Sarah Crunkhorn

Trial watch: PCSK9 antibody reduces LDL cholesterol

Preliminary Phase II trial results for REGN727 — a human proprotein convertase subtilisin/kexin type 9 (PCSK9)-specific monoclonal antibody co-developed by Regeneron and Sanofi — indicate that it effectively and safely reduces levels of low-density lipoprotein-cholesterol (LDL-C) in patients with hypercholesterolaemia undergoing statin therapy.

Anticancer drugs: Redesigning kinase inhibitors

highly successful in the treatment of both chronic myelogenous leukaemia (CML) and gastrointestinal stromal tumours (GISTs); however, it has been associated with uncommon, but severe, cardiotoxicity. Now, writing in JCI, Fernández and colleagues describe their novel approach of re-engineering imatinib to alter its kinase inhibition profile to specifically target GISTs, while reducing its toxic effects on the heart. Protein kinases are prime targets for anticancer therapies, but their close structural similarities can make achieving specificity for a particular kinase challenging, potentially leading to unwanted side effects. With this in mind, the authors aimed to redesign imatinib, focusing its specificity on C-Kit (also known as KIT) kinase inhibition (the therapeutic target for GIST) while reducing its effect on the kinase BCR–ABL (the therapeutic target for CML), which has also been linked to the cardiovascular side effects of the drug. Furthermore, they aimed to promote inhibition of JNK1, as previous research has indicated that this could ameliorate the adverse effects of imatinib on heart cells. Their methodology is based on the knowledge that protein binding sites are typically regions where it is most energetically advantageous to exclude surrounding water from the backbone hydrogen bonds. Therefore, they first set out to identify specific kinase residues within the imatinib–kinase interface that possess a local propensity for water removal. They pinpointed the residue pair C673–G676 within the imatinib–C-Kit interface, whose dehydration propensity is absent in BCR–ABL owing to differences in the local environment. Using structural and molecular dynamics analysis, they predicted that addition of a methyl group to imatinib to form the reengineered compound, WBZ_4, would promote dehydration of the C673–G676 residue pair upon binding to C-Kit and stabilize the complex, while hampering any association of WBZ_4 with BCR–ABL. Notably, this modification additionally favoured the affinity of WBZ_4 for JNK1. To confirm their theory, they screened WBZ_4 against 228 human kinases and identified selective affinities for both C-Kit and JNK1 with a 75% reduction in ABL affinity when compared with imatinib. Furthermore, in cancer cell lines, liposome-delivered WBZ_4 halted proliferation of C-Kit-expressing GIST882 cells, but, in contrast to imatinib, it had almost no effect on CML K562 cells. In a mouse model of GIST, WBZ_4 was shown to decrease tumour volume and weight to a similar extent to that observed with imatinib, but in contrast to imatinib, WBZ_4 did not affect levels of brain natriuretic peptide — a marker of myocardial hypertrophy and cardiac impairment — or affect left-ventricular cardiac function. JNK1/2 activation was reduced by WBZ_4 in mouse cardiomyocytes, contributing to the mechanistic basis for the lack of cardiotoxicity of this prototype compound. In summary, WBZ_4 could have potential as a novel therapy for GISTs, and the approach demonstrated in the study might also be applied to engineer the specificity of other kinase inhibitors with the aim of creating safer and more effective drugs.

Deal watch: Genentech dives deeper into the next wave of cancer immunotherapies

Genentech is partnering with NewLink Genetics on the development of NLG919 — a Phase I indoleamine 2,3‐dioxygenase (IDO) pathway inhibitor that could complement other cancer immunotherapies — by paying US

Antibacterial drugs: New antibiotics on the horizon?

150 million upfront and potentially more than

Trial watch: Success in amyloidosis trials supports potential of systemic RNAi

1 billion in milestones. The companies have also established a research collaboration for the discovery of further IDO inhibitors and tryptophan 2,3‐dioxygenase (TDO) inhibitors. Immunotherapies are among the hottest drugs in oncology, with agents that target immune checkpoints demonstrating impressive efficacy in a growing range of cancers and garnering blockbuster sales predictions (Nature Rev. Drug Discov. 13, 883–884; 2014). However, “we still have to learn how to activate antitumour T cells in a better and more specific manner,” says Benoît Van den Eynde, Director of The Ludwig Institute for Cancer Research, Brussels, Belgium, and co‐founder of iTeos Therapeutics. “The main challenge now is to combine current immunotherapy approaches with strategies to overcome the barriers put in place by many tumours to resist immune attack.” IDO inhibitors or TDO inhibitors may help to do just that. IDO and TDO — key enzymes in the tryptophan catabolism pathway — are overexpressed in a variety of cancers, leading to depletion of tryptophan and an accumulation of immunosuppressive tryptophan catabolites. “This pathway is an important mediator of cancer immune evasion, and so inhibiting IDO or TDO could enhance current immunotherapy strategies,” says Hatem Soliman, at the Moffitt Cancer Center, Tampa, Florida, USA. Indeed, preclinical studies suggest that small‐molecule IDO inhibitors may synergize with, and help overcome resistance to, existing clinical cancer immunotherapies, such as antibodies that target the immune checkpoint programmed cell‐death protein 1 (PD1) or its ligand PDL1 (Clin. Cancer Res. 20, 5290–5301; 2014). “Given their unique mechanism of action, IDO inhibitors and TDO inhibitors have the potential to synergize with any approach that stimulates antitumour T lymphocyte responses, including checkpoint inhibitors, cancer vaccines or adoptive transfer of T cells; as well as other, more classical approaches, including chemotherapy and radiotherapy, whose clinical efficacy also depends to some extent on the immune system,” says Van den Eynde. Soliman is excited about the ease with which these drugs can be combined with other therapies. “As a class, these drugs exhibit mild toxicity profiles that make them ideal combination partners and since they are oral agents, they can be conveniently dosed,” he says. Among the agents that Genentech could combine with NLG919 is its PDL1‐specific antibody MPDL3280A, which is in late‐stage development and under investigation in combination with an IDO inhibitor from Incyte, INCB24360, as agreed in a deal signed last year. iTeos Therapeutics, IOmet Pharma and Curadev Pharma are also developing IDO and TDO inhibitors, and NewLink has another IDO inhibitor, indoximod, in Phase II trials in combination with docetaxel in metastatic breast cancer. “The data with chemotherapy are showing some intriguing early signals, and we eagerly await completion of these trials to see if IDO inhibitors can build upon the therapeutic activity of these agents,” says Soliman. Sarah Crunkhorn N E W S & A N A LY S I S

Metabolic disease: New role for HDACs in glucose homeostasis

aureus is responsible for a variety of diseases ranging from skin and soft-tissue infections to pneumonia and sepsis. The increasing prevalence of multidrug-resistant bacterial strains and the lack of new classes of antibiotics have together made the effective treatment of such infections challenging. Now, two recent papers report novel strategies for combating this prevalent bacterial pathogen. S. aureus secretes several factors that are toxic to human immune cells, including several pore-forming leuko-toxins. However, the specific range of immune cells that are targeted by such leukotoxins and the host factors responsible for their selectivity remain unclear. Torres and colleagues therefore set out to identify potential leukotoxin receptors, with the aim of identifying novel therapeutic targets. The authors first purified recom-binant leukotoxins and assessed their ability to kill a set of human cell lines. They discovered that the leukotoxin LukED only displayed cytotoxicity to human T cell lines ectopically expressing CC chemokine receptor 5 (CCR5; a co-receptor required for HIV infection). Further in vitro experiments showed that LukED interacted directly with CCR5 and specifically targeted human T cells, macrophages and dendritic cells. Next, they assessed whether LukED-mediated cell killing could be prevented by blocking the CCR5–toxin interaction. They found that CCR5 antagonists, including the clinically approved HIV therapeutic maraviroc, blocked LukED-mediated pore formation and killing of CCR5 + T cells. Furthermore, S. aureus-mediated CCR5 + T cell cytotoxicity was dependent on LukED and completely blocked by maraviroc. Finally, they examined the contribution of CCR5 to S. aureus pathogenesis in vivo. S. aureus-elicited lymphocytes and macrophages from wild-type mice were highly susceptible to purified LukED, whereas those from Ccr5 –/– mice were markedly resistant. Moreover, Ccr5 –/– mice were largely resistant to lethal S. aureus infections, further supporting the potential of CCR5 blockade as a novel antibacterial strategy. Meanwhile, Oldfield and colleagues set out to inhibit the early steps of S. aureus cell wall peptidoglycan biosynthesis. Specifically, they targeted an enzyme in the isoprenoid biosynthesis pathway — undecaprenyl pyrophos-phate synthase (UppS), which cata-lyses the formation of undecaprenyl pyrophosphate. UppS inhibitors are predicted to synergize with existing cell wall biosynthesis inhibitors (such as vancomycin and methi cillin), which act in the later stages of peptidoglycan formation, potentially reducing toxicity or restoring drug sensitivity. Using in silico high-throughput screening and hit development, the authors produced a small series of benzoic, phosphonic and diketo acids, as well as a bisamidine and a bisamine, that were active …

Fungal infection: Protecting from Candida albicans

antitransthyretin small interfering RNA (siRNA) therapies indicate that these treatments safely and effectively reduce levels of transthyretin (TTR) in healthy volunteers and in patients with transthyretin-related amyloidosis (TTRA). TTRA is an inherited, debilitating, fatal disease caused by mutations in the TTR gene. These mutations decrease the stability of TTR protein tetramers, which ultimately form insoluble amyloid fibrils that accumulate in various tissues, causing progressive sensory, motor and autonomic impairment. There are two major forms of the disease: familial amyloidotic polyneuropathy (FAP) and familial amyloidotic cardiomyopathy (FAC). Aside from symptomatic treatments, therapeutic options are limited. “Orthotopic liver transplantation, which suppresses mutated TTR, has increased life expectancy in some FAC patients, but it does not prevent cardiac and ocular amyloid depositions. Another option is tafamidis, a small-molecule TTR stabilizer approved in the European Union, which seems to improve quality of life in early-stage FAP patients, but the long-term effects remain to be seen,” notes Gerard Said, Centre Hospitalier, Universitaire Pitié-Salpêtrière, Paris, France. There are no approved therapies for patients with FAC. “New treatments should block synthesis of amyloid precursors to prevent new amyloid deposits in peripheral nerves and in the T R I A L WAT C H

Metabolic disease: Exercise hormone fights metabolic disease.

of antidiabetic agents, achieving effective long-term blood sugar control is often challenging. Now, two papers have identified a novel mechanism that is involved in the regulation of glucose production by the liver, implicating class IIa histone deacetylases (HDACs) as promising therapeutic targets for the treatment of type 2 diabetes. The liver is responsible for the production of glucose (gluconeogenesis), a process that is tightly regulated by the hormones insulin and glucagon, in response to nutrient availability. These hormones differentially regulate a complex signalling cascade, which ultimately leads to the regulation of the forkhead box protein O (FOXO) transcription factors that control gluconeogenic gene transcription. Part of this signalling cascade involves the LKB1 (also known as STK11)–AMPactivated protein kinase (AMPK) pathway that acts to suppress gluconeogenesis. Although numerous AMPK substrates have now been identified, it is likely that others exist. With this in mind, Shaw and colleagues set out to identify novel substrates of AMPK and its related family members that are involved in glucose homeostasis. First, using a bioinformatics and proteomics screen for substrates of AMPK family kinases, they identified class IIa HDACs as direct targets of the AMPK pathway. Confirming this finding, deletion of LKB1 (an upstream AMPK activator) in mouse liver blocked basal phosphorylation of class IIa HDACs, whereas small molecule AMPK activation increased their phosphorylation. Next, they investigated whether class IIa HDACs might be involved in the regulation of glucose production. Fasting and refeeding of mice respectively reduced and increased class IIa HDAC phosphorylation in the liver. These effects were shown to be hormonally regulated — injection of the fasting hormone glucagon reduced class IIa HDAC phosphorylation, inducing their nuclear translocation. Once in the nucleus, these HDACs associated with gluconeogenic gene promotors and recruited HDAC3 (a class I HDAC), causing the deacetylation and activation of FOXO transcription factors and induction of gluconeogenic gene transcription. Intriguingly, further studies showed that class IIa HDACs contribute to hyperglycaemia in several mouse models of type 2 diabetes, with their suppression effectively restoring glucose homeostasis. Meanwhile, Montminy and colleagues revealed that the same regulatory mechanism is present in Drosophila melanogaster. Their initial studies were focused on identifying the precise role of an AMPK-related kinase family member — salt inducible kinase 3 (SIK3) — in energy balance. They found SIK3 to be both nutritionally and hormonally activated. During feeding, SIK3 was activated by insulin, and this resulted in the phosphorylation and cytoplasmic sequestration of HDAC4, a class IIa HDAC. In response to starvation, however, SIK3 was inactivated, leading to dephosphorylation of HDAC4 and its translocation to the nucleus, causing deacetylation of FOXOs and activation of catabolic enzymes. Importantly, they tested whether this pathway was conserved in mammals. Indeed, in mouse hepatocytes, SIK2 (the mouse SIK3 homologue) mediated HDAC4 phosphorylation in response to insulin, whereas glucagon induced HDAC4 dephosphorylation through SIK2 inhibition, allowing gluconeogenic gene transcription. Together, these studies have identified a novel role for class IIa HDACs in regulating liver glucose production. As many HDAC inhibitors are currently being developed as anticancer agents, it may be feasible to target this class of HDACs in the treatment of type 2 diabetes. Sarah Crunkhorn

Alzheimer disease: BACE1 inhibitor reduces [beta]-amyloid production in humans

The biological effects of urolithins (metabolites of ellagitannins, which are found in pomegranates, nuts and berries) are poorly characterized. Here, Ryu and colleagues report that urolithin A (UA) extends lifespan and improves fitness of Caenorhabditis elegans. These effects were mediated by the induction of mitophagy, which prevented the accumulation of dysfunctional mitochondria, thereby improving respiratory capacity, mobility and pharyngeal pumping. Dietary supplementation of UA similarly stimulated mitophagy in mice, resulting in improved muscle function and increased spontaneous exercise in aged mice and enhanced running endurance in young mice.

Metabolic disease: potassium channel blocker prevents obesity.

PPARγ co-activator 1α (PGC1α) has a central role in the regulation of cellular energy metabolism. Its expression is induced by exercise in muscle, where it mediates various beneficial effects. Now, writing in Nature, Boström and colleagues demonstrate that increased muscle PGC1α expression also positively affects adipose tissue — it stimulates the production and secretion of the novel hormone irisin from the muscle, which activates thermogenesis in fat, resulting in weight loss and improved glucose homeostasis in obese mice. Exercise improves metabolic status in obesity and type 2 diabetes, but the underlying molecular mechanisms are poorly understood. Given that muscle PGC1α expression is elevated upon exercise, and transgenic mice with mildly elevated muscle PGC1α (MCK-PGC1α mice) are resistant to age-related obesity and insulin resistance, Boström and colleagues set out to investigate a possible role of muscle PGC1α in the beneficial metabolic effects of exercise. First, they analysed the adipose tissue of MCK-PGC1α mice and discovered that mRNA levels of thermogenic genes characteristic of brown fat, including the brown adipocyte marker uncoupling protein 1 (UCP1), were significantly increased in subcutaneous white adipose tissue (WAT) — an effect termed ‘browning’. This thermogenic gene programme was similarly induced when control mice were exposed to wheel running or swimming in warm water. Next, they treated cultured primary subcutaneous adipocytes with media conditioned by myocytes expressing PGC1α, and found mRNA levels of several brown-fat-specific genes to be increased in the adipocytes. This suggested that the browning they observed in mice may be mediated by a molecule secreted from muscle under the regulation of PGC1α. To search for such a molecule, they analysed muscle from MCK-PGC1α mice using gene expression arrays and an algorithm that predicts protein secretion, and identified five candidate proteins. Applying these proteins directly to primary white adipocytes during differentiation revealed that one of them — fibronectin type III domain-containing protein 5 (FNDC5) — potently upregulated the expression of UCP1 and other brown fat genes, whereas it downregulated the expression of genes characteristic of WAT development. Importantly, FNDC5 mRNA expression was increased in muscle from mice and humans, following exercise. Further in vitro studies revealed that FNDC5 undergoes proteolytic cleavage and glycosylation to produce a highly conserved 112-amino-acid secreted polypeptide, which they named irisin. Irisin was detected in mouse and human plasma, and its levels were increased upon exercise. Finally, they assessed the biological and therapeutic effects of irisin. Mildly increasing irisin levels in mice by injecting adenoviral vectors expressing FNDC5 induced WAT browning; this resulted in increased energy expenditure, reduced body weight and improved glucose tolerance in obese, insulin-resistant mice. When anti-FNDC5 antibodies were injected into mice prior to swimming, this prevented browning, indicating a requirement for irisin in this exercise-associated effect. Together, these findings may have implications for the future treatment of metabolic disease. Indeed, Ember Therapeutics, co-founded by the lead author of this study, is currently generating variants of irisin in preparation for clinical trials. Sarah Crunkhorn