Neuroprotection in an Experimental Model of Multiple Sclerosis via Opening of Big Conductance, Calcium-Activated Potassium Channels.

Big conductance calcium-activated (BK) channel openers can inhibit pathologically driven neural hyperactivity to control symptoms via hyperpolarizing signals to limit neural excitability. We hypothesized that BK channel openers would be neuroprotective during neuroinflammatory, autoimmune disease. The neurodegenerative disease was induced in a mouse experimental autoimmune encephalomyelitis model with translational value to detect neuroprotection in multiple sclerosis. Following the treatment with the BK channel openers, BMS-204253 and VSN16R, neuroprotection was assessed using subjective and objective clinical outcomes and by quantitating spinal nerve content. Treatment with BMS-204253 and VSN16R did not inhibit the development of relapsing autoimmunity, consistent with minimal channel expression via immune cells, nor did it change leukocyte levels in rodents or humans. However, it inhibited the accumulation of nerve loss and disability as a consequence of autoimmunity. Therefore, in addition to symptom control, BK channel openers have the potential to save nerves from excitotoxic damage and could be useful as either stand-alone neuroprotective agents or as add-ons to current disease-modifying treatments that block relapsing MS but do not have any direct neuroprotective activity.

Therapeutic Validation of GEF-H1 Using a De Novo Designed Inhibitor in Models of Retinal Disease.

Inflammation and fibrosis are important components of diseases that contribute to the malfunction of epithelia and endothelia. The Rho guanine nucleotide exchange factor (GEF) GEF-H1/ARHGEF-2 is induced in disease and stimulates inflammatory and fibrotic processes, cell migration, and metastasis. Here, we have generated peptide inhibitors to block the function of GEF-H1. Inhibitors were designed using a structural in silico approach or by isolating an inhibitory sequence from the autoregulatory C-terminal domain. Candidate inhibitors were tested for their ability to block RhoA/GEF-H1 binding in vitro, and their potency and specificity in cell-based assays. Successful inhibitors were then evaluated in models of TGFß-induced fibrosis, LPS-stimulated endothelial cell-cell junction disruption, and cell migration. Finally, the most potent inhibitor was successfully tested in an experimental retinal disease mouse model, in which it inhibited blood vessel leakage and ameliorated retinal inflammation when treatment was initiated after disease diagnosis. Thus, an antagonist that blocks GEF-H1 signaling effectively inhibits disease features in in vitro and in vivo disease models, demonstrating that GEF-H1 is an effective therapeutic target and establishing a new therapeutic approach.

VEGFA, B, C: Implications of the C-Terminal Sequence Variations for the Interaction with Neuropilins.

Vascular endothelial growth factors (VEGFs) are the key regulators of blood and lymphatic vessels' formation and function. Each of the proteins from the homologous family VEGFA, VEGFB, VEGFC and VEGFD employs a core cysteine-knot structural domain for the specific interaction with one or more of the cognate tyrosine kinase receptors. Additional diversity is exhibited by the involvement of neuropilins-transmembrane co-receptors, whose b1 domain contains the binding site for the C-terminal sequence of VEGFs. Although all relevant isoforms of VEGFs that interact with neuropilins contain the required C-terminal Arg residue, there is selectivity of neuropilins and VEGF receptors for the VEGF proteins, which is reflected in the physiological roles that they mediate. To decipher the contribution made by the C-terminal sequences of the individual VEGF proteins to that functional differentiation, we determined structures of molecular complexes of neuropilins and VEGF-derived peptides and examined binding interactions for all neuropilin-VEGF pairs experimentally and computationally. While X-ray crystal structures and ligand-binding experiments highlighted similarities between the ligands, the molecular dynamics simulations uncovered conformational preferences of VEGF-derived peptides beyond the C-terminal arginine that contribute to the ligand selectivity of neuropilins. The implications for the design of the selective antagonists of neuropilins' functions are discussed.

A Series of Substituted Bis-Aminotriazines Are Activators of the Natriuretic Peptide Receptor C.

C-type natriuretic peptide (CNP) is involved in the regulation of vascular homeostasis, which is at least partly mediated through agonism of natriuretic peptide receptor C (NPR-C), and loss of this signaling has been associated with vascular dysfunction. As such, NPR-C is a novel therapeutic target to treat cardiovascular diseases. A series of novel small molecules have been designed and synthesized, and their structure-activity relationships were evaluated by a surface plasmon resonance binding assay. The biological activity of hit compounds was confirmed through organ bath assays measuring vascular relaxation and inhibition of cAMP production, which was shown to be linked to its NPR-C activity. Lead compound 1 was identified as a potent agonist (EC50 ∼ 1 µM) with promising in vivo pharmacokinetic properties.

Peptides Derived from Vascular Endothelial Growth Factor B Show Potent Binding to Neuropilin-1.

Vascular endothelial growth factors (VEGFs) regulate significant pathways in angiogenesis, myocardial and neuronal protection, metabolism, and cancer progression. The VEGF-B growth factor is involved in cell survival, anti-apoptotic and antioxidant mechanisms, through binding to VEGF receptor 1 and neuropilin-1 (NRP1). We employed surface plasmon resonance technology and X-ray crystallography to analyse the molecular basis of the interaction between VEGF-B and the b1 domain of NRP1, and developed VEGF-B C-terminus derived peptides to be used as chemical tools for studying VEGF-B - NRP1 related pathways. Peptide lipidation was used as a means to stabilise the peptides. VEGF-B-derived peptides containing a C-terminal arginine show potent binding to NRP1-b1. Peptide lipidation increased binding residence time and improved plasma stability. A crystal structure of a peptide with NRP1 demonstrated that VEGF-B peptides bind at the canonical C-terminal arginine binding site. VEGF-B C-terminus imparts higher affinity for NRP1 than the corresponding VEGF-A165 region. This tight binding may impact on the activity and selectivity of the full-length protein. The VEGF-B167 derived peptides were more effective than VEGF-A165 peptides in blocking functional phosphorylation events. Blockers of VEGF-B function have potential applications in diabetes and non-alcoholic fatty liver disease.

Reversal of behavioural phenotype by the cannabinoid-like compound VSN16R in fragile X syndrome mice.

Fragile X syndrome is the most common inherited intellectual disability and mono-genetic cause of autism spectrum disorder. It is a neurodevelopmental condition occurring due to a CGG trinucleotide expansion in the FMR1 gene. Polymorphisms and variants in large-conductance calcium-activated potassium channels are increasingly linked to intellectual disability and loss of FMR protein causes reduced large-conductance calcium-activated potassium channel activity leading to abnormalities in synapse function. Using the cannabinoid-like large-conductance calcium-activated potassium channel activator VSN16R we rescued behavioural deficits such as repetitive behaviour, hippocampal dependent tests of daily living, hyperactivity and memory in a mouse model of fragile X syndrome. VSN16R has been shown to be safe in a phase 1 study in healthy volunteers and in a phase 2 study in patients with multiple sclerosis with high oral bioavailability and no serious adverse effects reported. VSN16R could therefore be directly utilized in a fragile X syndrome clinical study. Moreover, VSN16R showed no evidence of tolerance, which strongly suggests that chronic VSN16R may have great therapeutic value for fragile X syndrome and autism spectrum disorder. This study provides new insight into the pathophysiology of fragile X syndrome and identifies a new pathway for drug intervention for this debilitating disorder.

Cannabidiol activates PINK1-Parkin-dependent mitophagy and mitochondrial-derived vesicles.

The PINK1/Parkin pathway plays an important role in maintaining a healthy pool of mitochondria. Activation of this pathway can lead to apoptosis, mitophagy, or mitochondrial-derived vesicle formation, depending on the nature of mitochondrial damage. The signaling by which PINK/Parkin activation leads to these different mitochondrial outcomes remains understudied. Here we present evidence that cannabidiol (CBD) activates the PINK1-Parkin pathway in a unique manner. CBD stimulates PINK1-dependent Parkin mitochondrial recruitment similarly to other well-studied Parkin activators but with a distinctive shift in the temporal dynamics and mitochondrial fates. The mitochondrial permeability transition pore inhibitor cyclosporine A exclusively diminished the CBD-induced PINK1/Parkin activation and its associated mitochondrial effects. Unexpectedly, CBD treatment also induced elevated production of mitochondrial-derived vesicles (MDV), a potential quality control mechanism that may help repair partial damaged mitochondria. Our results suggest that CBD may engage the PINK1-Parkin pathway to produce MDV and repair mitochondrial lesions via mitochondrial permeability transition pore opening. This work uncovered a novel link between CBD and PINK1/Parkin-dependent MDV production in mitochondrial health regulation.

Oligodendrocytes, BK channels and the preservation of myelin.

Oligodendrocytes wrap multiple lamellae of their membrane, myelin, around axons of the central nervous system (CNS), to improve impulse conduction. Myelin synthesis is specialised and dynamic, responsive to local neuronal excitation. Subtle pathological insults are sufficient to cause significant neuronal metabolic impairment, so myelin preservation is necessary to safeguard neural networks. Multiple sclerosis (MS) is the most prevalent demyelinating disease of the CNS. In MS, inflammatory attacks against myelin, proposed to be autoimmune, cause myelin decay and oligodendrocyte loss, leaving neurons vulnerable. Current therapies target the prominent neuroinflammation but are mostly ineffective in protecting from neurodegeneration and the progressive neurological disability. People with MS have substantially higher levels of extracellular glutamate, the main excitatory neurotransmitter. This impairs cellular homeostasis to cause excitotoxic stress. Large conductance Ca2 +-activated K + channels (BK channels) could preserve myelin or allow its recovery by protecting cells from the resulting excessive excitability. This review evaluates the role of excitotoxic stress, myelination and BK channels in MS pathology, and explores the hypothesis that BK channel activation could be a therapeutic strategy to protect oligodendrocytes from excitotoxic stress in MS. This could reduce progression of neurological disability if used in parallel to immunomodulatory therapies.

Hepatitis C virus exploits cyclophilin A to evade PKR.

Counteracting innate immunity is essential for successful viral replication. Host cyclophilins (Cyps) have been implicated in viral evasion of host antiviral responses, although the mechanisms are still unclear. Here, we show that hepatitis C virus (HCV) co-opts the host protein CypA to aid evasion of antiviral responses dependent on the effector protein kinase R (PKR). Pharmacological inhibition of CypA rescues PKR from antagonism by HCV NS5A, leading to activation of an interferon regulatory factor-1 (IRF1)-driven cell intrinsic antiviral program that inhibits viral replication. These findings further the understanding of the complexity of Cyp-virus interactions, provide mechanistic insight into the remarkably broad antiviral spectrum of Cyp inhibitors, and uncover novel aspects of PKR activity and regulation. Collectively, our study identifies a novel antiviral mechanism that harnesses cellular antiviral immunity to suppress viral replication.

The ß4-Subunit of the Large-Conductance Potassium Ion Channel KCa1.1 Regulates Outflow Facility in Mice.

Purpose: The large-conductance calcium-activated potassium channel KCa1.1 (BKCa, maxi-K) influences aqueous humor outflow facility, but the contribution of auxiliary ß-subunits to KCa1.1 activity in the outflow pathway is unknown. Methods: Using quantitative polymerase chain reaction, we measured expression of ß-subunit genes in anterior segments of C57BL/6J mice (Kcnmb1-4) and in cultured human trabecular meshwork (TM) and Schlemm's canal (SC) cells (KCNMB1-4). We also measured expression of Kcnma1/KCNMA1 that encodes the pore-forming α-subunit. Using confocal immunofluorescence, we visualized the distribution of ß4 in the conventional outflow pathway of mice. Using iPerfusion, we measured outflow facility in enucleated mouse eyes in response to 100 or 500 nM iberiotoxin (IbTX; N = 9) or 100 nM martentoxin (MarTX; N = 12). MarTX selectively blocks ß4-containing KCa1.1 channels, whereas IbTX blocks KCa1.1 channels that lack ß4. Results: Kcnmb4 was the most highly expressed ß-subunit in mouse conventional outflow tissues, expressed at a level comparable to Kcnma1. ß4 was present within the juxtacanalicular TM, appearing to label cellular processes connecting to SC cells. Accordingly, KCNMB4 was the most highly expressed ß-subunit in human TM cells, and the sole ß-subunit in human SC cells. To dissect functional contribution, MarTX decreased outflow facility by 35% (27%, 42%; mean, 95% confidence interval) relative to vehicle-treated contralateral eyes, whereas IbTX reduced outflow facility by 16% (6%, 25%). Conclusions: The ß4-subunit regulates KCa1.1 activity in the conventional outflow pathway, significantly influencing outflow function. Targeting ß4-containing KCa1.1 channels may be a promising approach to lower intraocular pressure to treat glaucoma.

Discovery of a novel fluorescent chemical probe suitable for evaluation of neuropilin-1 binding of small molecules.

Neuropilin-1 (NRP1) is emerging as an important molecule in immune signaling where it has been shown to modulate the actions of TGF-ß1 in macrophages and regulatory T cells. The development of cost-effective and reliable assays for NRP1 binding is therefore important. We synthesized three new NRP1 small molecule fluorophores and examined their performance as fluorescent polarization probes. One molecule DS108 exhibited favorable binding and fluorescent characteristics and allowed us to establish a simple assay suitable for medium to high throughput screening of small molecules.

Critical Care Network in the State of Qatar.

Critical care is a multidisciplinary and interprofessional specialty providing comprehensive care to patients in an acute life-threatening, but treatable condition.1 The aim is to prevent further physiological deterioration while the failing organ is treated. Patients admitted to a critical care unit normally need constant attention from specialist nursing and therapy staff at an appropriate ratio, continuous, uninterrupted physiological monitoring supervised by staff that are able to interpret and immediately act on the information, continuous clinical direction and care from a specialist consultant-led medical team trained and able to provide appropriate cover for each critical care unit, and artificial organ support and advanced therapies which are only safe to administer in the above environment. It is an important aspect of medical care within a hospital as it is an underpinning service without which a hospital would not be able to conduct most or all of its planned and unplanned activities. As such, critical care requires a very intensive input of human, physical, and financial resources.2 It occupies a proportionately large fraction of a hospital's estate and infrastructure for a small number of patients. The resources that are invested into a critical care bed should therefore be valued against the activities and care throughout the hospital that the availability of that bed allows to happen. Given that demand for critical care beds will continue to grow, providing more critical care beds is unlikely to work on its own since experience has shown that additional capacity is soon absorbed within routine provision.3 Attention must therefore be given to maximising the efficient and effective use of existing critical care beds, necessitating an ability to cope with peaks in demand. Historically the world over, the development of critical care units has been unplanned and haphazard and largely relied on the interest of local clinicians to drive development. However, there is now an eminent body of opinion that supports an alternative approach to critical care provision - namely through a managed Critical Care Network with an agenda of integrated working and the focus on facilitating safe quality care that is cost-effective and patient-focused for acutely and critically ill patients across the various constituent organisations of a healthcare system. The Critical Care Service in Hamad Medical Corporation (HMC) has developed rapidly to address the increasing demand linked to the population growth in the State of Qatar with the aim of meeting the vision of the National Health Strategy (NHS). It is paralleled with HMC's vision to improve the delivery of critical care to patients and their families in a way that meets the highest international standards such as those set by the Joint Commission International by whom the Corporation has been accredited since 2007.4 For this reason, the organisation took the lead to perform a gap analysis with expert auditors from the United States of America and the United Kingdom who have experience in critical care service provision. The aim was to assess the Critical Care Service within HMC and identify potential short-term, medium-term, and long-term opportunities for improvement. This assessment focused on a very broad range of aspects such as: bed capacity, facilities and equipment, medical, nursing and allied healthcare staffing levels and their education, career development pathways, patient safety, quality metrics, clinical governance structure, clinical protocols and pathways, critical care outreach, and future planning for critical care at HMC. As a result of extensive review for the Critical Care Services at HMC, the Critical Care Network (CCNW) in the State of Qatar was established in 2014. It is a strategic and operational delivery network, which includes 12 hospitals across the country. The network functions through a combination of strategic programmes, working groups, and large multidisciplinary governance and professional development events. Through collaborative working with the leadership of the various facilities and critical care clinicians, the network reviews services and makes improvements where they are required, ensuring delivery of patient-focused care by appropriately educated and trained healthcare professionals as well as the appropriate utilisation of critical care beds for those patients who require such care. Detailed involvement and engagement from the clinical membership at every event and in the various working groups ensures that all decisions, reports, and improvement programmes are clinically-focused and benefit from a diversity of opinions that can be considered for implementation. All of this is carefully aligned to the requirements of the latest Qatar National Health Strategy.5 It aims to adopt evidence-based best practices to deliver the safest, most effective and most compassionate care to our critical care patients by setting the most appropriate care pathway to transform Critical Care Services across HMC hospitals. The key aims of the CCNW as stated in its Terms of Reference document are listed in Table 1.6 This enhances the quality and safety of patient care across HMC, promotes staff satisfaction, and improves customer service and patient outcome. The CCNW is structured in a way that involves all Critical Care Service stakeholders to maintain the stability and sustainability of delivering the best care to critically ill patients. The CCNW is steered by a multidisciplinary committee (Figure 1) that is empowered with the generative, managerial, and fiscal responsibilities to enable the required changes to take place. The committee oversees the HMC Critical Care Services through coordinating and standardising their activities and governance arrangements across the complete HMC healthcare system. It provides HMC clinical and managerial leadership at a corporate and local level, the opportunity to jointly develop critical care standards, policies, and operating procedures. In doing so, the CCNW decides on and implements recommendations on how to best plan and deliver critical care services using evidence-based practice set against the context of national and international practices. The HMC CCNW gives recommendations to various committees to improve the services in the following areas: 1. Defining the level of care and critical care core standards for HMC: The CCNW standardises critical care across the Corporation regardless of where it is being delivered. As such it develops the critical care core standards for the critical care units and gives recommendations regarding future critical care core facility planning within HMC. The CCNW helps the Ministry of Public Health (MoPH) develop the National Critical Care Core Standards. 2. Quality and safety: The CCNW works collaboratively with HMC leaders to ensure a culture of quality is embedded within all critical care services delivered within HMC. There is a continuous evaluation process in place to measure the quality of care for high performance critical care which is the goal. This is based upon ongoing observations, robust data collection and analysis, and a change management strategy implemented as required. 3. Clinical pathways, guidelines, and protocols: The CCNW develops, according to international best practice, clinical care pathways, guidelines, and protocols that govern critical care units throughout HMC. Critical care clinical practice is audited against these standards, compared with the international benchmark, and updated as required to ensure currency of all patient care aspects. 4. Transfer and transportation of critically ill patients: The CCNW develops HMC-wide criteria for patient intramural, extramural, and international transfers, and sets standards of care during transportation in collaboration with the HMC Ambulance Service Transfer and Retrieval team. This includes HMC-wide bed management consideration with the senior consultants on call, review of the patient's condition and medical needs, and assessment of the mission associated risks and mitigating strategies. This involves significant planning on the part of the team, clear communication and handovers, and the use of checklists at several stages to ensure the provision of safe and efficient patient transfers. 5. Education: The CCNW develops educational plans and ensures corresponding courses accredited by the Qatar Council for Healthcare Practitioners (QCHP) are designed and delivered to address the training needs of clinicians. The portfolio of courses is regularly reviewed to meet identified needs so clinicians always possess the appropriate knowledge and skills to manage critically ill patients. 6. Research and Critical Care Data Registry development: Being a key player in an Academic Health System, HMC fosters a relatively young but growing research environment4 of which the CCNW forms an integral part. Creating opportunities for epidemiological research and also fulfilling the needs for quality monitoring and benchmarking, the CCNW has enabled the creation of critical care data registries. Such registries provide a valuable source of information and have already been exploited at HMC to better understand the type of patients a service cares for and patient outcomes with respects various factors.7 The establishment of a CCNW at a corporate level (with membership from local leaders across HMC) has provided a level of oversight and leadership which has significantly contributed to optimizing and reshaping the way acutely ill patients are cared for. It has enabled the adoption of evidence-based best practices across the various critical care services of HMC as well as created a multidisciplinary forum for dialogue and collaboration. Innovative work focusing on providing effective, up-to-date, and patient-focused care are ongoing as well as HMC's pursuit of various internationalaccreditation awards by prestigious organisations and professional bodies.

A new patient-derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan.

Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of α-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of α-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.

The Cannabinoid-Like Compound, VSN16R, Acts on Large Conductance, Ca2+-Activated K+ Channels to Modulate Hippocampal CA1 Pyramidal Neuron Firing.

Large conductance, Ca2+-activated K+ (BKCa) channels are widely expressed in the central nervous system, where they regulate action potential duration, firing frequency and consequential neurotransmitter release. Moreover, drug action on, mutations to, or changes in expression levels of BKCa can modulate neuronal hyperexcitability. Amongst other potential mechanisms of action, cannabinoid compounds have recently been reported to activate BKCa channels. Here, we examined the effects of the cannabinoid-like compound (R,Z)-3-(6-(dimethylamino)-6-oxohex-1-en-1-yl)-N-(1-hydroxypropan-2-yl) benzamide (VSN16R) at CA1 pyramidal neurons in hippocampal ex vivo brain slices using current clamp electrophysiology. We also investigated effects of the BKCa channel blockers iberiotoxin (IBTX) and the novel 7-pra-martentoxin (7-Pra-MarTx) on VSN16R action. VSN16R (100 µM) increased first and second fast after-hyperpolarization (fAHP) amplitude, decreased first and second inter spike interval (ISI) and shortened first action potential (AP) width under high frequency stimulation protocols in mouse hippocampal pyramidal neurons. IBTX (100 nM) decreased first fAHP amplitude, increased second ISI and broadened first and second AP width under high frequency stimulation protocols; IBTX also broadened first and second AP width under low frequency stimulation protocols. IBTX blocked effects of VSN16R on fAHP amplitude and ISI. 7-Pra-MarTx (100 nM) had no significant effects on fAHP amplitude and ISI but, unlike IBTX, shortened first and second AP width under high frequency stimulation protocols; 7-Pra-MarTx also shortened second AP width under low frequency stimulation protocols. However, in the presence of 7-Pra-MarTx, VSN16R retained some effects on AP waveform under high frequency stimulation protocols; moreover, VSN16R effects were revealed under low frequency stimulation protocols. These findings demonstrate that VSN16R has effects in native hippocampal neurons consistent with its causing an increase in initial firing frequency via activation of IBTX-sensitive BKCa channels. The differential pharmacological effects described suggest that VSN16R may differentially target BKCa channel subtypes.

N10 -carbonyl-substituted phenothiazines inhibiting lipid peroxidation and associated nitric oxide consumption powerfully protect brain tissue against oxidative stress.

During some investigations into the mechanism of nitric oxide consumption by brain preparations, several potent inhibitors of this process were identified. Subsequent tests revealed the compounds act by inhibiting lipid peroxidation, a trigger for a form of regulated cell death known as ferroptosis. A quantitative structure-activity study together with XED (eXtended Electron Distributions) field analysis allowed a qualitative understanding of the structure-activity relationships. A representative compound N-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)-10H-phenothiazine-10-carboxamide (DT-PTZ-C) was able to inhibit completely oxidative damage brought about by two different procedures in organotypic hippocampal slice cultures, displaying a 30- to 100-fold higher potency than the standard vitamin E analogue, Trolox or edaravone. The compounds are novel, small, drug-like molecules of potential therapeutic use in neurodegenerative disorders and other conditions associated with oxidative stress.

Emerging small-molecule treatments for multiple sclerosis: focus on B cells.

Multiple sclerosis (MS) is a major cause of disability in young adults. Following an unknown trigger (or triggers), the immune system attacks the myelin sheath surrounding axons, leading to progressive nerve cell death. Antibodies and small-molecule drugs directed against B cells have demonstrated good efficacy in slowing progression of the disease. This review focusses on small-molecule drugs that can affect B-cell biology and may have utility in disease management. The risk genes for MS are examined from the drug target perspective. Existing small-molecule therapies for MS with B-cell actions together with new drugs in development are described. The potential for experimental molecules with B-cell effects is also considered. Small molecules can have diverse actions on B cells and be cytotoxic, anti-inflammatory and anti-viral. The current B cell-directed therapies often kill B-cell subsets, which can be effective but lead to side effects and toxicity. A deeper understanding of B-cell biology and the effect on MS disease should lead to new drugs with better selectivity, efficacy, and an improved safety profile. Small-molecule drugs, once the patent term has expired, provide a uniquely sustainable form of healthcare.

Biophysical screening methods for extracellular domain peptide receptors, application to natriuretic peptide receptor C ligands.

Endothelium-derived C-type natriuretic peptide possesses cytoprotective and anti-atherogenic functions that regulate vascular homeostasis. The vasoprotective effects of C-type natriuretic peptide are somewhat mediated by the natriuretic peptide receptor C, suggesting that this receptor represents a novel therapeutic target for the treatment of cardiovascular diseases. In order to facilitate our drug discovery efforts, we have optimized an array of biophysical methods including surface plasmon resonance, fluorescence polarization and thermal shift assays to aid in the design, assessment and characterization of small molecule agonist interactions with natriuretic peptide receptors. Assay conditions are investigated to explore the feasibility and dynamic range of each method, and peptide-based agonists and antagonists are used as controls to validate these conditions. Once established, each technique was compared and contrasted with respect to their drug discovery utility. We foresee that such techniques will facilitate the discovery and development of potential therapeutic agents for NPR-C and other large extracellular domain membrane receptors.

Mitochondrial Permeability Transition: A Molecular Lesion with Multiple Drug Targets.

Mitochondrial permeability transition, as the consequence of opening of a mitochondrial permeability transition pore (mPTP), is a cellular catastrophe. Initiating bioenergetic collapse and cell death, it has been implicated in the pathophysiology of major human diseases, including neuromuscular diseases of childhood, ischaemia-reperfusion injury, and age-related neurodegenerative disease. Opening of the mPTP represents a major therapeutic target, as it can be mitigated by a number of compounds. However, clinical studies have so far been disappointing. We therefore address the prospects and challenges faced in translating in vitro findings to clinical benefit. We review the role of mPTP opening in disease, discuss recent findings defining the putative structure of the mPTP, and explore strategies to identify novel, clinically useful mPTP inhibitors, highlighting key considerations in the drug discovery process.

Identification of Kinases and Phosphatases That Regulate ATG4B Activity by siRNA and Small Molecule Screening in Cells.

Autophagy protease ATG4B is a key regulator of the LC3/GABARAP conjugation system required for autophagosome formation, maturation and closure. Members of the ATG4 and the LC3/GABARAP family have been implicated in various diseases including cancer, and targeting the ATG4B protease has been suggested as a potential therapeutic anti-cancer strategy. Recently, it has been demonstrated that ATG4B is regulated by multiple post-translational modifications, including phosphorylation and de-phosphorylation. In order to identify regulators of ATG4B activity, we optimized a cell-based luciferase assay based on ATG4B-dependent release of Gaussia luciferase. We applied this assay in a proof-of-concept small molecule compound screen and identified activating compounds that increase cellular ATG4B activity. Next, we performed a high-throughput screen to identify kinases and phosphatases that regulate cellular ATG4B activity using siRNA mediated knockdown and cDNA overexpression. Of these, we provide preliminary evidence that the kinase AKT2 enhances ATG4B activity in cells. We provide all raw and processed data from the screens as a resource for further analysis. Overall, our findings provide novel insights into the regulation of ATG4B and highlight the importance of post-translational modifications of ATG4B.