Emerging approaches to CDK inhibitor development, a structural perspective.

Aberrant activity of the cyclin-dependent kinase family is frequently noted in a number of diseases identifying them as potential targets for drug development. However, current CDK inhibitors lack specificity owing to the high sequence and structural conservation of the ATP binding cleft across family members, highlighting the necessity of finding novel modes of CDK inhibition. The wealth of structural information regarding CDK assemblies and inhibitor complexes derived from X-ray crystallographic studies has been recently complemented through the use of cryo-electron microscopy. These recent advances have provided insights into the functional roles and regulatory mechanisms of CDKs and their interaction partners. This review explores the conformational malleability of the CDK subunit, the importance of SLiM recognition sites in CDK complexes, the progress made in chemically induced CDK degradation and how these studies can contribute to CDK inhibitor design. Additionally, fragment-based drug discovery can be utilised to identify small molecules that bind to allosteric sites on the CDK surface employing interactions which mimic those of native protein-protein interactions. These recent structural advances in CDK inhibitor mechanisms and in chemical probes which do not occupy the orthosteric ATP binding site can provide important insights for targeted CDK therapies.

Mapping Ligand Interactions of Bromodomains BRD4 and ATAD2 with FragLites and PepLites─Halogenated Probes of Druglike and Peptide-like Molecular Interactions.

The development of ligands for biological targets is critically dependent on the identification of sites on proteins that bind molecules with high affinity. A set of compounds, called FragLites, can identify such sites, along with the interactions required to gain affinity, by X-ray crystallography. We demonstrate the utility of FragLites in mapping the binding sites of bromodomain proteins BRD4 and ATAD2 and demonstrate that FragLite mapping is comparable to a full fragment screen in identifying ligand binding sites and key interactions. We extend the FragLite set with analogous compounds derived from amino acids (termed PepLites) that mimic the interactions of peptides. The output of the FragLite maps is shown to enable the development of ligands with leadlike potency. This work establishes the use of FragLite and PepLite screening at an early stage in ligand discovery allowing the rapid assessment of tractability of protein targets and informing downstream hit-finding.

Single Amino Acid Changes in the Ryanodine Receptor in the Human Population Have Effects In Vivo on Caenorhabditis elegans Neuro-Muscular Function.

The ryanodine receptor mediates intracellular calcium ion release with excitation of nerve and muscle cells. Ryanodine receptor missense variants cause a number of myopathologies, such as malignant hyperthermia, and have been linked with various neuropathologies, including Alzheimer's disease. We characterized the consequences of ryanodine receptor variants in vivo. Eight Caenorhabditis elegans strains, with ryanodine receptor modifications equivalent to human myopathic RYR1 variants, were generated by genome editing. In humans, these variants are rare and confer sensitivity to the inhalational anaesthetic halothane when heterozygous. Increased sensitivity to halothane was found in both homozygous and heterozygous C. elegans. Close analysis revealed distinct subtle locomotion defects, due to the different single amino acid residue changes, even in the absence of the external triggering agent. Distinct pre- and postsynaptic consequences of the variants were characterized through the responses to cholinergic pharmacological agents. The range of phenotypes reflects the complexity of the regulatory inputs to the ryanodine receptor and the criticality of the calcium ion channel opening properties, in different cell types and with age. Ryanodine receptors with these single amino acid residue changes still function as calcium ion channels, but with altered properties which are likely to have subtle consequences for human carriers of such variants. The long-term consequences of subtly altered calcium ion signalling could be cumulative and may be focussed in the smaller nerve cells rather than the more robust muscle cells. It was important to assess phenotypes in vivo to properly appreciate consequences for a whole organism.

Aging Effects of Caenorhabditis elegans Ryanodine Receptor Variants Corresponding to Human Myopathic Mutations.

Delaying the decline in skeletal muscle function will be critical to better maintenance of an active lifestyle in old age. The skeletal muscle ryanodine receptor, the major intracellular membrane channel through which calcium ions pass to elicit muscle contraction, is central to calcium ion balance and is hypothesized to be a significant factor for age-related decline in muscle function. The nematode Caenorhabditis elegans is a key model system for the study of human aging, and strains were generated with modified C. elegans ryanodine receptors corresponding to human myopathic variants linked with malignant hyperthermia and related conditions. The altered response of these strains to pharmacological agents reflected results of human diagnostic tests for individuals with these pathogenic variants. Involvement of nerve cells in the C. elegans responses may relate to rare medical symptoms concerning the central nervous system that have been associated with ryanodine receptor variants. These single amino acid modifications in C. elegans also conferred a reduction in lifespan and an accelerated decline in muscle integrity with age, supporting the significance of ryanodine receptor function for human aging.

Stressful environments can indirectly select for increased longevity.

Longevity is modulated by a range of conserved genes in eukaryotes, but it is unclear how variation in these genes contributes to the evolution of longevity in nature. Mutations that increase life span in model organisms typically induce trade-offs which lead to a net reduction in fitness, suggesting that such mutations are unlikely to become established in natural populations. However, the fitness consequences of manipulating longevity have rarely been assessed in heterogeneous environments, in which stressful conditions are encountered. Using laboratory selection experiments, we demonstrate that long-lived, stress-resistant Caenorhabditis elegans age-1(hx546) mutants have higher fitness than the wild-type genotype if mixed genotype populations are periodically exposed to high temperatures when food is not limited. We further establish, using stochastic population projection models, that the age-1(hx546) mutant allele can confer a selective advantage if temperature stress is encountered when food availability also varies over time. Our results indicate that heterogeneity in environmental stress may lead to altered allele frequencies over ecological timescales and indirectly drive the evolution of longevity. This has important implications for understanding the evolution of life-history strategies.

The significance of alternative transcripts for Caenorhabditis elegans transcription factor genes, based on expression pattern analysis.

BACKGROUND: Sequence-specific DNA-binding proteins, with their paramount importance in the regulation of expression of the genetic material, are encoded by approximately 5% of the genes in an animal's genome. But it is unclear to what extent alternative transcripts from these genes may further increase the complexity of the transcription factor complement. RESULTS: Of the 938 potential C. elegans transcription factor genes, 197 were annotated in WormBase as encoding at least two distinct isoforms. Evaluation of prior evidence identified, with different levels of confidence, 50 genes with alternative transcript starts, 23 with alternative transcript ends, 35 with alternative splicing and 34 with alternative transcripts generated by a combination of mechanisms, leaving 55 that were discounted. Expression patterns were determined for transcripts for a sample of 29 transcription factor genes, concentrating on those with alternative transcript starts for which the evidence was strongest. Seamless fosmid recombineering was used to generate reporter gene fusions with minimal modification to assay expression of specific transcripts while maintaining the broad genomic DNA context and alternative transcript production. Alternative transcription factor gene transcripts were typically expressed with identical or substantially overlapping distributions rather than in distinct domains. CONCLUSIONS: Increasingly sensitive sequencing technologies will reveal rare transcripts but many of these are clearly non-productive. The majority of the transcription factor gene alternative transcripts that are productive may represent tolerable noise rather than encoding functionally distinct isoforms.

The Caenorhabditis elegans homeobox gene ceh-19 is required for MC motorneuron function.

Simplicity has made C. elegans pharyngeal development a particularly well-studied subject. Nevertheless, here we add the previously uncharacterized homeobox gene F20D12.6/ceh-19 to the set of transcription factor genes involved. GFP reporter assays revealed that ceh-19 is expressed in three pairs of neurons, the pharyngeal pace-maker neurons MC, the amphid neurons ADF and the phasmid neurons PHA. ceh-19(tm452) mutants are viable and fertile, but grow slightly slower, produce less progeny over a prolonged period, and live longer than the wild type. These phenotypes are likely due to the moderately reduced pharyngeal pumping speed arising from the impairment of MC activity. MC neurons are still born in the ceh-19 mutants but display various morphological defects. ceh-19 expression in MC is completely lost in progeny from animals subject to RNAi for pha-4, which encodes an organ-specifying forkhead transcription factor. CEH-19 is required for the activation in MCs of the excitatory FMRFamide-like neuropeptide-encoding gene flp-2. A regulatory pathway from pha-4 through ceh-19 to flp-2 is thereby defined. The resilience of MC identity in the absence of CEH-19 may reflect the buffering qualities of transcription factor regulatory networks.

A simplified counter-selection recombineering protocol for creating fluorescent protein reporter constructs directly from C. elegans fosmid genomic clones.

BACKGROUND: Recombineering is a genetic engineering tool that enables facile modification of large episomal clones, e.g. BACs, fosmids. We have previously adapted this technology to generate, directly from fosmid-based genomic clones, fusion gene reporter constructs designed to investigate gene expression patterns in C. elegans. In our adaptation a rpsL-tet(A) positive/negative-selection cassette (RT-cassette) is first inserted and then, under negative selection, seamlessly replaced with the desired sequence. We report here on the generation and application of a resource comprising two sets of constructs designed to facilitate this particular recombineering approach. RESULTS: Two complementary sets of constructs were generated. The first contains different fluorescent protein reporter coding sequences and derivatives while the second set of constructs, based in the copy-number inducible vector pCC1Fos, provide a resource designed to simplify RT-cassette-based recombineering. These latter constructs are used in pairs the first member of which provides a template for PCR-amplification of an RT-cassette while the second provides, as an excised restriction fragment, the desired fluorescent protein reporter sequence. As the RT-cassette is flanked by approximately 200 bp from the ends of the reporter sequence the subsequent negative selection replacement step is highly efficient. Furthermore, use of a restriction fragment minimizes artefacts negating the need for final clone sequencing. Utilizing this resource we generated single-, double- and triple-tagged fosmid-based reporters to investigate expression patterns of three C. elegans genes located on a single genomic clone. CONCLUSIONS: We describe the generation and application of a resource designed to facilitate counter-selection recombineering of fosmid-based C. elegans genomic clones. By choosing the appropriate pair of 'insertion' and 'replacement' constructs recombineered products, devoid of artefacts, are generated at high efficiency. Gene expression patterns for three genes located on the same genomic clone were investigated via a set of fosmid-based reporter constructs generated with the modified protocol.

Expression pattern analysis of regulatory transcription factors in Caenorhabditis elegans.

Expression pattern data are fundamental to understanding transcriptional regulatory networks and the biological significance of such networks. For Caenorhabditis elegans, expression pattern analysis of transcription factor genes, with cellular resolution, typically involves generation of transcription factor gene/reporter gene fusions. This is followed by the creation of C. elegans strains transgenic for, and determination of expression patterns driven by, these fusions. Physiologically relevant regulatory relationships between transcription factors are both inferred from their expression patterns, in combination with protein-DNA interaction data, and evidenced from alterations of expression patterns when networks are disturbed.

A regulatory cascade of three transcription factors in a single specific neuron, DVC, in Caenorhabditis elegans.

Homeobox proteins are critical regulators of developmental gene transcription and cell specification. Many insights into transcriptional regulation have been gained from studies in the nematode Caenorhabditis elegans. We investigated the expression and regulation of the C. elegans homeobox gene ceh-63, which encodes a single-homeodomain transcription factor of 152 amino acids. ceh-63 is expressed in the interneuron DVC in both sexes, from late embryogenesis through adulthood, and two pairs of uterine cells in reproductive hermaphrodites only. A reporter gene fusion, encoding GFP fused to the full-length CEH-63, also drove weak inconsistent expression in additional unidentified cells in the head and tail. A potential ceh-63 null mutant had no obvious abnormalities, except for a possible increase in subtle defects of the DVC axon projection. No behavioural responses were observed upon either laser ablation of DVC or activation of DVC through light stimulation of channelrhodopsin-2 specifically expressed in this neuron. The function of DVC therefore remains enigmatic. A transcriptional regulatory cascade operating in DVC was defined from the LIM-homeodomain protein CEH-14 through CEH-63 to the helix-turn-helix transcription factor MBR-1. Both CEH-14 and CEH-63 individually bound the mbr-1 promoter in a yeast one-hybrid assay. A model is proposed suggesting that CEH-14 activates ceh-63 and then along with CEH-63 co-ordinately activates mbr-1.

DAF-16 and Δ9 desaturase genes promote cold tolerance in long-lived Caenorhabditis elegans age-1 mutants.

In Caenorhabditis elegans, mutants of the conserved insulin/IGF-1 signalling (IIS) pathway are long-lived and stress resistant due to the altered expression of DAF-16 target genes such as those involved in cellular defence and metabolism. The three Δ(9) desaturase genes, fat-5, fat-6 and fat-7, are included amongst these DAF-16 targets, and it is well established that Δ(9) desaturase enzymes play an important role in survival at low temperatures. However, no assessment of cold tolerance has previously been reported for IIS mutants. We demonstrate that long-lived age-1(hx546) mutants are remarkably resilient to low temperature stress relative to wild type worms, and that this is dependent upon daf-16. We also show that cold tolerance following direct transfer to low temperatures is increased in wild type worms during the facultative, daf-16 dependent, dauer stage. Although the cold tolerant phenotype of age-1(hx546) mutants is predominantly due to the Δ(9) desaturase genes, additional transcriptional targets of DAF-16 are also involved. Surprisingly, survival of wild type adults following a rapid temperature decline is not dependent upon functional daf-16, and cellular distributions of a DAF-16::GFP fusion protein indicate that DAF-16 is not activated during low temperature stress. This suggests that cold-induced physiological defences are not specifically regulated by the IIS pathway and DAF-16, but expression of DAF-16 target genes in IIS mutants and dauers is sufficient to promote cross tolerance to low temperatures in addition to other forms of stress.

Distinct mechanisms for delimiting expression of four Caenorhabditis elegans transcription factor genes encoding activators or repressors.

Regulatory transcription factors operate in networks, conferring biological robustness that makes dissection of such gene control processes difficult. The nematode Caenorhabditis elegans is a powerful molecular genetic system that allows the close scrutiny needed to understand these processes in an animal, in vivo. Strikingly lower levels of gene expression were observed when a gfp reporter was inserted into C. elegans transcription factor genes, in their broader genomic context, in comparison to when the reporter was fused to just the promoter regions. The lower level of expression is more consistent with endogenous levels of the gene products, based on independent protein and transcript assays. Through successive precise manipulations of the reporter fusion genes, elements essential for the lower level of expression were localised to the protein-coding region. With a closer focus on four transcription factor genes, the expression of both genes encoding transcriptional activators was found to be restricted by a post-transcriptional mechanism while expression of both genes encoding transcriptional repressors was delimited by transcriptional repression. An element through which the transcriptional repression acts for unc-4 was localised to a 30 base-pair region of a protein-encoding exon, with potentially wider implications for how homeobox genes operate. The hypothesis that the distinction in mechanisms delimiting expression of the two types of transcription factor genes, as observed here, may apply more widely is raised. This leads to observations concerning the implications of these different mechanisms on stochastic noise in gene expression and the consequent significance for developmental decisions in general.

DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO.

Insulin/IGF-1 signaling controls metabolism, stress resistance and aging in Caenorhabditis elegans by regulating the activity of the DAF-16/FoxO transcription factor (TF). However, the function of DAF-16 and the topology of the transcriptional network that it crowns remain unclear. Using chromatin profiling by DNA adenine methyltransferase identification (DamID), we identified 907 genes that are bound by DAF-16. These were enriched for genes showing DAF-16-dependent upregulation in long-lived daf-2 insulin/IGF-1 receptor mutants (P=1.4e(-11)). Cross-referencing DAF-16 targets with these upregulated genes (daf-2 versus daf-16; daf-2) identified 65 genes that were DAF-16 regulatory targets. These 65 were enriched for signaling genes, including known determinants of longevity, but not for genes specifying somatic maintenance functions (e.g. detoxification, repair). This suggests that DAF-16 acts within a relatively small transcriptional subnetwork activating (but not suppressing) other regulators of stress resistance and aging, rather than directly regulating terminal effectors of longevity. For most genes bound by DAF-16::DAM, transcriptional regulation by DAF-16 was not detected, perhaps reflecting transcriptionally non-functional TF 'parking sites'. This study demonstrates the efficacy of DamID for chromatin profiling in C. elegans.

Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host.

BACKGROUND: Escherichia coli strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, oriV, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate trans-replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer oriV-equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a PBAD-driven trfA gene generating strain MW005 that supports, independently, both recombineering and copy-number induction. RESULTS: The PBAD-driven copy of cre in EL350 was replaced seamlessly with a copy of trfA, PCR-amplified from EPI300 chromosomal DNA, to generate MW005. This new strain has been used to both generate, via recombineering, a number of reporter gene fusions directly from pCC1FOS-based Caenorhabditis elegans genomic clones and to transiently induce copy-number of fosmid and BAC clones prior to DNA preparation. CONCLUSIONS: By retrofitting EL350, an established 'recombineering' E. coli strain, with a tightly regulated copy of trfA we have produced a new strain, MW005, which combines recombineering capacity with the useful ability to transiently induce copy-number of oriV-equipped clones. By coupling these two steps in a single strain, use of MW005 will enable the more rapid recombineering-mediated production of recombinant clones in the yield and quality necessary for many downstream purposes.

Determination of the mobility of novel and established Caenorhabditis elegans sarcomeric proteins in vivo.

A screen was instigated to identify novel protein components of the Caenorhabditis elegans sarcomere. The subcellular localisation of full-length GFP fusion proteins was examined, in transgenic animals, for 62 essentially uncharacterized genes thought to be expressed within bodywall muscle cells. Three genes, T03G6.3, C46G7.2 and K04A8.6, were identified for further study. K04A8.6::GFP only displayed a regular sarcomeric distribution sporadically. However, C46G7.2::GFP localised to the centre of A-bands and dense bodies and T03G6.3::GFP localised in the I-band, of the bodywall muscle sarcomeres, consistently. This success with such a small screen suggests that there are further minor components of the C. elegans sarcomere yet to be discovered. Fluorescence Recovery After Photobleaching (FRAP) was applied to live transgenic individuals to assess the mobility of T03G6.3 and C46G7.2 and other well-known constituents of the sarcomere in vivo. Proteins associated with the thin filaments showed dynamic exchange whilst those associated with thick filaments appeared more static. This is the first demonstration that there are sarcomeric proteins in C. elegans muscle cells in dynamic exchange and that the rates of exchange in vivo correspond in general terms with observations in other experimental systems.

The Caenorhabditis elegans sirtuin gene, sir-2.1, is widely expressed and induced upon caloric restriction.

INTRODUCTION: As in yeast, flies and mammals, over-expression of the Caenorhabditis elegans sirtuin gene sir-2.1 leads to extension of lifespan and deletion of the gene shortens lifespan. The sir-2.1 gene, however, is located in an operon, an organization not taken into account in previous studies of this gene's expression. MATERIALS AND METHODS: Recombineering allowed insertion of both a mCherry and a gfp reporter gene precisely at the end of the two protein-coding regions of the 4.5kb sir-2.1 operon within a 29.3kb genomic DNA fosmid clone. RESULTS AND DISCUSSION: In C. elegans transgenic for this recombineered fosmid, with abundant food, the sir-2.1::mCherry distribution indicated that sir-2.1 is indeed expressed in the hypodermis and many nerve cells, as previously described, but also in the intestine and in muscles. This broader expression of sir-2.1, which would fit with an expectation that SIR2.1 function in influencing lifespan might be required in most cell types, arises from transcription starting with the gene upstream of sir-2.1 in the operon. Importantly, the expression of both genes in the operon increases upon starvation, this induction also depending on the operon promoter. Furthermore, SIR-2.1::mCherry undergoes a dynamic subcellular relocalization through starvation.

Forward locomotion of the nematode C. elegans is achieved through modulation of a single gait.

The ability of an animal to locomote through its environment depends crucially on the interplay between its active endogenous control and the physics of its interactions with the environment. The nematode worm Caenorhabditis elegans serves as an ideal model system for studying the respective roles of neural control and biomechanics, as well as the interaction between them. With only 302 neurons in a hard-wired neural circuit, the worm's apparent anatomical simplicity belies its behavioural complexity. Indeed, C. elegans exhibits a rich repertoire of complex behaviors, the majority of which are mediated by its adaptive undulatory locomotion. The conventional wisdom is that two kinematically distinct C. elegans locomotion behaviors-swimming in liquids and crawling on dense gel-like media-correspond to distinct locomotory gaits. Here we analyze the worm's motion through a series of different media and reveal a smooth transition from swimming to crawling, marked by a linear relationship between key locomotion metrics. These results point to a single locomotory gait, governed by the same underlying control mechanism. We further show that environmental forces play only a small role in determining the shape of the worm, placing conditions on the minimal pattern of internal forces driving locomotion.

Large-scale gene expression pattern analysis, in situ, in Caenorhabditis elegans.

Observation of gene expression in situ provides a direct connection between the genetic information in the genome sequence and the fully determined developmental cell lineage of Caenorhabditis elegans. Green Fluorescent Protein (GFP) reporters have been fused with many C. elegans genes, in large-scale projects, by conventional DNA ligation, PCR stitching, Gateway recombination and recombineering. These reporter gene fusions have then been used in C. elegans transformation either by microinjection or microprojectile bombardment. So far, the developmental distributions of GFP, as driven by the C. elegans DNA to which the reporter gene has been attached, have been determined simply from direct examination of the transgenic strains by epifluorescence microscopy. Automation of GFP expression pattern determination promises improvements in both quality and quantity of this data type, facilitating the handling of such expression pattern data within computer databases. As with the descriptions of the developmental cell lineage and the genome sequence, a complete description of gene expression patterns will provide a vital knowledge framework through which a full understanding of the development of this animal can emerge.

Genome-scale analysis of in vivo spatiotemporal promoter activity in Caenorhabditis elegans.

Differential regulation of gene expression is essential for cell fate specification in metazoans. Characterizing the transcriptional activity of gene promoters, in time and in space, is therefore a critical step toward understanding complex biological systems. Here we present an in vivo spatiotemporal analysis for approximately 900 predicted C. elegans promoters (approximately 5% of the predicted protein-coding genes), each driving the expression of green fluorescent protein (GFP). Using a flow-cytometer adapted for nematode profiling, we generated 'chronograms', two-dimensional representations of fluorescence intensity along the body axis and throughout development from early larvae to adults. Automated comparison and clustering of the obtained in vivo expression patterns show that genes coexpressed in space and time tend to belong to common functional categories. Moreover, integration of this data set with C. elegans protein-protein interactome data sets enables prediction of anatomical and temporal interaction territories between protein partners.