DNA-Encoded Macrocyclic Peptide Libraries Enable the Discovery of a Neutral MDM2-p53 Inhibitor.

Synthetic macrocyclic peptides are an emerging molecular class for both targeting intracellular protein-protein interactions (PPIs) and providing an oral modality for drug targets typically addressed by biologics. Display technologies, such as mRNA and phage display, often yield peptides that are too large and too polar to achieve passive permeability or oral bioavailability without substantial off-platform medicinal chemistry. Herein, we use DNA-encoded cyclic peptide libraries to discover a neutral nonapeptide, UNP-6457, that inhibits MDM2-p53 interaction with an IC50 of 8.9 nM. X-ray structural analysis of the MDM2-UNP-6457 complex revealed mutual binding interactions and identified key ligand modification points which may be tuned to enhance its pharmacokinetic profile. These studies showcase how tailored DEL libraries can directly yield macrocyclic peptides benefiting from low MW, TPSA, and HBD/HBA counts that are capable of potently inhibiting therapeutically relevant protein-protein interactions.

Identifying the Cellular Target of Cordyheptapeptide A and Synthetic Derivatives.

Cordyheptapeptide A is a lipophilic cyclic peptide from the prized Cordyceps fungal genus that shows potent cytotoxicity in multiple cancer cell lines. To better understand the bioactivity and physicochemical properties of cordyheptapeptide A with the ultimate goal of identifying its cellular target, we developed a solid-phase synthesis of this multiply N-methylated cyclic heptapeptide which enabled rapid access to both side chain- and backbone-modified derivatives. Removal of one of the backbone amide N-methyl (N-Me) groups maintained bioactivity, while membrane permeability was also preserved due to the formation of a new intramolecular hydrogen bond in a low dielectric solvent. Based on its cytotoxicity profile in the NCI-60 cell line panel, as well as its phenotype in a microscopy-based cytological assay, we hypothesized that cordyheptapeptide was acting on cells as a protein synthesis inhibitor. Further studies revealed the molecular target of cordyheptapeptide A to be the eukaryotic translation elongation factor 1A (eEF1A), a target shared by other lipophilic cyclic peptide natural products. This work offers a strategy to study and improve cyclic peptide natural products while highlighting the ability of these lipophilic compounds to effectively inhibit intracellular disease targets.

Pharmacological Targeting of Vacuolar H+-ATPase via Subunit V1G Combats Multidrug-Resistant Cancer.

Multidrug resistance (MDR) in cancer remains a major challenge for the success of chemotherapy. Natural products have been a rich source for the discovery of drugs against MDR cancers. Here, we applied high-throughput cytotoxicity screening of an in-house natural product library against MDR SGC7901/VCR cells and identified that the cyclodepsipeptide verucopeptin demonstrated notable antitumor potency. Cytological profiling combined with click chemistry-based proteomics revealed that ATP6V1G directly interacted with verucopeptin. ATP6V1G, a subunit of the vacuolar H+-ATPase (v-ATPase) that has not been previously targeted, was essential for SGC7901/VCR cell growth. Verucopeptin exhibited strong inhibition of both v-ATPase activity and mTORC1 signaling, leading to substantial pharmacological efficacy against SGC7901/VCR cell proliferation and tumor growth in vivo. Our results demonstrate that targeting v-ATPase via its V1G subunit constitutes a unique approach for modulating v-ATPase and mTORC1 signaling with great potential for the development of therapeutics against MDR cancers.

Macrophage Cytological Profiling and Anti-Inflammatory Drug Discovery.

Millions of people are affected by diseases and conditions related to the immune system. Unfortunately, our current supply of approved anti-inflammatory medicine is very limited and only treats a small fraction of inflammatory diseases. Nearly half of the drugs on the market today are natural products and natural product derivatives. The long-term objective of my research is to continue efforts toward the discovery of diverse chemical compounds and their mechanism of action (MOA) to inspire the next generation of novel therapeutics. This project approaches this objective by creating a robust platform for the in-depth phenotypic profiling of complex natural product samples with respect to their effect on pathways related to the innate immune response. This approach has the potential to elucidate the MOAs of novel natural products relevant to inflammation and accelerate the pace of drug discovery in this therapeutic area.

Genome-Based Identification of a Plant-Associated Marine Bacterium as a Rich Natural Product Source.

The large number of sequenced bacterial genomes provides the opportunity to bioinformatically identify rich natural product sources among previously neglected microbial groups. Testing this discovery strategy, unusually high biosynthetic potential was suggested for the Oceanospirillales member Gynuella sunshinyii, a Gram-negative marine bacterium from the rhizosphere of the halophilic plant Carex scabrifolia. Its genome contains numerous unusual biosynthetic gene clusters for diverse types of metabolites. Genome-guided isolation yielded representatives of four different natural product classes, of which only alteramide A was known. Cytotoxic lacunalides were identified as products of a giant trans-acyltransferase polyketide synthase gene cluster, one of six present in this strain. Cytological profiling against HeLa cells suggested that lacunalide A disrupts CDK signaling in the cell cycle. In addition, chemical studies on model compounds were conducted, suggesting the structurally unusual ergoynes as products of a conjugated diyne-thiourea cyclization reaction.

The Bioactive Lipid (S)-Sebastenoic Acid Impacts Motility and Dispersion in Vibrio cholerae.

Although Gram-negative bacterial pathogens continue to impart a substantial burden on global healthcare systems, much remains to be understood about aspects of basic physiology in these organisms. In recent years, cyclic-diguanylate (c-di-GMP) has emerged as a key regulator of a number of important processes related to pathogenicity, including biofilm formation, motility and virulence. In an effort to discover chemical genetic probes for studying V. cholerae we have developed a new motility-based high-throughput screen to identify compounds that modulate c-di-GMP levels. Using this new screening platform, we tested a library of microbially-derived marine natural products extracts, leading to the discovery of the bioactive lipid (S)-sebastenoic acid. Evaluation of the effect of this new compound on bacterial motility, vpsL expression and biofilm formation implied that (S)-sebastenoic acid may alter phenotypes associated to c-di-GMP signaling in V. cholerae.

Naphthablins B and C, Meroterpenoids Identified from the Marine Sediment-Derived Streptomyces sp. CP26-58 Using HeLa Cell-Based Cytological Profiling.

HeLa cell-based cytological profiling (CP) was applied to an extract library of marine sediment-derived actinomycetes to discover new cytotoxic secondary metabolites. Among the hit strains, Streptomyces sp. CP26-58 was selected for further investigation to identify its cytotoxic metabolites. CP revealed that the known ionophore tetronasin (1) was responsible for the cytotoxic effect found in the extract. Furthermore, three naphthoquinone meroterpenoids, naphthablin A (2) and two new derivatives designated as naphthablins B (3) and C (4), were isolated from other cytotoxic fractions. The structures of the new compounds were elucidated based on analysis of their HRESIMS and comprehensive NMR data. The absolute configurations of the new compounds were deduced by simulating ECD spectra and calculating potential energies for the model compounds using density function theory (DFT) calculations. Compound 1 showed a significant cytotoxic effect against HeLa cells with an IC50 value of 0.23 µM, and CP successfully clustered 1 with calcium ionophores.

Biofilm Formation and Detachment in Gram-Negative Pathogens Is Modulated by Select Bile Acids.

Biofilms are a ubiquitous feature of microbial community structure in both natural and host environments; they enhance transmission and infectivity of pathogens and provide protection from human defense mechanisms and antibiotics. However, few natural products are known that impact biofilm formation or persistence for either environmental or pathogenic bacteria. Using the combination of a novel natural products library from the fish microbiome and an image-based screen for biofilm inhibition, we describe the identification of taurine-conjugated bile acids as inhibitors of biofilm formation against both Vibrio cholerae and Pseudomonas aeruginosa. Taurocholic acid (1) was isolated from the fermentation broth of the fish microbiome-derived strain of Rhodococcus erythropolis and identified using standard NMR and MS methods. Screening of the twelve predominant human steroidal bile acid components revealed that a subset of these compounds can inhibit biofilm formation, induce detachment of preformed biofilms under static conditions, and that these compounds display distinct structure-activity relationships against V. cholerae and P. aeruginosa. Our findings highlight the significance of distinct bile acid components in the regulation of biofilm formation and dispersion in two different clinically relevant bacterial pathogens, and suggest that the bile acids, which are endogenous mammalian metabolites used to solubilize dietary fats, may also play a role in maintaining host health against bacterial infection.

A Strategy for Direct Chemical Activation of the Retinoblastoma Protein.

The retinoblastoma (Rb) tumor suppressor protein negatively regulates cell proliferation by binding and inhibiting E2F transcription factors. Rb inactivation occurs in cancer cells upon cyclin-dependent kinase (Cdk) phosphorylation, which induces E2F release and activation of cell cycle genes. We present a strategy for activating phosphorylated Rb with molecules that bind Rb directly and enhance affinity for E2F. We developed a fluorescence polarization assay that can detect the effect of exogenous compounds on modulating affinity of Rb for the E2F transactivation domain. We found that a peptide capable of disrupting the compact inactive Rb conformation increases affinity of the repressive Rb-E2F complex. Our results demonstrate the feasibility of discovering novel molecules that target the cell cycle and proliferation through directly targeting Rb rather than upstream kinase activity.

Phenotype-Guided Natural Products Discovery Using Cytological Profiling.

Phenotype-guided natural products discovery is emerging as a useful new discovery tool that addresses challenges in early, unbiased natural product biological annotation. These high-content approaches yield screening results that report directly on the impact of test compounds on cellular processes in target organisms and can be used to predict the modes of action of bioactive constituents from primary screening data. In this study we explored the use of our recently implemented cytological profiling platform for the isolation of compounds with a specific, predefined mode of action, namely, induction of mitotic arrest. Screening of a microbially derived extract library revealed six extracts whose cytological profiles clustered closely with those of known antimitotic agents from the pure compound training set. Subsequent examination of one of these extracts revealed the presence of two separate bioactive constituents, each of which possessed a unique cytological profile. The first, diketopiperazine XR334 (3), recapitulated the observed antimitotic phenotype of the original extract, demonstrating that cytological profiling can be used for the targeted isolation of compounds with specific modes of action. The second, nocapyrone L (6), possessed a cytological profile that clustered with known calcium channel modulators, in line with previous published activities for this compound class, indicating that cytological profiling is a flexible and powerful platform for the de novo characterization of compound modes of action.

Borrelidin B: isolation, biological activity, and implications for nitrile biosynthesis.

Borrelidin (1) is a nitrile-containing bacterially derived polyketide that is a potent inhibitor of bacterial and eukaryotic threonyl-tRNA synthetases. We now report the discovery of borrelidin B (2), a tetrahydro-borrelidin derivative containing an aminomethyl group in place of the nitrile functionality in borrelidin. The discovery of this new metabolite has implications for both the biosynthesis of the nitrile group and the bioactivity of the borrelidin compound class. Screening in the SToPS assay for tRNA synthetase inhibition revealed that the nitrile moiety is essential for activity, while profiling using our in-house image-based cytological profiling assay demonstrated that 2 retains biological activity by causing a mitotic stall, even in the absence of the nitrile motif.

Confirmation of the cellular targets of benomyl and rapamycin using next-generation sequencing of resistant mutants in S. cerevisiae.

Investigating the mechanisms of action (MOAs) of bioactive compounds and the deconvolution of their cellular targets is an important and challenging undertaking. Drug resistance in model organisms such as S. cerevisiae has long been a means for discovering drug targets and MOAs. Strains are selected for resistance to a drug of interest, and the resistance mutations can often be mapped to the drug's molecular target using classical genetic techniques. Here we demonstrate the use of next generation sequencing (NGS) to identify mutations that confer resistance to two well-characterized drugs, benomyl and rapamycin. Applying NGS to pools of drug-resistant mutants, we develop a simple system for ranking single nucleotide polymorphisms (SNPs) based on their prevalence in the pool, and for ranking genes based on the number of SNPs that they contain. We clearly identified the known targets of benomyl (TUB2) and rapamycin (FPR1) as the highest-ranking genes under this system. The highest-ranking SNPs corresponded to specific amino acid changes that are known to confer resistance to these drugs. We also found that by screening in a pdr1Δ null background strain that lacks a transcription factor regulating the expression of drug efflux pumps, and by pre-screening mutants in a panel of unrelated anti-fungal agents, we were able to mitigate against the selection of multi-drug resistance (MDR) mutants. We call our approach "Mutagenesis to Uncover Targets by deep Sequencing", or "MUTseq", and show through this proof-of-concept study its potential utility in characterizing MOAs and targets of novel compounds.

Coherence between cellular responses and in vitro splicing inhibition for the anti-tumor drug pladienolide B and its analogs.

Pladienolide B (PB) is a potent cancer cell growth inhibitor that targets the SF3B1 subunit of the spliceosome. There is considerable interest in the compound as a potential chemotherapeutic, as well as a tool to study SF3B1 function in splicing and cancer development. The molecular structure of PB, a bacterial natural product, contains a 12-member macrolide ring with an extended epoxide-containing side chain. Using a novel concise enantioselective synthesis, we created a series of PB structural analogs and the structurally related compound herboxidiene. We show that two methyl groups in the PB side chain, as well as a feature of the macrolide ring shared with herboxidiene, are required for splicing inhibition in vitro. Unexpectedly, we find that the epoxy group contributes only modestly to PB potency and is not absolutely necessary for activity. The orientations of at least two chiral centers off the macrolide ring have no effect on PB activity. Importantly, the ability of analogs to inhibit splicing in vitro directly correlated with their effects in a series of cellular assays. Those effects likely arise from inhibition of some, but not all, endogenous splicing events in cells, as previously reported for the structurally distinct SF3B1 inhibitor spliceostatin A. Together, our data support the idea that the impact of PB on cells is derived from its ability to impair the function of SF3B1 in splicing and also demonstrate that simplification of the PB scaffold is feasible.

Image-based 384-well high-throughput screening method for the discovery of skyllamycins A to C as biofilm inhibitors and inducers of biofilm detachment in Pseudomonas aeruginosa.

To date, most antibiotics have primarily been developed to target bacteria in the planktonic state. However, biofilm formation allows bacteria to develop tolerance to antibiotics and provides a mechanism to evade innate immune systems. Therefore, there is a significant need to identify small molecules to prevent biofilm formation and, more importantly, to disperse or eradicate preattached biofilms, which are a major source of bacterial persistence in nosocomial infections. We now present a modular high-throughput 384-well image-based screening platform to identify Pseudomonas aeruginosa biofilm inhibitors and dispersal agents. Biofilm coverage measurements were accomplished using non-z-stack epifluorescence microscopy to image a constitutively expressing green fluorescent protein (GFP)-tagged strain of P. aeruginosa and quantified using an automated image analysis script. Using the redox-sensitive dye XTT, bacterial cellular metabolic activity was measured in conjunction with biofilm coverage to differentiate between classical antibiotics and nonantibiotic biofilm inhibitors/dispersers. By measuring biofilm coverage and cellular activity, this screen identifies compounds that eradicate biofilms through mechanisms that are disparate from traditional antibiotic-mediated biofilm clearance. Screening of 312 natural-product prefractions identified the cyclic depsipeptide natural products skyllamycins B and C as nonantibiotic biofilm inhibitors with 50% effective concentrations (EC50s) of 30 and 60 µM, respectively. Codosing experiments of skyllamycin B and azithromycin, an antibiotic unable to clear preattached biofilms, demonstrated that, in combination, these compounds were able to eliminate surface-associated biofilms and depress cellular metabolic activity. The skyllamycins represent the first known class of cyclic depsipeptide biofilm inhibitors/dispersers.

An NF-κB-based high-throughput screen identifies piericidins as inhibitors of the Yersinia pseudotuberculosis type III secretion system.

The type III secretion system (T3SS) is a bacterial appendage used by dozens of Gram-negative pathogens to subvert host defenses and cause disease, making it an ideal target for pathogen-specific antimicrobials. Here, we report the discovery and initial characterization of two related natural products with T3SS-inhibitory activity that were derived from a marine actinobacterium. Bacterial extracts containing piericidin A1 and the piericidin derivative Mer-A 2026B inhibited Yersinia pseudotuberculosis from triggering T3SS-dependent activation of the host transcription factor NF-κB in HEK293T cells but were not toxic to mammalian cells. As the Yersinia T3SS must be functional in order to trigger NF-κB activation, these data indicate that piericidin A1 and Mer-A 2026B block T3SS function. Consistent with this, purified piericidin A1 and Mer-A 2026B dose-dependently inhibited translocation of the Y. pseudotuberculosis T3SS effector protein YopM inside CHO cells. In contrast, neither compound perturbed bacterial growth in vitro, indicating that piericidin A1 and Mer-A 2026B do not function as general antibiotics in Yersinia. In addition, when Yersinia was incubated under T3SS-inducing culture conditions in the absence of host cells, Mer-A 2026B and piericidin A1 inhibited secretion of T3SS cargo as effectively as or better than several previously described T3SS inhibitors, such as MBX-1641 and aurodox. This suggests that Mer-A 2026B and piericidin A1 do not block type III secretion by blocking the bacterium-host cell interaction, but rather inhibit an earlier stage, such as T3SS needle assembly. In summary, the marine-derived natural products Mer-A 2026B and piericidin A1 possess previously uncharacterized activity against the bacterial T3SS.

Large-scale cytological profiling for functional analysis of bioactive compounds.

Cytological profiling (CP) is an unbiased image-based screening technique that uses automated microscopy and image analysis to profile compounds based on numerous quantifiable phenotypic features. We used CP to evaluate a library of nearly 500 compounds with documented mechanisms of action (MOAs) spanning a wide range of biological pathways. We developed informatics techniques for generating dosage-independent phenotypic "fingerprints" for each compound, and for quantifying the likelihood that a compound's CP fingerprint corresponds to its annotated MOA. We identified groups of features that distinguish classes with closely related phenotypes, such as microtubule poisons vs. HSP90 inhibitors, and DNA synthesis vs. proteasome inhibitors. We tested several cases in which cytological profiles indicated novel mechanisms, including a tyrphostin kinase inhibitor involved in mitochondrial uncoupling, novel microtubule poisons, and a nominal PPAR-gamma ligand that acts as a proteasome inhibitor, using independent biochemical assays to confirm the MOAs predicted by the CP signatures. We also applied maximal-information statistics to identify correlations between cytological features and kinase inhibitory activities by combining the CP fingerprints of 24 kinase inhibitors with published data on their specificities against a diverse panel of kinases. The resulting analysis suggests a strategy for probing the biological functions of specific kinases by compiling cytological data from inhibitors of varying specificities.

A high-throughput splicing assay identifies new classes of inhibitors of human and yeast spliceosomes.

The spliceosome is the macromolecular machine responsible for pre-mRNA splicing, an essential step in eukaryotic gene expression. During splicing, myriad subunits join and leave the spliceosome as it works on the pre-mRNA substrate. Strikingly, there are very few small molecules known to interact with the spliceosome. Splicing inhibitors are needed to capture transient spliceosome conformations and probe important functional components. Such compounds may also have chemotherapeutic applications, as links between splicing and cancer are increasingly uncovered. To identify new splicing inhibitors, we developed a high-throughput assay for in vitro splicing using a reverse transcription followed by quantitative PCR readout. In a pilot screen of 3080 compounds, we identified three small molecules that inhibit splicing in HeLa extract by interfering with different stages of human spliceosome assembly. Two of the compounds similarly affect spliceosomes in yeast extracts, suggesting selective targeting of conserved components. By examining related molecules, we identified chemical features required for the activity of two of the splicing inhibitors. In addition to verifying our assay procedure and paving the way to larger screens, these studies establish new compounds as chemical probes for investigating the splicing machinery.

Mechanism of action-based classification of antibiotics using high-content bacterial image analysis.

Image-based screening has become a mature field over the past decade, largely due to the detailed information that can be obtained about compound mode of action by considering the phenotypic effects of test compounds on cellular morphology. However, very few examples exist of extensions of this approach to bacterial targets. We now report the first high-throughput, high-content platform for the prediction of antibiotic modes of action using image-based screening. This approach employs a unique feature segmentation and extraction protocol to quantify key size and shape metrics of bacterial cells over a range of compound concentrations, and matches the trajectories of these metrics to those of training set compounds of known molecular target to predict the test compound's mode of action. This approach has been used to successfully predict the modes of action of a panel of known antibiotics, and has been extended to the evaluation of natural products libraries for the de novo prediction of compound function directly from primary screening data.

"Function-first" lead discovery: mode of action profiling of natural product libraries using image-based screening.

Cytological profiling is a high-content image-based screening technology that provides insight into the mode of action (MOA) for test compounds by directly measuring hundreds of phenotypic cellular features. We have extended this recently reported technology to the mechanistic characterization of unknown natural products libraries for the direct prediction of compound MOAs at the primary screening stage. By analyzing a training set of commercial compounds of known mechanism and comparing these profiles to those obtained from natural product library members, we have successfully annotated extracts based on MOA, dereplicated known compounds based on biological similarity to the training set, and identified and predicted the MOA of a unique family of iron siderophores. Coupled with traditional analytical techniques, cytological profiling provides an avenue for the creation of "function-first" approaches to natural products discovery.