We combined reverse and chemical genetics to identify targets and compounds modulating blood vessel development. Through transcript profiling in mice, we identified 150 potentially druggable microvessel-enriched gene products. Orthologs of 50 of these were knocked down in a reverse genetic screen in zebrafish, demonstrating that 16 were necessary for developmental angiogenesis. In parallel, 1280 pharmacologically active compounds were screened in a human cell-based assay, identifying 28 compounds selectively inhibiting endothelial sprouting. Several links were revealed between the results of the reverse and chemical genetic screens, including the serine/threonine (S/T) phosphatases ppp1ca, ppp1cc, and ppp4c and an inhibitor of this gene family; Endothall. Our results suggest that the combination of reverse and chemical genetic screens, in vertebrates, is an efficient strategy for the identification of drug targets and compounds that modulate complex biological systems, such as angiogenesis.
The serine esterase monoacylglycerol lipase (MGL) is primarily responsible for deactivating the signaling lipid 2-arachidonoylglycerol (2-AG), an endocannabinoid with full agonist activity at both principal cannabinoid receptors. Although MGL is recognized as a potential therapeutic target, the paucity of structural information on this enzyme has hindered development of MGL-selective inhibitors. Previously, we overexpressed and purified human MGL as the hexa-histidine-tagged recombinant protein (hMGL) and showed that it catalyzed the hydrolysis of both 2-AG and novel fluorogenic reporters. We now characterize by mass spectroscopy the hMGL active site using two chemically distinct inhibitors as direct probes: 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701) and N-arachidonylmaleimide (NAM). Suitable conditions were established for hMGL inhibition by AM6701, and the inhibitor-treated enzyme was subjected to trypsin digestion. The tryptic digest of AM6701-inhibited hMGL was analyzed by MALDI-TOF and tandem MS, which showed that AM6701 had carbamylated the serine in a GXSXG motif of the putative MGL catalytic triad. These results provide the first direct confirmation of the essential role of this serine residue for catalysis and establish the mechanism of AM6701 as a high-affinity, covalent hMGL inhibitor. When applied to NAM-treated hMGL, our direct, ligand-assisted approach revealed that partial alkylation of cysteine residues 215 and/or 249 was sufficient to achieve ~ 80% hMGL inhibition. Further alkylation at cysteine 39 did not increase the extent of enzyme inhibition. Although Cys215 and/or Cys249 mutations to alanine(s) did not affect hMGL’s ability to hydrolyze reporter substrate, as compared to nonmutated hMGL the C215A mutant was more sensitive to NAM, whereas the C249A mutation reduced the enzyme’s sensitivity to NAM. These data conclusively demonstrate a sulfhydryl-based mechanism underlying MGL inhibition by this fatty alkyl-maleimide substrate analog in which Cys249 is of paramount importance. Identification of amino acids critical to catalysis by and pharmacological modulation of hMGL provides information useful in the design of selective MGL inhibitors as potential drugs.
Structural data on mammalian proteins are often difficult to obtain by conventional NMR approaches because of an inability to produce samples with uniform isotope labeling in bacterial expression hosts. Proteins with sparse isotope labels can be produced in eukaryotic hosts by using isotope-labeled forms of specific amino acids, but structural analysis then requires information from experiments other than nuclear Overhauser effects. One source of alternate structural information is distance-dependent perturbation of spin relaxation times by nitroxide spin-labeled analogs of natural protein ligands. Here, we introduce spin-labeled analogs of sugar nucleotide donors for sialyltransferases, specifically, CMP-TEMPO (CMP-4-O-[2,2,6,6-tetramethylpiperidine-1-oxyl]) and CMP-4carboxyTEMPO (CMP-4-O-[4-carboxy-2,2,6,6-tetramethylpiperidinine-1-oxyl]). An ability to identify resonances from active site residues and produce distance constraints is illustrated on a 15N phenylalanine-labeled version of the structurally uncharacterized, α-2,6-linked sialyltransferase, ST6Gal I.
Fragment-based ligand design (FBLD) approaches have become more widely used in drug discovery projects from both academia and industry, and are even often preferred to traditional high-throughput screening (HTS) of large collection of compounds (>105). A key advantage of FBLD approaches is that these often rely on robust biophysical methods such as NMR spectroscopy for detection of ligand binding, hence are less prone to artifacts that too often plague the results from HTS campaigns. In this article, we introduce a screening strategy that takes advantage of both the robustness of protein NMR spectroscopy as the detection method, and the basic principles of combinatorial chemistry to enable the screening of large libraries of fragments (>105 compounds) preassembled on a common backbone. We used the method to identify compounds that target protein-protein interactions.
Identification of unique leads represents a significant challenge in drug discovery. This hurdle is magnified in neglected diseases such as tuberculosis. We have leveraged public high-throughput screening (HTS) data, to experimentally validate virtual screening approach employing Bayesian models built with bioactivity information (single-event model) as well as bioactivity and cytotoxicity information (dual-event model). We virtually screen a commercial library and experimentally confirm actives with hit rates exceeding typical HTS results by 1-2 orders of magnitude. The first dual-event Bayesian model identified compounds with antitubercular whole-cell activity and low mammalian cell cytotoxicity from a published set of antimalarials. The most potent hit exhibits the in vitro activity and in vitro/in vivo safety profile of a drug lead. These Bayesian models offer significant economies in time and cost to drug discovery.
Lysosomal storage diseases (LSDs) are often caused by mutations compromising lysosomal enzyme folding in the endoplasmic reticulum (ER), leading to degradation and loss of function. Mass spectrometry analysis of Gaucher fibroblasts treated with mechanistically distinct molecules that increase LSD enzyme folding, trafficking and function resulted in the identification of 9 commonly down-regulated and 2 jointly up-regulated proteins, which we hypothesized would be critical proteostasis network components for ameliorating loss-of-function diseases. LIMP-2 and FK506 binding protein 10 (FKBP10) were validated as such herein. Increased FKBP10 levels accelerated mutant glucocerebrosidase (GC) degradation over folding and trafficking, whereas decreased ER FKBP10 concentration led to more LSD enzyme partitioning into the calnexin profolding pathway, enhancing folding and activity to levels thought to ameliorate LSDs. Thus, targeting FKBP10 appears to be a heretofore unrecognized therapeutic strategy to ameliorate LSDs.
endoplasmic reticulum associated degradation; lysosomal storage diseases; protein homeostasis
We present a novel approach for fluorescent in situ detection of short, single-copy sequences within genomic DNA in human cells. The single copy sensitivity and single base specificity of our method is achieved due to the combination of three components. First, a peptide nucleic acid (PNA) probe locally opens a chosen target site, which allows a padlock DNA probe to access the site and become ligated. Second, rolling circle amplification (RCA) generates thousands of single-stranded copies of the target sequence. Finally, fluorescent in situ hybridization (FISH) is used to visualize the amplified DNA. We validate this new technique by successfully detecting six unique target sites on human mitochondrial and autosomal DNA. We also demonstrate the high specificity of this method by detecting X- and Y- specific sequences on human sex chromosomes and by simultaneously detecting three unique target sites. Finally, we discriminate two target sites that differ by two nucleotides. The PNA-RCA-FISH approach is a unique in situ hybridization method capable of multi-target visualization within human chromosomes and nuclei that does not require DNA denaturation and is extremely sequence specific.
Neuromyelitis optica (NMO) is an autoimmune inflammatory disorder of the central nervous system. In most NMO patients, autoantibodies to the water channel protein Aquaporin 4 (AQP4) are present at high levels and are thought to drive pathology by mediating complement-dependent destruction of astrocytes. Here we apply recently developed chemical library screening technology to identify a synthetic peptoid that binds anti-AQP4 antibodies in the serum of NMO patients. This finding validates, in a well-defined human disease, that synthetic, unnatural ligands for the antigen-binding site of a disease-linked antibody can be isolated by high-throughput screening.
Because proteostasis networks manage the cellular proteome, their pharmacological manipulation might correct pathologies associated with numerous protein misfolding diseases. In this issue of Chemistry & Biology, Tong Ong et al. identify a novel biosynthetic juncture for glucocerebrosidase as a site for therapeutic intervention in Gaucher’s disease.
There are no approved therapeutics for the most deadly nonsegmented negative-strand (NNS) RNA viruses, including Ebola (EBOV). To identify new chemical scaffolds for development of broad-spectrum antivirals, we undertook a prototype-based lead identification screen. Using the prototype NNS virus, vesicular stomatitis virus (VSV), multiple inhibitory compounds were identified. Three compounds were investigated for broad-spectrum activity, and inhibited EBOV infection. The most potent, CMLDBU3402, was selected for further study. CMLDBU3402 did not show significant activity against segmented negative-strand RNA viruses suggesting proscribed broad-spectrum activity. Mechanistic analysis indicated that CMLDBU3402 blocked VSV viral RNA synthesis and inhibited EBOV RNA transcription, demonstrating a consistent mechanism of action against genetically distinct viruses. The identification of this chemical backbone as a broad-spectrum inhibitor of viral RNA synthesis offers significant potential for the development of new therapies for highly pathogenic viruses.
Protein kinases are a large family of approximately 530 highly conserved enzymes that transfer a γ-phosphate group from ATP to a variety of amino acid residues such as tyrosine, serine and threonine which serves as a ubiquitous mechanism for cellular signal transduction. The clinical success of a number of kinase-directed drugs and the frequent observation of disease causing mutations in protein kinases suggest that a large number of kinases may represent therapeutically relevant targets. To-date the majority of clinical and preclinical kinase inhibitors are ATP-competitive, non-covalent inhibitors that achieve selectivity through recognition of unique features of particular protein kinases. Recently there has been renewed interest in the development of irreversible inhibitors that form covalent bonds with cysteine or other nucleophilic residues in the ATP-binding pocket. Irreversible kinase inhibitors have a number of potential advantages including prolonged pharmacodynamics, suitability for rational design, high potency and ability to validate pharmacological specificity through mutation of the reactive cysteine residue. Here we review recent efforts to develop cysteine-targeted irreversible protein kinase inhibitors and discuss their modes of recognizing the ATP-binding pocket and their biological activity profiles. In addition, we provided an informatics assessment of the potential ‘kinase-cysteinome’ and discuss strategies for the efficient development of new covalent inhibitors.
Protein Kinases; Irreversible Kinase inhibitors
Resolvins are a new family of n-3 lipid mediators initially identified in resolving inflammatory exudates that temper inflammatory responses to promote catabasis. Here, temporal metabololipidomics with self-limited resolving exudates revealed that resolvin (Rv) D3 has a distinct time frame from other lipid mediators, appearing late in resolution phase. Using synthetic materials prepared by stereocontrolled total organic synthesis and metabololipidomics, we established complete stereochemistry of RvD3 and its aspirin-triggered 17R-epimer (AT-RvD3). Both synthetic resolvins potently regulated neutrophils and mediators, reducing murine peritonitis and dermal inflammation. RvD3 and AT-RvD3 displayed leukocyte-directed actions, e.g. blocking human neutrophil transmigration and enhancing macrophage phagocytosis and efferocytosis. These results position RvD3 uniquely within the inflammation-resolution time frame to vantage and contribute to the beneficial actions of aspirin and essential n-3 fatty acids.
Purine nucleoside phosphorylase (PNP) is a target for leukemia, gout and autoimmune disorders. Dynamic motion of catalytic site loops has been implicated in catalysis, but experimental evidence was lacking. We replaced catalytic site groups His257 or His64 with 6-fluoro-tryptophan (6FW) as site-specific NMR probes. Conformational adjustments in the 6FW-His257-helical and His64-6FW-loop regions were characterized in PNP phosphate bound enzyme and in complexes with catalytic site ligands, including transition state analogues. Chemical shift and line-shape changes associated with these complexes revealed dynamic coexistence of several conformational states in these regions in phosphate bound enzyme and altered or single conformations in other complexes. These conformations were also characterized by X-ray crystallography. Specific 19F-Trp labels and X-ray crystallography provide multidimensional characterization of conformational states for free, catalytic and inhibited complexes of human PNP.
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 mode of action, dereplicated known compounds based on biological similarity to the training set, and identified and predicted the MOA of a family of new iron siderophores. Coupled with traditional analytical techniques, cytological profiling provides a new avenue for the creation of ‘function-first’ platforms for natural products discovery.
Protein kinases may function more like variable rheostats, rather than two-state switches. However, we lack approaches to properly analyze this aspect of kinase-dependent regulation. To address this we develop a strategy in which a kinase inhibitor is identified using genetics-based screens, kinase mutations that confer resistance are characterized, and dose-dependent responses of isogenic drug-sensitive and -resistant cells to inhibitor treatments are compared. This approach has the advantage that function of wild-type kinase, rather than mutants, is examined. To develop this approach we focus on Ark1, the fission yeast member of the conserved Aurora kinase family. Applying this approach reveals that proper chromosome compaction in fission yeast needs high Ark1 activity, while other processes depend on significantly lower activity levels. Our strategy is general and can be used to examine the functions of other molecular rheostats.
Small molecules that perturb protein homeostasis are used as cancer therapeutics and as antibiotics to treat bacterial infections. Kannan et al. (Cell 2012) describe an intriguing mechanism that enables ribosome-targeted macrolides to selectively remodel the bacterial proteome. This finding suggests the exciting possibility of targeting additional proteostasis regulators in a substrate-selective manner.
Innovative strategies are needed to combat drug resistance associated with methicillin-resistant Staphylococcus aureus (MRSA). Here, we investigate the potential of wall teichoic acid (WTA) biosynthesis inhibitors as combination agents to restore β-lactam efficacy against MRSA. Performing a whole cell pathway-based screen we identified a series of WTA inhibitors (WTAIs) targeting the WTA transporter protein, TarG. Whole genome sequencing of WTAI resistant isolates across two methicillin-resistant Staphylococci spp. revealed TarG as their common target, as well as a broad assortment of drug resistant bypass mutants mapping to earlier steps of WTA biosynthesis. Extensive in vitro microbiological analysis and animal infection studies provide strong genetic and pharmacological evidence of the potential effectiveness of WTAIs as anti-MRSA β-lactam combination agents. This work also highlights the emerging role of whole genome sequencing in antibiotic mode-of-action and resistance studies.
Staphylococcus aureus; MRSA; MRSE; imipenem; wall teichoic acid; antibiotic resistance; β-lactam potentiation; combination agent; chemical biology; Next Generation Sequencing
The Bloom’s syndrome protein, BLM, is a member of the conserved RecQ helicase family. Although cell lines lacking BLM exist, these exhibit progressive genomic instability that makes distinguishing primary from secondary effects of BLM loss problematic. In order to be able to acutely disable BLM function in cells, we undertook a high throughput screen of a chemical compound library for small molecule inhibitors of BLM. We present ML216, a potent inhibitor of the DNA unwinding activity of BLM. ML216 shows cell-based activity, and can induce sister chromatid exchanges, enhance to the toxicity of aphidicolin and exert anti-proliferative activity in cells expressing BLM, but not in those lacking BLM. These data indicate that ML216 shows strong selectively for BLM in cultured cells. We discuss the potential utility of such a BLM-targeting compound as an anticancer agent.
In the oceans, toxic secondary metabolites often protect otherwise poorly defended, soft-bodied invertebrates such as shell-less mollusks from predation. The origins of these metabolites are largely unknown, but many of them are thought to be made by symbiotic bacteria. In contrast, mollusks with thick shells and toxic venoms are thought to lack these secondary metabolites due to reduced defensive needs. Here, we show that heavily defended cone snails also occasionally contain abundant secondary metabolites, γ-pyrones known as nocapyrones, and that these pyrones are synthesized by symbiotic bacteria. This study shows that symbiotic bacteria can produce metabolites isolated from gastropod mollusks. The symbiotic bacteria, Nocardiopsis alba CR167, are closely related to potentially widespread actinomycetes that we propose to be casual symbionts of invertebrates on land and in the sea. The natural roles of nocapyrones are not known, but they are active in neurological assays at low micromolar levels, revealing that mollusks with external shells are an overlooked source of secondary metabolite diversity.
Computational prediction of protein function is frequently error-prone and incomplete. In Mycobacterium tuberculosis (Mtb), ~25% of all genes have no predicted function and are annotated as hypothetical proteins, severely limiting our understanding of Mtb pathogenicity. Here, we utilize a high throughput, quantitative, activity-based protein profiling (ABPP) platform to probe, annotate, and validate ATP-binding proteins in Mtb. We experimentally validate prior in silico predictions of >250 proteins and identify 72 hypothetical proteins as novel ATP binders. ATP interacts with proteins with diverse and unrelated sequences, providing a new and expanded view of adenosine nucleotide binding in Mtb. Several hypothetical ATP binders are essential or taxonomically limited, suggesting specialized functions in mycobacterial physiology and pathogenicity.
Bacteria establish stable communities, known as biofilms, that are resistant to antimicrobials. Biofilm robustness is due to the presence of an extracellular matrix, which for several species - among them Bacillus subtilis - includes amyloid-like protein fibers. In this work, we show that B. subtilis biofilms can be a simple and reliable tool for screening of molecules with anti-amyloid activity. We identified two molecules, AA-861 and parthenolide, which efficiently inhibited biofilms by preventing the formation of amyloid-like fibers. We found that parthenolide also disrupted pre-established biofilms. These molecules also impeded the formation of biofilms of other bacterial species that secrete amyloid proteins, such as Bacillus cereus and Escherichia coli. Furthermore, the identified molecules decreased the conversion of the yeast protein New1 to the prion state in a heterologous host, indicating the broad range of activity of the molecules.
HIV-1 Nef, a critical AIDS progression factor, represents an important target protein for antiretroviral drug discovery. Because Nef lacks intrinsic enzymatic activity, we developed an assay that couples Nef to the activation of Hck, a Src-family member and Nef effector protein. Using this assay, we screened a large, diverse chemical library and identified small molecules that block Nef-dependent Hck activity with low micromolar potency. Of these, a diphenylpyrazolo compound demonstrated sub-micromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound binds directly to Nef via a pocket formed by the Nef dimerization interface and disrupts Nef dimerization in cells. Coupling of non-enzymatic viral accessory factors to host cell effector proteins amenable to high-throughput screening may represent a general strategy for the discovery of new antimicrobial agents.
Small molecule inhibitors of amyloid aggregation have potential as treatment for a variety of conditions. In this issue of Chemistry & Biology, Romero et al. (2013) use amyloid-dependent B. subtilis biofilm formation to screen for amyloid inhibitors, identifying compounds that not only inhibit B. subtilis biofilm formation but also ones that disrupt preformed biofilms.
The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in kcat/Km with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in kcat/Km values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the “nonribosomal code” of A-domains.
MutY and its human ortholog, MUTYH, repair a specific form of DNA damage:
adenine mis-paired with the oxidatively modified form of deoxyguanosine,
8-oxo-7,8-dihydro-2′-deoxyguanosine. In a recent issue of
Chemistry & Biology, Brinkmeyer et al. utilized
mutant forms of MutY to reveal the critical residues in MutY that are required
for this selectivity and specificity.