabilized the RORγ ligand-binding domain and induced coactivator recruitment. Genetic deletion of metabolic enzymes upstream of the RORγt-ligand(s) affected the development of lymph nodes and Th17 cells. Our data suggest that CBIs play a role in lymphocyte development potentially through regulation of RORγt.

Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth.

Ergosterol biosynthesis and homeostasis in the parasitic protozoan Trypanosoma brucei was analyzed by RNAi silencing and inhibition of sterol C24ß-methyltransferase (TbSMT) and sterol 14α-demethylase [TbSDM (TbCYP51)] to explore the functions of sterols in T. brucei growth. Inhibition of the amount or activity of these enzymes depletes ergosterol from cells at <6 fg/cell for procyclic form (PCF) cells or <0.01 fg/cell for bloodstream form (BSF) cells and reduces infectivity in a mouse model of infection. Silencing of TbSMT expression by RNAi in PCF or BSF in combination with 25-azalanosterol (AZA) inhibited parasite growth and this inhibition was restored completely by adding synergistic cholesterol (7.8 µM from lipid-depleted media) with small amounts of ergosterol (1.2 µM) to the medium. These observations are consistent with the proposed requirement for ergosterol as a signaling factor to spark cell proliferation while imported cholesterol or the endogenously formed cholesta-5,7,24-trienol act as bulk membrane components. To test the potential chemotherapeutic importance of disrupting ergosterol biosynthesis using pairs of mechanism-based inhibitors that block two enzymes in the post-squalene segment, parasites were treated with AZA and itraconazole at 1 µM each (ED50 values) resulting in parasite death. Taken together, our results demonstrate that the ergosterol pathway is a prime drug target for intervention in T. brucei infection.

Characterization, mutagenesis and mechanistic analysis of an ancient algal sterol C24-methyltransferase: Implications for understanding sterol evolution in the green lineage.

Sterol C24-methyltransferases (SMTs) constitute a group of sequence-related proteins that catalyze the pattern of sterol diversity across eukaryotic kingdoms. The only gene for sterol alkylation in green algae was identified and the corresponding catalyst from Chlamydomonas reinhardtii (Cr) was characterized kinetically and for product distributions. The properties of CrSMT were similar to those predicted for an ancient SMT expected to possess broad C3-anchoring requirements for substrate binding and formation of 24ß-methyl/ethyl Δ(25(27))-olefin products typical of primitive organisms. Unnatural Δ(24(25))-sterol substrates, missing a C4ß-angular methyl group involved with binding orientation, convert to product ratios in favor of Δ(24(28))-products. Remodeling the active site to alter the electronics of Try110 (to Leu) results in delayed timing of the hydride migration from methyl attack of the Δ(24)-bond, that thereby produces metabolic switching of product ratios in favor of Δ(25(27))-olefins or impairs the second C1-transfer activity. Incubation of [27-(13)C]lanosterol or [methyl-(2)H3]SAM as co-substrates established the CrSMT catalyzes a sterol methylation pathway by the "algal" Δ(25(27))-olefin route, where methylation proceeds by a conserved SN2 reaction and de-protonation proceeds from the pro-Z methyl group on lanosterol corresponding to C27. This previously unrecognized catalytic competence for an enzyme of sterol biosynthesis, together with phylogenomic analyses, suggest that mutational divergence of a promiscuous SMT produced substrate- and phyla-specific SMT1 (catalyzes first biomethylation) and SMT2 (catalyzes second biomethylation) isoforms in red and green algae, respectively, and in the case of SMT2 selection afforded modification in reaction channeling necessary for the switch in ergosterol (24ß-methyl) biosynthesis to stigmasterol (24α-ethyl) biosynthesis during the course of land plant evolution.

C-24-methylation of 26-fluorocycloartenols by recombinant sterol C-24-methyltransferase from soybean: evidence for channel switching and its phylogenetic implications.

The tightly coupled nature of the electrophilic alkylation reaction sequence catalysed by 24-SMT (sterol C-24-methyltransferase) of land plants and algae can be distinguished by the formation of cationic intermediates that yield phyla-specific product profiles. C-24-methylation of the cycloartenol substrate by the recombinant Glycine max (soybean) 24-SMT proceeds to a single product 24(28)-methylenecycloartanol, whereas the 24-SMT from green algae converts cycloartenol into two products cyclolaudenol [∆(25(27))-olefin] and 24(28)-methylenecycloartanol [(∆24(28))-olefin]. Substrate analogues that differed in the steric-electronic features at either end of the molecule, 26-homocycloartenol or 3ß-fluorolanostadiene, were converted by G. max SMT into a single 24(28)-methylene product. Alternatively, incubation of the allylic 26-fluoro cyclosteroid with G. max SMT afforded a bound intermediate that converted in favour of the ∆(25(27))-olefin product via the cyclolaudenol cation formed initially during the C-24-methylation reaction. A portion of the 26-fluorocycloartenol substrate was also intercepted by the enzyme and the corresponding hydrolysis product identified by GC-MS as 26-fluoro-25-hydroxy-24-methylcycloartanol. Finally, the 26-fluorocycloartenols are competitive inhibitors for the methylation of cycloartenol and 26-monofluorocycloartenol generated timedependent inactivation kinetics exhibiting a kinact value of 0.12 min(-1). The ability of soybean 24-SMT to generate a 25-hydroxy alkylated sterol and fluorinated ∆(25(27))-olefins is consistent with our hypothesis that (i) achieving the cyclolaudenyl cation intermediate by electrophilic alkylation of cycloartenol is significant to the overall reaction rate, and (ii) the evolution of variant sterol C-24-methylation patterns is driven by competing reaction channels that have switched in algae from formation of primarily ∆(25(27)) products that convert into ergosterol to, in land plants, formation of ∆(24(28)) products that convert into sitosterol.

Evolutionarily conserved Delta(25(27))-olefin ergosterol biosynthesis pathway in the alga Chlamydomonas reinhardtii.

Ergosterol is the predominant sterol of fungi and green algae. Although the biosynthetic pathway for sterol synthesis in fungi is well established and is known to use C24-methylation-C24 (28)-reduction (Δ(24(28))-olefin pathway) steps, little is known about the sterol pathway in green algae. Previous work has raised the possibility that these algae might use a novel pathway because the green alga Chlamydomonas reinhardtii was shown to possess a mevalonate-independent methylerythritol 4-phosphate not present in fungi. Here, we report that C. reinhardtii synthesizes the protosterol cycloartenol and converts it to ergosterol (C24ß-methyl) and 7-dehydroporiferasterol (C24ß-ethyl) through a highly conserved sterol C24- methylation-C25-reduction (Δ(25(27))-olefin) pathway that is distinct from the well-described acetate-mevalonate pathway to fungal lanosterol and its conversion to ergosterol by the Δ(24(28))-olefin pathway. We isolated and characterized 23 sterols by a combination of GC-MS and proton nuclear magnetic resonance spectroscopy analysis from a set of mutant, wild-type, and 25-thialanosterol-treated cells. The structure and stereochemistry of the final C24-alkyl sterol side chains possessed different combinations of 24ß-methyl/ethyl groups and Δ(22(23))E and Δ(25(27))-double bond constructions. When incubated with [methyl-(2)H(3)]methionine, cells incorporated three (into ergosterol) or five (into 7-dehydroporiferasterol) deuterium atoms into the newly biosynthesized 24ß-alkyl sterols, consistent only with a Δ(25(27))-olefin pathway. Thus, our findings demonstrate that two separate isoprenoid-24-alkyl sterol pathways evolved in fungi and green algae, both of which converge to yield a common membrane insert ergosterol.