Identified genetic interaction offers possible new target for glaucoma therapy
Scientists at the University of California, San Diego School of Medicine have elucidated a genetic interaction that may prove key to the development and progression of glaucoma, a blinding neurodegenerative disease that affects tens of millions of people worldwide and is a leading cause of irreversible blindness.
The findings, published in the September 10 online issue of Molecular Cell, suggest a new therapeutic target for treating the eye disease.
Primary open-angle glaucoma (POAG) is the most common form of glaucoma, affecting more than 3 million Americans, primarily after the age of 50. Pressure inside the eye (known as intraocular pressure) and age are the leading risk factors for POAG, resulting in progressive degeneration of retinal ganglion cells, optic nerve damage and eventual vision loss.
Genetics also plays a role. Recent genome-wide association studies have identified two genes – SIX1-SIX6 and p16INK4a – as strongly associated with POAG. SIX6 is required for proper eye development. P16INK4a irreversibly arrests cell growth, a phenomenon called senescence.
In their new paper, principal investigator Kang Zhang, MD, PhD, professor of ophthalmology and chief of Ophthalmic Genetics at Shiley Eye Institute at UC San Diego Health, and colleagues report that some variants of SIX6 boost expression of p16INK4a, which in turn accelerates senescence and death of retinal ganglion cells.
“We also show that high intraocular pressure in glaucoma increases expression of p16INK4a, making it a key integrator of inherent genetic and environmental risk factors that can result in glaucoma,” said Zhang.
The findings suggest that inhibiting p16INK4a could offer a new therapeutic approach for glaucoma, which is currently treated by drugs that lower intraocular pressure. “Although lowering intraocular pressure can slow worsening of the disease, it does not stop it and prevent further cell death or possible blindness,” said co-author Robert N. Weinreb, MD, Distinguished Professor of Ophthalmology and director of the Shiley Eye Institute.
The authors note that earlier studies in mouse models have shown that selective elimination of p16INK4a-positive senescent cells can prevent or delay age-related tissue deterioration.
According to the UC San Diego team, the next step is to conduct preclinical studies to assess the efficacy and safety of antisense oligonucleotides – strands of synthesized DNA or RNA that can prevent transfer of genetic information – which might inhibit p16INK4a expression and prevent worsening of glaucoma. “If they are effective, we may contemplate a human clinical trial in the future,” Zhang said.
Funding for this research came, in part, from the 973 Program, the State Key Laboratory of Ophthalmology, the National Institutes of Health and Research to Prevent Blindness.
University of California, San Diego School of Medicine
Boulder Peptide Society announced the 2015 Roche Meienhofer award recipient: Richard DiMarchi
Richard DiMarchi, Ph.D. the Standiford H. Cox Distinguished Professor of Chemistry and the Linda & Jack Gill Chair in Biomolecular Sciences at Indiana University has been named as the 2015 recipient of the Meienhofer Award for Excellence in Peptide Sciences. The award was presented to Dr. DiMarchi on September 30, 2015 at the Boulder Peptide Symposium, which took place from September 28 to October 1, 2015 in Boulder, Colorado, where Dr. DiMarchi gave a presentation of highlights from his prodigious record of research achievements.
Dr. DiMarchi's contributions in peptide & protein sciences consist of three decades of work in academia, the pharmaceutical industry and biotechnology companies. He is a co-founder of Ambrx, Inc., Marcadia Biotech, Assembly, Calibrium and MB2 Biotech. He has served as a scientific advisor to multiple pharmaceutical companies (Kai, Ferring, Lilly, Merck, and Roche), three venture funds; 5AM, TMP, Twilight and a former board member of BIO, Isis and Millenium-Bio. He is currently Chairman of the Peptide Therapeutics Foundation and external board member at Assembly Biosciences and On-Target Therapeutics.
Dr. DiMarchi earned his Ph.D. from Indiana University in 1979. He completed post-doctoral training at Rockefeller University under the mentorship of Noble Laureate Prof. Bruce Merrifield after which he began a twenty-two year career as a scientist and executive at Eli Lilly and Company. For more than two decades at Lilly Research Laboratories he provided leadership in biotechnology, endocrine research and product development, retiring as Group Vice President in 2003. The focus of much of Dr. DiMarchi’s research has been the chemical biology of endocrine hormones, and more specifically those pertaining to diabetes and obesity. He is readily recognized for discovery and development of rDNA derived Humalog® (LisPro-human insulin) and the advancement of mixed incretin agonists for treatment of the metabolic syndrome. As scientist and executive, Dr. DiMarchi also significantly contributed to the commercial development of Humulin®, Humatrope®, rGlucagon®, Evista®, and Forteo®. His current research is focused on developing macromolecules with enhanced therapeutic properties through biochemical and chemical optimization, an approach he has termed chemical-biotechnology. He is the author of nearly two hundred scientific publications and holds more than a hundred patents.
Dr. DiMarchi is the recipient of numerous awards including the 2005 AAPS Career Research Achievement Award in Biotechnology, the 2006 ACS Barnes Award for Leadership in Chemical Research Management, the 2006 ACS Esselen Award for Chemistry in the Service of Public Interest, the 2007 Carothers Award for Excellence in Polymer Sciences, the 2009 Watanabe Award for Life Sciences Research, the 2011 Merrifield Award for Career Contributions in Peptide Sciences, the 2012 Phillip Nelson Innovation Award, the 2014 Erwin Schrödinger-Preis, a 2014 inductee to the National Inventors Hall of Fame, a 2015 winner of the Patient Advocacy award of the philanthropic society Cures within Reach, a 2015 inductee to the National Association of Inventors.
Jammed up cellular highways may initiate Dementia and ALS
Molecular therapy partially relieves havoc wreaked by gene mutation in human and fly cells
Johns Hopkins researchers say they have discovered some of the first steps in how a very common gene mutation causes the brain damage associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
They report that the altered C9orf72 gene, located on human chromosome 9, causes RNA molecules to block critical pathways for protein transport, causing a molecular traffic jam outside brain cell nuclei and affecting their operations and survival. In a proof-of-concept experiment, the researchers also say that a molecular therapy eased the jam and restored molecular flow into the cell’s core.
A report on the work was published online on Aug. 26 in the journal Nature.
“The discovery several years ago of this mutation — the most common one linked to ALS and FTD — was really a game changer for the field because it wasn’t a typical genetic mutation,” says Jeffrey Rothstein, M.D., Ph.D., a professor of neurology and director of the Brain Science Institute and the Robert Packard Center for ALS Research at the Johns Hopkins University School of Medicine. “Now we have some information about what it is doing early on to damage brain and spinal cord cells.”
The mutation, the most common of the known genetic risk factors for the diseases, is associated with 40 percent of inherited ALS cases, 25 percent of inherited FTD and around 10 percent of noninherited cases of each disease. Both diseases are characterized by degeneration of nerve cells over time. In the case of FTD, the damage causes problems with speech, understanding language and processing emotions. In ALS, the degeneration affects cells in the spinal cord as well as the brain, and patients gradually lose the ability to control their muscles.
According to Rothstein, researchers knew that the C9orf72 mutation, rather than changing one building block of DNA to another, caused a stretch of six DNA nucleotides to repeat hundreds of times. Based on the mutated DNA, affected cells create long strands of repetitive RNA, genetic material normally responsible for transferring DNA’s genetic code outside the nuclei, to the machinery that translates it into proteins.
In 2013, Rothstein’s lab identified more than 400 proteins in the cell with which the repetitive RNA strands might directly interact. Now, that research group, along with that of Thomas Lloyd, M.D., Ph.D., an associate professor of neurology at Johns Hopkins, have homed in on one of those proteins, RanGAP, as key to mediating the mutated RNA’s effect on cells.
“The key breakthrough came from using a fruit fly model of human ALS and FTD that allowed us to screen these 400 candidates for ones that block brain cell death in a living organism,” says Lloyd. “This work identified RanGAP as a critical target of the C9orf72 repeats that could prevent brain cell death when its function was restored.”
In healthy cells, RanGAP helps transport molecules through nuclear pores that connect a cell’s cytoplasm — the liquid that fills most of a cell — and the nucleus — the central compartment containing genetic material.
But in their experiments with both fly and human brain cells made from patients with the ALS-associated C9orf72 mutation, Rothstein and Lloyd discovered RanGAP is clumped up outside the nucleus. Moreover, proteins that rely on RanGAP for transportation into the nucleus don’t flow through the nuclear pores.
“The group had the data in human stem cells and a fly model, but we really wanted to know whether we could see this in the brains of patients,” says Rothstein. “So we went to our autopsy bank of human brain tissue and started looking.”
Examinations of slices of brain tissue from patients with ALS and FTD showed similar clumps of RanGAP and other proteins — including some vital to neuron function — stuck outside the nuclei of brain cells. “Now, flies, human stem cells and autopsied brains are all telling us the same story here, that this is a fundamental defect causing disease,” says Rothstein.
In another set of experiments using the fly and human stem cells, the scientists added antisense oligonucleotides, bits of RNA designed to bind to the repetitive RNA strands, blocking them from interacting with the RanGAP protein. The jammed up nuclear pores reopened, they report, and key proteins once again moved into the nucleus.
Rothstein has launched a collaboration with California-based Isis Pharmaceuticals to pursue the development of a drug that could do the same for patients with ALS and FTD. He cautioned, however, that further studies must be done to confirm this potential, and a commercially available drug is many years off.
“We still don’t know every step between the C9orf72 mutation and cellular death in the brain,” says Rothstein. “But our belief is that this is what starts it off, and this is certainly a good therapeutic target.”
Funding for the study was provided by grants from the National Institute of Neurological Disorders and Stroke (R01 NS085207, NS091046, R01 NS082563, R01 NS074324, NS089616, NS091486), the National Cancer Institute (CA009110), the Brain Science Institute, the Robert Packard Center for ALS Research at Johns Hopkins, the Muscular Dystrophy Association, the Alzheimer’s Drug Discovery Foundation, the Judith and Jean Pape Adams Charitable Foundation, the Alzheimer’s Disease Research Center - Johns Hopkins, Maryland TEDCO, the Target ALS Springboard Fellowship, the William and Ella Owens Foundation and the ALS Association.
Johns Hopkins Medicine
An in silico platform for predicting, screening and designing of antihypertensive peptides
High blood pressure or hypertension is an affliction that threatens millions of lives worldwide. Peptides from natural origin have been shown recently to be highly effective in lowering blood pressure. In the present study, we have framed a platform for predicting and designing novel antihypertensive peptides. Due to a large variation found in the length of antihypertensive peptides, we divided these peptides into four categories (i) Tiny peptides, (ii) small peptides, (iii) medium peptides and (iv) large peptides. First, we developed SVM based regression models for tiny peptides using chemical descriptors and achieved maximum correlation of 0.701 and 0.543 for dipeptides and tripeptides, respectively. Second, classification models were developed for small peptides and achieved maximum accuracy of 76.67%, 72.04% and 77.39% for tetrapeptide, pentapeptide and hexapeptides, respectively. Third, we have developed a model for medium peptides using amino acid composition and achieved maximum accuracy of 82.61%. Finally, we have developed a model for large peptides using amino acid composition and achieved maximum accuracy of 84.21%. Based on the above study, a web-based platform has been developed for locating antihypertensive peptides in a protein, screening of peptides and designing of antihypertensive peptides.
Gajendra P.S. Raghava et al., Scientific Reports 5, Article number: 12512 (2015) doi:10.1038/srep12512
A biomimetic approach for enhancing the in vivo half-life of peptides
The tremendous therapeutic potential of peptides has not yet been realized, mainly owing to their short in vivo half-life. Although conjugation to macromolecules has been a mainstay approach for enhancing protein half-life, the steric hindrance of macromolecules often harms the binding of peptides to target receptors, compromising the in vivo efficacy. Here we report a new strategy for enhancing the in vivo half-life of peptides without compromising their potency. Our approach involves endowing peptides with a small molecule that binds reversibly to the serum protein transthyretin. Although there are a few molecules that bind albumin reversibly, we are unaware of designed small molecules that reversibly bind other serum proteins and are used for half-life extension in vivo. We show here that our strategy was effective in enhancing the half-life of an agonist for GnRH receptor while maintaining its binding affinity, which was translated into superior in vivo efficacy.
Mamoun M Alhamadsheh et al. Nature Chemical Biology (2015) doi:10.1038/nchembio.1907
Improving on nature: making a cyclic heptapeptide orally bioavailable
The use of peptides in medicine is limited by low membrane permeability, metabolic instability, high clearance, and negligible oral bioavailability. The prediction of oral bioavailability of drugs relies on physicochemical properties that favor passive permeability and oxidative metabolic stability, but these may not be useful for peptides. Here we investigate effects of heterocyclic constraints, intramolecular hydrogen bonds, and side chains on the oral bioavailability of cyclic heptapeptides. NMR-derived structures, amide H-D exchange rates, and temperature-dependent chemical shifts showed that the combination of rigidification, stronger hydrogen bonds, and solvent shielding by branched side chains enhances the oral bioavailability of cyclic heptapeptides in rats without the need for N-methylation.
Fairlie, D. P. et al. , Angew. Chem. Int. Ed., 53: 12059–12063. doi: 10.1002/anie.201405364
Role of electrostatic interactions for ligand recognition and specificity of peptide transporters
Peptide transporters are membrane proteins that mediate the cellular uptake of di- and tripeptides, and of peptidomimetic drugs such as b-lactam antibiotics, antiviral drugs and antineoplastic agents. In spite of their high physiological and pharmaceutical importance, the molecular recognition by these transporters of the amino acid side chains of short peptides and thus the mechanisms for substrate binding and specificity are far from being understood.
The X-ray crystal structure of the peptide transporter YePEPT from the bacterium Yersinia enterocolitica together with functional studies have unveiled the molecular bases for recognition, binding and specificity of dipeptides with a charged amino acid residue at the N-terminal position. In wild-type YePEPT, the significant specificity for the dipeptides Asp-Ala and Glu-Ala is defined by electrostatic interaction between the in the structure identified positively charged Lys314 and the negatively charged amino acid side chain of these dipeptides. Mutagenesis of Lys314 into the negatively charged residue Glu allowed tuning of the substrate specificity of YePEPT for the positively charged dipeptide Lys-Ala. Importantly, molecular insights acquired from the prokaryotic peptide transporter YePEPT combined with mutagenesis and functional uptake studies with human PEPT1 expressed in Xenopus oocytes also allowed tuning of human PEPT1’s substrate specificity, thus improving our understanding of substrate recognition and specificity of this physiologically and pharmaceutically important peptide transporter.
This study provides the molecular bases for recognition, binding and specificity of peptide transporters for dipeptides with a charged amino acid residue at the N-terminal position.
Dimitrios Fotiadis et al., BMC Biology 2015, 13:58 doi:10.1186/s12915
Self-sorting heterodimeric coiled coil peptides with defined and tuneable self-assembly properties
Coiled coils with defined assembly properties and dissociation constants are highly attractive components in synthetic biology and for fabrication of peptide-based hybrid nanomaterials and nanostructures. Complex assemblies based on multiple different peptides typically require orthogonal peptides obtained by negative design. Negative design does not necessarily exclude formation of undesired species and may eventually compromise the stability of the desired coiled coils. This work describe a set of four promiscuous 28-residue de novo designed peptides that heterodimerize and fold into parallel coiled coils. The peptides are non-orthogonal and can form four different heterodimers albeit with large differences in affinities. The peptides display dissociation constants for dimerization spanning from the micromolar to the picomolar range. The significant differences in affinities for dimerization make the peptides prone to thermodynamic social self-sorting as shown by thermal unfolding and fluorescence experiments, and confirmed by simulations. The peptides self-sort with high fidelity to form the two coiled coils with the highest and lowest affinities for heterodimerization. The possibility to exploit self-sorting of mutually complementary peptides could hence be a viable approach to guide the assembly of higher order architectures and a powerful strategy for fabrication of dynamic and tuneable nanostructured materials.
Daniel Aili et al., Scientific Reports 5, Article number: 14063 (2015) doi:10.1038/srep14063
Pharmaceutical optimization of peptide toxins for Ion channel targets: potent, selective, and long-lived antagonists of Kv1.3
To realize the medicinal potential of peptide toxins-naturally occurring disulfide-rich peptides-as ion channel antagonists, more efficient pharmaceutical optimization technologies must be developed. Here we show that the therapeutic properties of multiple cysteine toxin peptides can be rapidly and substantially improved by combining direct chemical strategies with high-throughput electrophysiology. We applied whole-molecule, brute-force, structure-activity analoging to ShK, a peptide toxin from the sea anemone Stichodactyla helianthus that inhibits the voltage-gated potassium ion channel Kv1.3, to effectively discover critical structural changes for 15x selectivity against the closely related neuronal ion channel Kv1.1. Subsequent site-specific polymer conjugation resulted in an exquisitely selective Kv1.3 antagonist (>1000x over Kv1.1) with picomolar functional activity in whole blood and a pharmacokinetic profile suitable for weekly administration in primates. The pharmacological potential of the optimized toxin peptide was demonstrated by potent and sustained inhibition of cytokine secretion from T cells, a therapeutic target for autoimmune diseases, in cynomolgus monkeys.
Miranda LP et al., J Med Chem, 2015, 58(17):6784-802. doi: 10.1021/acs.jmedchem.5b00495
Blocking of the PD-1/PD-L1 interaction by a D-peptide antagonist for cancer immunotherapy
Blockade of the protein-protein interaction between the transmembrane protein programmed cell death protein 1 (PD-1) and its ligand PD-L1 has emerged as a promising immunotherapy for treating cancers. Using the technology of mirror-image phage display, we developed the first hydrolysis-resistant D-peptide antagonists to target the PD-1/PD-L1 pathway. The optimized compound D PPA-1 could bind PD-L1 at an affinity of 0.51 μM in vitro. A blockade assay at the cellular level and tumor-bearing mice experiments indicated that D PPA-1 could also effectively disrupt the PD-1/PD-L1 interaction in vivo. Thus D-peptide antagonists may provide novel low-molecular-weight drug candidates for cancer immunotherapy.
Gao YF et al., Angew Chem Int Ed Engl, 2015 doi: 10.1002/anie.201506225
Cross-linking strategies to study peptide ligand-receptor interactions
Experiments are described that allowed cross-linking of analogs of a 13-amino acid peptide into the binding site of a model G protein-coupled receptor. Syntheses of peptide analogs that were used for photochemical or chemical cross-linking were carried out using solid-phase peptide synthesis. Chemical cross-linking utilized 3,4-dihydroxy-l-phenylalanine-incorporated peptides and subsequent periodate-mediated activation, whereas photochemical cross-linking was mediated by p-benzoyl-l-phenylalanine (Bpa)-labeled peptides and UV-initiated activation. Mass spectrometry was employed to locate the site(s) in the receptor that formed the cross-links to the ligand. We also describe a method called unnatural amino acid replacement that allowed capture of a peptide ligand into the receptor. In this method, the receptor was genetically modified by replacement of a natural amino acid with Bpa. The modified receptor was UV-irradiated to capture the ligand. The approaches described are applicable to other peptide-binding proteins and can reveal the ligand-binding site in atomic detail.
Becker JM, Naider F, Methods Enzymol. 2015, 556:527-47. doi: 10.1016/bs.mie.2014.12.001
Predicting the sequence specificities of DNA- and RNA-binding proteins by deep learning
Knowing the sequence specificities of DNA- and RNA-binding proteins is essential for developing models of the regulatory processes in biological systems and for identifying causal disease variants. Here we show that sequence specificities can be ascertained from experimental data with 'deep learning' techniques, which offer a scalable, flexible and unified computational approach for pattern discovery. Using a diverse array of experimental data and evaluation metrics, we find that deep learning outperforms other state-of-the-art methods, even when training on in vitro data and testing on in vivo data. We call this approach DeepBind and have built a stand-alone software tool that is fully automatic and handles millions of sequences per experiment. Specificities determined by DeepBind are readily visualized as a weighted ensemble of position weight matrices or as a 'mutation map' that indicates how variations affect binding within a specific sequence.
Brendan J Frey et al., Nature Biotechnology 33, 831–838 (2015) doi:10.1038/nbt.3300