Jeffrey M. Axten

Oral Treatment Targeting the Unfolded Protein Response Prevents Neurodegeneration and Clinical Disease in Prion-Infected Mice

Pharmacological inhibition of PERK, the key kinase of the unfolded protein response that mediates translational shutdown, restores protein synthesis in prion-infected mice, thus preventing neurodegeneration and clinical disease. Perking Up Prion Disease Therapy There are no effective treatments for neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and prion disease. These diseases share common features, including the accumulation of misfolded disease-specific proteins in the brain, leading to neuronal loss, which is ultimately fatal. In addition, the brains of patients with these neurodegenerative diseases show overactivation of a cellular defense pathway for dealing with misfolded proteins called the unfolded protein response (UPR). The UPR deals with the misfolded protein load in a number of ways including transiently switching off translation. Moreno et al. now report that the buildup of misfolded prion protein in mice with prion disease causes sustained overactivation of this pathway. This results in long-term translational inhibition, causing a critical decline in key proteins needed for neuronal survival. The authors used a newly described specific inhibitor of a key UPR kinase mediating translational shutdown to test if pharmacological inhibition would be neuroprotective. The compound prevented neurodegeneration and the emergence of clinical disease in prion-infected mice, whereas untreated animals all succumbed to disease. These data suggest that the UPR may represent a new therapeutic target for drug development to treat prion disease and possibly other neurodegenerative diseases as well. During prion disease, an increase in misfolded prion protein (PrP) generated by prion replication leads to sustained overactivation of the branch of the unfolded protein response (UPR) that controls the initiation of protein synthesis. This results in persistent repression of translation, resulting in the loss of critical proteins that leads to synaptic failure and neuronal death. We have previously reported that localized genetic manipulation of this pathway rescues shutdown of translation and prevents neurodegeneration in a mouse model of prion disease, suggesting that pharmacological inhibition of this pathway might be of therapeutic benefit. We show that oral treatment with a specific inhibitor of the kinase PERK (protein kinase RNA–like endoplasmic reticulum kinase), a key mediator of this UPR pathway, prevented UPR-mediated translational repression and abrogated development of clinical prion disease in mice, with neuroprotection observed throughout the mouse brain. This was the case for animals treated both at the preclinical stage and also later in disease when behavioral signs had emerged. Critically, the compound acts downstream and independently of the primary pathogenic process of prion replication and is effective despite continuing accumulation of misfolded PrP. These data suggest that PERK, and other members of this pathway, may be new therapeutic targets for developing drugs against prion disease or other neurodegenerative diseases where the UPR has been implicated.

Characterization of a Novel PERK Kinase Inhibitor with Antitumor and Antiangiogenic Activity

The unfolded protein response (UPR) is a signal transduction pathway that coordinates cellular adaptation to microenvironmental stresses that include hypoxia, nutrient deprivation, and change in redox status. These stress stimuli are common in many tumors and thus targeting components of the UPR signaling is an attractive therapeutic approach. We have identified a first-in-class, small molecule inhibitor of the eukaryotic initiation factor 2-alpha kinase 3 (EIF2AK3) or PERK, one of the three mediators of UPR signaling. GSK2656157 is an ATP-competitive inhibitor of PERK enzyme activity with an IC(50) of 0.9 nmol/L. It is highly selective for PERK with IC(50) values >100 nmol/L against a panel of 300 kinases. GSK2656157 inhibits PERK activity in cells with an IC(50) in the range of 10-30 nmol/L as shown by inhibition of stress-induced PERK autophosphorylation, eIF2α substrate phosphorylation, together with corresponding decreases in ATF4 and CAAT/enhancer binding protein homologous protein (CHOP) in multiple cell lines. Oral administration of GSK2656157 to mice shows a dose- and time-dependent pharmacodynamic response in pancreas as measured by PERK autophosphorylation. Twice daily dosing of GSK2656157 results in dose-dependent inhibition of multiple human tumor xenografts growth in mice. Altered amino acid metabolism, decreased blood vessel density, and vascular perfusion are potential mechanisms for the observed antitumor effect. However, despite its antitumor activity, given the on-target pharmacologic effects of PERK inhibition on pancreatic function, development of any PERK inhibitor in human subjects would need to be cautiously pursued in cancer patients.

Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1

PDK1 (3-phosphoinositide-dependent protein kinase 1) activates a group of protein kinases belonging to the AGC [PKA (protein kinase A)/PKG (protein kinase G)/PKC (protein kinase C)]-kinase family that play important roles in mediating diverse biological processes. Many cancer-driving mutations induce activation of PDK1 targets including Akt, S6K (p70 ribosomal S6 kinase) and SGK (serum- and glucocorticoid-induced protein kinase). In the present paper, we describe the small molecule GSK2334470, which inhibits PDK1 with an IC₅₀ of ~10 nM, but does not suppress the activity of 93 other protein kinases including 13 AGC-kinases most related to PDK1 at 500-fold higher concentrations. Addition of GSK2334470 to HEK (human embryonic kidney)-293, U87 or MEF (mouse embryonic fibroblast) cells ablated T-loop residue phosphorylation and activation of SGK isoforms and S6K1 induced by serum or IGF1 (insulin-like growth factor 1). GSK2334470 also inhibited T-loop phosphorylation and activation of Akt, but was more efficient at inhibiting Akt in response to stimuli such as serum that activated the PI3K (phosphoinositide 3-kinase) pathway weakly. GSK2334470 inhibited activation of an Akt1 mutant lacking the PH domain (pleckstrin homology domain) more potently than full-length Akt1, suggesting that GSK2334470 is more effective at inhibiting PDK1 substrates that are activated in the cytosol rather than at the plasma membrane. Consistent with this, GSK2334470 inhibited Akt activation in knock-in embryonic stem cells expressing a mutant of PDK1 that is unable to interact with phosphoinositides more potently than in wild-type cells. GSK2334470 also suppressed T-loop phosphorylation and activation of RSK2 (p90 ribosomal S6 kinase 2), another PDK1 target activated by the ERK (extracellular-signal-regulated kinase) pathway. However, prolonged treatment of cells with inhibitor was required to observe inhibition of RSK2, indicating that PDK1 substrates possess distinct T-loop dephosphorylation kinetics. Our data define how PDK1 inhibitors affect AGC signalling pathways and suggest that GSK2334470 will be a useful tool for delineating the roles of PDK1 in biological processes.

Novel amino-piperidines as potent antibacterials targeting bacterial type IIA topoisomerases.

We have identified a series of amino-piperidine antibacterials with a good broad spectrum potency. We report the investigation of various subunits in this series and advanced studies on compound 8. Compound 8 possesses good pharmacokinetics, broad spectrum antibacterial activity and demonstrates oral efficacy in a rat lung infection model.

Discovery of a new series of Aurora inhibitors through truncation of GSK1070916.

Novel Aurora inhibitors were identified truncating clinical candidate GSK1070916. Many of these truncated compounds retained potent activity against Aurora B with good antiproliferative activity. Mechanistic studies suggested that these compounds, depending on the substitution pattern, may or may not exert their antiproliferative effects via inhibition of Aurora B. The SAR results from this investigation will be presented with an emphasis on the impact structural changes have on the cellular phenotype.

Conversion of 2-deoxyglucose-induced growth inhibition to cell death in normoxic tumor cells

BackgroundInhibition of glucose metabolism has recently become an attractive target for cancer treatment. Accordingly, since 2-deoxyglucose (2-DG) competes effectively with glucose, it has come under increasing scrutiny as a therapeutic agent. The initial response of tumor cells to 2-DG is growth inhibition, which is thought to conserve energy and consequently protect cells from its ATP-lowering effects as a glycolytic inhibitor. However, since 2-DG also mimics mannose and thereby interferes with N-linked glycosylation, the question is raised of how this sugar analog inhibits tumor cell growth and whether the mechanism by which it protects cells can be manipulated to convert 2-DG-induced growth inhibition to cell death.MethodsCell growth and death were measured via counting viable and dead cells based on trypan blue exclusion. Markers of ATP reduction and the unfolded protein response (UPR) were detected by Western blot. Protein functions were manipulated through chemical compounds, siRNA and the use of gene-specific wild-type and knock-out mouse embryonic fibroblasts (MEFs).ResultsAt 2-DG concentrations that can be achieved in human plasma without causing significant side effects, we find (a) It induces growth inhibition predominantly by interference with glycosylation, which leads to accumulation of unfolded proteins in the endoplasmic reticulum activating the UPR; (b) Inhibition of PERK (but not ATF6 or IRE1), a major component of the UPR, leads to conversion of 2-DG-induced growth inhibition to cell death and (c) secondarily to PERK, inhibition of GCN2, a kinase that is activated in response to low intracellular glutamine, increases 2-DG’s cytotoxic effects in PERK −/− MEFs.ConclusionsOverall, these findings present a novel anticancer strategy that can be translated into therapeutic gain as they uncover the metabolic target PERK, and to a lesser degree GCN2, that when inhibited convert 2-DG’s static effect to a toxic one in tumor cells growing under normoxia.

Targeting PERK signaling with the small molecule GSK2606414 prevents neurodegeneration in a model of Parkinson's disease

Parkinsons disease (PD) is the second most common neurodegenerative disorder, leading to the progressive decline of motor control due to the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Accumulating evidence suggest that altered proteostasis is a salient feature of PD, highlighting perturbations to the endoplasmic reticulum (ER), the main compartment involved in protein folding and secretion. PERK is a central ER stress sensor that enforces adaptive programs to recover homeostasis through a block of protein translation and the induction of the transcription factor ATF4. In addition, chronic PERK signaling results in apoptosis induction and neuronal dysfunction due to the repression in the translation of synaptic proteins. Here we confirmed the activation of PERK signaling in postmortem brain tissue derived from PD patients and three different rodent models of the disease. Pharmacological targeting of PERK by the oral administration of GSK2606414 demonstrated efficient inhibition of the pathway in the SNpc after experimental ER stress stimulation. GSK2606414 protected nigral-dopaminergic neurons against a PD-inducing neurotoxin, improving motor performance. The neuroprotective effects of PERK inhibition were accompanied by an increase in dopamine levels and the expression of synaptic proteins. However, GSK2606414 treated animals developed secondary effects possibly related to pancreatic toxicity. This study suggests that strategies to attenuate ER stress levels may be effective to reduce neurodegeneration in PD.

Protein kinase R(PKR)–like endoplasmic reticulum kinase (PERK) inhibitors: a patent review (2010-2015)

ABSTRACT Introduction: PKR-like endoplasmic reticulum kinase (PERK) is an essential component of the unfolded protein response (UPR) and a critical regulator of protein synthesis during endoplasmic reticulum (ER) stress. Transient PERK activation is protective; however, chronic ER stress and sustained PERK activation can be detrimental to cell health. Many diseases are associated with PERK over-activation, suggestive that small molecule PERK inhibitors may provide new opportunities for treating cancer and neurodegenerative diseases, among others. Areas covered: This review covers the therapeutic potential of PERK modulation and will focus more specifically on small molecule inhibitors of PERK disclosed in the patent literature from 2010–2015. During this time period the first PERK inhibitor patents appeared disclosing novel, potent, and selective inhibitors of PERK. Expert opinion: Compelling preclinical and clinical evidence supports the potential use of PERK modulators for a variety of diseases, particularly cancer and neurodegenerative disease. Potent and selective PERK inhibitors have been characterized pharmacologically and are available for further study. Despite high therapeutic potential, the future clinical use of PERK inhibitors will require thorough safety and toxicity evaluation to gauge therapeutic index and develop a framework for risk-benefit assessment.

Acylprolinamides: a new class of peptide deformylase inhibitors with in vivo antibacterial activity.

A new class of PDF inhibitor with potent, broad spectrum antibacterial activity is described. Optimization of blood stability and potency provided compounds with improved pharmacokinetics that were suitable for in vivo experiments. Compound 5c, which has robust antibacterial activity, demonstrated efficacy in two respiratory tract infection models.

PERK inhibition delays neurodegeneration and improves motor function in a mouse model of Marinesco-Sjögren syndrome

Marinesco-Sjögren syndrome (MSS) is a rare, early onset, autosomal recessive multisystem disorder characterized by cerebellar ataxia, cataracts and myopathy. Most MSS cases are caused by loss-of-function mutations in the gene encoding SIL1, a nucleotide exchange factor for the molecular chaperone BiP which is essential for correct protein folding in the endoplasmic reticulum. Woozy mice carrying a spontaneous Sil1 mutation recapitulate key pathological features of MSS, including cerebellar atrophy with degeneration of Purkinje cells and progressive myopathy. Because the PERK branch of the unfolded protein response is activated in degenerating neurons of woozy mice, and inhibiting PERK-mediated translational attenuation has shown protective effects in protein-misfolding neurodegenerative disease models, we tested the therapeutic efficacy of GSK2606414, a potent inhibitor of PERK. Mice were chronically treated with GSK2606414 starting from a presymptomatic stage, and the effects were evaluated on biochemical, histopathological and clinical readouts. GSK2606414 delayed Purkinje cell degeneration and the onset of motor deficits, prolonging the asymptomatic phase of the disease; it also reduced the skeletal muscle abnormalities and improved motor performance during the symptomatic phase. The protein but not the mRNA level of ORP150, a nucleotide exchange factor which can substitute for SIL1, was increased in the cerebellum of GSK2606414-treated woozy mice, suggesting that translational recovery promoted the synthesis of this alternative BiP co-factor. Targeting PERK signaling may have beneficial disease-modifying effects in carriers of SIL1 mutations.