Lipodistrophy: a paradigm for understanding the consequences of "overloading" adipose tissue.

Lipodystrophies have been recognized since at least the nineteenth century and, despite their rarity, tended to attract considerable medical attention because of the severity and somewhat paradoxical nature of the associated metabolic disease that so closely mimics that of obesity. Within the last 20 yr most of the monogenic subtypes have been characterized, facilitating family genetic screening and earlier disease detection as well as providing important insights into adipocyte biology and the systemic consequences of impaired adipocyte function. Even more recently, compelling genetic studies have suggested that subtle partial lipodystrophy is likely to be a major factor in prevalent insulin-resistant type 2 diabetes mellitus (T2DM), justifying the longstanding interest in these disorders. This progress has also underpinned novel approaches to treatment that, in at least some patients, can be of considerable therapeutic benefit.

Characterization of mitochondrion-targeted GTPases in Plasmodium falciparum.

Ribosome assembly is critical for translation and regulating the response to cellular events and requires a complex interplay of ribosomal RNA and proteins with assembly factors. We investigated putative participants in the biogenesis of the reduced organellar ribosomes of Plasmodium falciparum and identified homologues of two assembly GTPases - EngA and Obg that were found in mitochondria. Both are indispensable in bacteria and P. berghei EngA is among the 'essential' parasite blood stage proteins identified recently. PfEngA and PfObg1 interacted with parasite mitoribosomes in vivo. GTP stimulated PfEngA interaction with the 50S subunit of Escherichia coli surrogate ribosomes. Although PfObg1-ribosome interaction was independent of nucleotide binding, GTP hydrolysis by PfObg1 was enhanced upon ribosomal association. An additional function for PfObg1 in mitochondrial DNA transactions was suggested by its specific interaction with the parasite mitochondrial genome in vivo. Deletion analysis revealed that the positively-charged OBG (spoOB-associated GTP-binding protein) domain mediates DNA-binding. A role for PfEngA in mitochondrial genotoxic stress response was indicated by its over-expression upon methyl methanesulfonate-induced DNA damage. PfEngA had lower sensitivity to an E. coli EngA inhibitor suggesting differences with bacterial counterparts. Our results show the involvement of two important GTPases in P. falciparum mitochondrial function, with the first confirmed localization of an EngA homologue in eukaryotic mitochondria.

Conserved Amphipathic Helices Mediate Lipid Droplet Targeting of Perilipins 1-3.

Perilipins (PLINs) play a key role in energy storage by orchestrating the activity of lipases on the surface of lipid droplets. Failure of this activity results in severe metabolic disease in humans. Unlike all other lipid droplet-associated proteins, PLINs localize almost exclusively to the phospholipid monolayer surrounding the droplet. To understand how they sense and associate with the unique topology of the droplet surface, we studied the localization of human PLINs inSaccharomyces cerevisiae,demonstrating that the targeting mechanism is highly conserved and that 11-mer repeat regions are sufficient for droplet targeting. Mutations designed to disrupt folding of this region into amphipathic helices (AHs) significantly decreased lipid droplet targetingin vivoandin vitro Finally, we demonstrated a substantial increase in the helicity of this region in the presence of detergent micelles, which was prevented by an AH-disrupting missense mutation. We conclude that highly conserved 11-mer repeat regions of PLINs target lipid droplets by folding into AHs on the droplet surface, thus enabling PLINs to regulate the interface between the hydrophobic lipid core and its surrounding hydrophilic environment.

Targeting and function of proteins mediating translation initiation in organelles of Plasmodium falciparum.

The malaria parasite Plasmodium falciparum has two translationally active organelles - the apicoplast and mitochondrion, which import nuclear-encoded translation factors to mediate protein synthesis. Initiation of translation is a complex step wherein initiation factors (IFs) act in a regulated manner to form an initiation complex. We identified putative organellar IFs and investigated the targeting, structure and function of IF1, IF2 and IF3 homologues encoded by the parasite nuclear genome. A single PfIF1 is targeted to the apicoplast. Apart from its critical ribosomal interactions, PfIF1 also exhibited nucleic-acid binding and melting activities and mediated transcription anti-termination. This suggests a prominent ancillary function for PfIF1 in destabilisation of DNA and RNA hairpin loops encountered during transcription and translation of the A+T rich apicoplast genome. Of the three putative IF2 homologues, only one (PfIF2a) was an organellar protein with mitochondrial localisation. We additionally identified an IF3 (PfIF3a) that localised exclusively to the mitochondrion and another protein, PfIF3b, that was apicoplast targeted. PfIF3a exhibited ribosome anti-association activity, and monosome splitting by PfIF3a was enhanced by ribosome recycling factor (PfRRF2) and PfEF-G(Mit). These results fill a gap in our understanding of organellar translation in Plasmodium, which is the site of action of several anti-malarial compounds.

Structure-Based Optimization of a Series of Covalent, Cell Active Bfl-1 Inhibitors.

Bfl-1, a member of the Bcl-2 family of proteins, plays a crucial role in apoptosis regulation and has been implicated in cancer cell survival and resistance to venetoclax therapy. Due to the unique cysteine residue in the BH3 binding site, the development of covalent inhibitors targeting Bfl-1 represents a promising strategy for cancer treatment. Herein, the optimization of a covalent cellular tool from a lead-like hit using structure based design is described. Informed by a reversible X-ray fragment screen, the strategy to establish interactions with a key glutamic acid residue (Glu78) and optimize binding in a cryptic pocket led to a 1000-fold improvement in biochemical potency without increasing reactivity of the warhead. Compound (R,R,S)-26 has a kinact/KI of 4600 M-1 s-1, shows <1 µM caspase activation in a cellular assay and cellular target engagement, and has good physicochemical properties and a promising in vivo profile.

A mouse model of human mitofusin-2-related lipodystrophy exhibits adipose-specific mitochondrial stress and reduced leptin secretion.

Mitochondrial dysfunction has been reported in obesity and insulin resistance, but primary genetic mitochondrial dysfunction is generally not associated with these, arguing against a straightforward causal relationship. A rare exception, recently identified in humans, is a syndrome of lower body adipose loss, leptin-deficient severe upper body adipose overgrowth, and insulin resistance caused by the p.Arg707Trp mutation in MFN2, encoding mitofusin 2. How the resulting selective form of mitochondrial dysfunction leads to tissue- and adipose depot-specific growth abnormalities and systemic biochemical perturbation is unknown. To address this, Mfn2R707W/R707W knock-in mice were generated and phenotyped on chow and high fat diets. Electron microscopy revealed adipose-specific mitochondrial morphological abnormalities. Oxidative phosphorylation measured in isolated mitochondria was unperturbed, but the cellular integrated stress response was activated in adipose tissue. Fat mass and distribution, body weight, and systemic glucose and lipid metabolism were unchanged, however serum leptin and adiponectin concentrations, and their secretion from adipose explants were reduced. Pharmacological induction of the integrated stress response in wild-type adipocytes also reduced secretion of leptin and adiponectin, suggesting an explanation for the in vivo findings. These data suggest that the p.Arg707Trp MFN2 mutation selectively perturbs mitochondrial morphology and activates the integrated stress response in adipose tissue. In mice, this does not disrupt most adipocyte functions or systemic metabolism, whereas in humans it is associated with pathological adipose remodelling and metabolic disease. In both species, disproportionate effects on leptin secretion may relate to cell autonomous induction of the integrated stress response.

Discovery of AZD4747, a Potent and Selective Inhibitor of Mutant GTPase KRASG12C with Demonstrable CNS Penetration.

The glycine to cysteine mutation at codon 12 of Kirsten rat sarcoma (KRAS) represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 14, AZD4747, a clinical development candidate for the treatment of KRASG12C-positive tumors, including the treatment of central nervous system (CNS) metastases. Building on our earlier discovery of C5-tethered quinazoline AZD4625, excision of a usually critical pyrimidine ring yielded a weak but brain-penetrant start point which was optimized for potency and DMPK. Key design principles and measured parameters that give high confidence in CNS exposure are discussed. During optimization, divergence between rodent and non-rodent species was observed in CNS exposure, with primate PET studies ultimately giving high confidence in the expected translation to patients. AZD4747 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.

Accelerating the Validation of Endogenous On-Target Engagement and In Cellulo Kinetic Assessment for Covalent Inhibitors of KRASG12C in Early Drug Discovery.

Covalent inhibition is a valuable modality in drug discovery because of its potential ability in decoupling pharmacokinetics from pharmacodynamics by prolonging the residence time of the drug on the target of interest. This increase in target occupancy is limited only by the rate of target turnover. However, a limitation in such studies is to translate the in vitro inhibition assessment to the appropriate in cellulo target engagement parameter by covalent probes. Estimation of such parameters is often impeded by the low-throughput nature of current probe-free approaches. In this study, an ultra-performance liquid chromatography-multiple reaction monitoring mass spectrometry platform was utilized to develop a targeted proteomics workflow that can evaluate cellular on-target engagement of covalent molecules in an increased throughput manner. This workflow enabled a throughput increase of 5-10 fold when compared to traditional nanoLC-based proteomics studies. To demonstrate the applicability of the method, KRASG12C was used as a model system to investigate the interaction of an irreversible covalent small molecule, compound 25, both in vitro and in cellulo. Initial biochemical studies confirmed that the small molecule forms an adduct with the targeted cysteine on the protein, as assessed at the level of both intact protein and on the target peptide. In cellulo studies were carried out to quantify target engagement and allele selectivity assessment for the small molecule in the heterozygous NCI-H358 cell line for KRASG12C with respect to the WT type protein. The workflow enabled evaluation of in vitro and in cellulo target engagement kinetics, providing mechanistic insights into the irreversible mode of inhibition. In summary, the method has the potential for target agnostic application in the assessment of on-target engagement of covalent probes compatible with the high-throughput requirements of early drug discovery.

Combined genetic deletion of GDF15 and FGF21 has modest effects on body weight, hepatic steatosis and insulin resistance in high fat fed mice.

OBJECTIVES: Obesity in humans and mice is associated with elevated levels of two hormones responsive to cellular stress, namely GDF15 and FGF21. Over-expression of each of these is associated with weight loss and beneficial metabolic changes but where they are secreted from and what they are required for physiologically in the context of overfeeding remains unclear. METHODS: Here we used tissue selective knockout mouse models and human transcriptomics to determine the source of circulating GDF15 in obesity. We then generated and characterized the metabolic phenotypes of GDF15/FGF21 double knockout mice. RESULTS: Circulating GDF15 and FGF21 are both largely derived from the liver, rather than adipose tissue or skeletal muscle, in obese states. Combined whole body deletion of FGF21 and GDF15 does not result in any additional weight gain in response to high fat feeding but it does result in significantly greater hepatic steatosis and insulin resistance than that seen in GDF15 single knockout mice. CONCLUSIONS: Collectively the data suggest that overfeeding activates a stress response in the liver which is the major source of systemic rises in GDF15 and FGF21. These hormones then activate pathways which reduce this metabolic stress.

Phenotypic characterization of Adig null mice suggests roles for adipogenin in the regulation of fat mass accrual and leptin secretion.

Adipogenin (Adig) is an adipocyte-enriched transmembrane protein. Its expression is induced during adipogenesis in rodent cells, and a recent genome-wide association study associated body mass index (BMI)-adjusted leptin levels with the ADIG locus. In order to begin to understand the biological function of Adig, we studied adipogenesis in Adig-deficient cultured adipocytes and phenotyped Adig null (Adig-/-) mice. Data from Adig-deficient cells suggest that Adig is required for adipogenesis. In vivo, Adig-/- mice are leaner than wild-type mice when fed a high-fat diet and when crossed with Ob/Ob hyperphagic mice. In addition to the impact on fat mass accrual, Adig deficiency also reduces fat-mass-adjusted plasma leptin levels and impairs leptin secretion from adipose explants, suggesting an additional impact on the regulation of leptin secretion.

PCYT1A Regulates Phosphatidylcholine Homeostasis from the Inner Nuclear Membrane in Response to Membrane Stored Curvature Elastic Stress.

Cell and organelle membranes consist of a complex mixture of phospholipids (PLs) that determine their size, shape, and function. Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic membranes, yet how cells sense and regulate its levels in vivo remains unclear. Here we show that PCYT1A, the rate-limiting enzyme of PC synthesis, is intranuclear and re-locates to the nuclear membrane in response to the need for membrane PL synthesis in yeast, fly, and mammalian cells. By aligning imaging with lipidomic analysis and data-driven modeling, we demonstrate that yeast PCYT1A membrane association correlates with membrane stored curvature elastic stress estimates. Furthermore, this process occurs inside the nucleus, although nuclear localization signal mutants can compensate for the loss of endogenous PCYT1A in yeast and in fly photoreceptors. These data suggest an ancient mechanism by which nucleoplasmic PCYT1A senses surface PL packing defects on the inner nuclear membrane to control PC homeostasis.

Reduced ribosomes of the apicoplast and mitochondrion of Plasmodium spp. and predicted interactions with antibiotics.

Apicomplexan protists such as Plasmodium and Toxoplasma contain a mitochondrion and a relic plastid (apicoplast) that are sites of protein translation. Although there is emerging interest in the partitioning and function of translation factors that participate in apicoplast and mitochondrial peptide synthesis, the composition of organellar ribosomes remains to be elucidated. We carried out an analysis of the complement of core ribosomal protein subunits that are encoded by either the parasite organellar or nuclear genomes, accompanied by a survey of ribosome assembly factors for the apicoplast and mitochondrion. A cross-species comparison with other apicomplexan, algal and diatom species revealed compositional differences in apicomplexan organelle ribosomes and identified considerable reduction and divergence with ribosomes of bacteria or characterized organelle ribosomes from other organisms. We assembled structural models of sections of Plasmodium falciparum organellar ribosomes and predicted interactions with translation inhibitory antibiotics. Differences in predicted drug-ribosome interactions with some of the modelled structures suggested specificity of inhibition between the apicoplast and mitochondrion. Our results indicate that Plasmodium and Toxoplasma organellar ribosomes have a unique composition, resulting from the loss of several large and small subunit proteins accompanied by significant sequence and size divergences in parasite orthologues of ribosomal proteins.