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.
The Plasmodium proteasome has been suggested to be a potential anti-malarial drug target, however toxicity of inhibitors has prevented validation of this enzyme in vivo. We report here a screen of a library of 670 analogs of the recently FDA approved inhibitor, carfilzomib, to identify compounds that selectively kill parasites. We identified one compound, PR3, that has significant parasite killing activity in vitro but dramatically reduced toxicity in host cells. We found that this parasite-specific toxicity is not due to selective targeting of the Plasmodium proteasome over the host proteasome, but instead is due to a lack of activity against one of the human proteasome subunits. Subsequently, we used PR3 to significantly reduce parasite load in P. berghei infected mice without host toxicity, thus validating the proteasome as a viable anti-malarial drug target.
Melanins are a broad class of darkly-pigmented macromolecules formed by oxidative polymerization of phenolic monomers. In fungi, melanins are known virulence factors that contribute to pathogenicity. Their biosynthesis generally involves polymerization of 1,8-dihydroxynaphthalene via a 1,3,6,8- tetrahydroxynaphthalene (THN) precursor assembled by multidomain, nonreducing polyketide synthases. Multiple, convergent routes to THN have evolved in fungi. Parallel heptaketide and hexaketide pathways exist that utilize conventional C-terminal thioesterase/Claisen cyclase domains and separate side-chain deacylases. Here, in vitro characterization of Pks1 from Colletotrichum lagenarium establishes a true THN synthase with a bifunctional thioesterase (TE) catalyzing both cyclization and deacetylation of an enzyme-bound hexaketide substrate. Chimeric TE domains were generated by swapping lid regions of active sites between classes of melanin TEs to gain insight into this unprecedented catalysis of carbon–carbon bond making and breaking by an α/β-hydrolase fold enzyme.
In the ubiquitin proteasome system, the E3 ligase SCF-Skp2 and its accessory protein Cks1 promote proliferation largely by inducing the degradation of the CDK inhibitor p27. Overexpression of Skp2 in human cancers correlates with poor prognosis, and deregulation of SCF-Skp2-Cks1 promotes tumorigenesis in animal models. We identified small molecule inhibitors specific to SCF-Skp2 activity using in silico screens targeted to the binding interface for p27. These compounds selectively inhibited Skp2-mediated p27 degradation by reducing p27 binding through key compound-receptor contacts. In cancer cells, the compounds induced p27 accumulation in a Skp2-dependent manner and promoted cell-type specific blocks in the G1 or G2/M phases. Designing SCF-Skp2 specific inhibitors may be a novel strategy to treat cancers dependent on the Skp2-p27 axis.
Bacterial biofilms are involved in a multitude of serious chronic infections. In recent years, modeling biofilm infection in vitro led to the identification of microbial determinants governing biofilm development. However, we lack information as to whether biofilm formation mechanisms identified in vitro have relevance for biofilm-associated infection. Here, we discuss the molecular basis of biofilm formation using staphylococci and Pseudomonas aeruginosa to illustrate key points, as their biofilm development process is well-studied. We will focus on in-vivo findings such as obtained in animal infection models, and critically evaluate in-vivo relevance of in-vitro findings. Although results on the role of quorum-sensing in biofilm formation have been conflicting, we now argue that integration of in-vitro and in-vivo studies allows a differentiated view of this mechanism as it relates to biofilm infection.
Biofilms; Staphylococcus aureus; Staphylococcus epidermidis; Pseudomonas aeruginosa; quorum-sensing
Thiopeptide antibiotics exhibit a profound level of chemical diversity that is installed through cascades of posttranslational modifications on ribosomal peptides. Here we present a technique to rapidly explore the chemical space of the thiopeptide GE37468 through codon randomization, yielding insights into thiopeptide maturation as well as structure and activity relationships. In this incarnation of the methodology, we randomized 7 residues of the prepeptide coding region, enabling the generation of 133 potential thiopeptide variants. Variant libraries were subsequently queried in two ways. First, high through-put MALDI-TOF mass spectrometry was applied to colony-level expressions to sample mutants which permitted full maturation of the antibiotic. Second, the activity of producing mutants was detected in an antibiotic overlay assay. In total, 29 of the 133 variants were found to produce mature compound, 12 of which retained antibiotic activity and one which had improved activity against Methicillin-resistant Staphylococcus aureus (MRSA).
The glyoxylate shunt plays an important role in fatty-acid metabolism, and has been shown to be critical to survival of several pathogens involved in chronic infections. For Mycobacterium tuberculosis (Mtb), a strain with a defective glyoxylate shunt was previously shown to be unable to establish infection in a mouse model. We report the development of novel phenyl-diketo acid (PDKA) inhibitors of malate synthase (GlcB), one of two glyoxylate shunt enzymes, using structure-based methods. PDKA inhibitors were active against Mtb grown on acetate, and over-expression of GlcB ameliorated this inhibition. Crystal structures of complexes of GlcB with PDKA inhibitors were used to guide optimization of potency. A selected PDKA compound demonstrated efficacy in a mouse model of tuberculosis. The discovery of these PDKA derivatives provides chemical validation of GlcB as an attractive target for tuberculosis therapeutics.
There is an urgent need to develop new drugs for treatment of tuberculosis, particularly against latent/persistent forms of the causative agent, Mycobacterium tuberculosis. In this issue of Chemistry & Biology, Krieger and colleagues use a structure-guided approach to develop novel inhibitors of malate synthase, a target in the glyoxylate shunt that is critical for pathogen survival in chronic infection.
The marine natural product symplostatin 4 (Sym4) has been recognized as a potent antimalarial agent. However, its mode of action and, in particular, direct targets have to date remained elusive. We report a chemical synthesis of Sym4 and show that Sym4-treatment of P. falciparum-infected red blood cells (RBCs) results in the generation of a swollen food vacuole phenotype and a reduction of parasitemia at nanomolar concentrations. We furthermore demonstrate that Sym4 is a nanomolar inhibitor of the P. falciparum falcipains in infected RBCs, suggesting inhibition of the hemoglobin degradation pathway as Sym4’s mode of action. Finally, we reveal a critical influence of the unusual methylmethoxypyrrolinone (mmp) group of Sym4 for potent inhibition, indicating that Sym4 derivatives with such a mmp moiety might represent viable lead structures for the development of antimalarial falcipain inhibitors.
Posttranslational modification with ubiquitin (Ub) controls many cellular processes, and aberrant ubiquitination can contribute to cancer, immunopathology, and neurodegeneration. The versatility arises from the ability of Ub to form polymer chains with eight distinct linkages via lysine side chains and the N terminus. In this study, we engineered Di-Ub probes mimicking all eight different poly-Ub linkages and profiled the deubiquitinating enzyme (DUB) selectivity for recognizing Di-Ub moieties in cellular extracts. Mass spectrometric profiling revealed that most DUBs examined have broad selectivity, whereas a subset displays a clear preference for recognizing noncanonical over K48/K63 Ub linkages. Our results expand knowledge of Ub processing enzyme functions in cellular contexts that currently depends largely on using recombinant enzymes and substrates.
•Synthesis of Di-ubiquitin-based active site probes representing all eight linkages•Mass spectrometric profiling of DUB-Ub linkage preference in whole cell extracts•Activity-based Di-Ub probe screen for DUB specificity toward poly-Ub linkages•DUBs detected with a preference for noncanonical linkages over K48/K63-linked Ub
Ubiquitination controls many cellular processes. McGouran et al. engineered Di-Ub probes mimicking all eight different poly-Ub linkages and profiled the deubiquitinating enzyme (DUB) selectivity. Most DUBs examined have broad selectivity while a subset displays a preference for recognizing noncanonical linkages.