Ehrlich II –2nd World Conference on Magic Bullets

Authors’ disclosure statement (not counting towards the character count):

Gary R Smith is a founding director of Perses Biosystems Ltd. The goals of the company are to drive laboratory and clinical research into the role of angiotensin receptors in disease management. Although we envisage these activities to be humanitarian (non-profit making) in nature, our long-term ambition is to identify additional drug targets and agents that could work in combination with ARBs to treat most diseases.

Finding a New Vaccine in a Ricin Protein Fold SMITH LA Senior Research Scientist, Medical Countermeasures Technology, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702-5011, USA, Telephone: [phone] FAX: [phone] E-Mail: [e-mail] Background: Early attempts to develop a subunit vaccine against ricin were impeded by safety concerns arising from residual toxicity and the unwanted aggregation or precipitation caused by exposure of hydrophobic surfaces on the ricin A-chain (RTA) in the absence of its natural B-chain component. A structure-based solution to the problem was undertaken to arrive at an optimized protein formulation that shows greater resistance to thermal denaturation, increased solubility and stability, and elimination of catalytic (N-glycosidase) activity. Methods: A structure-structure alignment between ricin A-chain and pokeweed antiviral protein (PAP) was performed to determine the relative hydrophobicity of the carboxyl terminal regions. Computational analysis of the structures provided insight into the hydrophobic effect causing the ricin A-chain subunit to aggregate and precipitate. C-terminal ricin A-chain truncation constructs were expressed in E. coli and purified by using conventional chromatography. Mice and non-human primates (NHP) were used to evaluate the efficacy of this vaccine. Results: This comparative analysis between RTA and PAP pointed to a partitioning of function between the two domains of RTA, with the N-terminal sequence of RTA optimized to serve as an “anchoring” fold. In this model, the C-terminal RTA domain is a later functional modification that contributes to ribosome-inactivating protein (RIP) activity, and in two-chain RIP also provides the hydrophobic interfacial region with the lectin subunit. We exploited this structural hierarchy of ricin by splitting the functions to eliminate the undesirable hydrophobic surface of the C-terminus, while preserving the integrity of the B-cell and T-cell epitopes. Computer models of the RTA-RNA assembly led to removing residues 199-267 to obtain truncated RTA 1-198. Further modeling of solvent effects for RTA 1-198 led to the removal of a loop region (RTA 34-43) that unfavorably increased overall solvent accessibility of the protein. The creation of the polypeptide, rRTA 1-33/44-198 (RVEc), further optimized the compactness of the structure, thereby disfavoring protein unfolding and aggregation. The RVEc vaccine showed no detectable RIP activity, thermal stability greater than its parent RTA molecule, no detectable precipitation unlike previous candidates, no detectable vascular leak activity in an in vitro cell assay, and protected vaccinated mice and NHP against lethal challenge with aerosolized ricin Conclusion(s): Protein engineering can be used to increase stability and solubility of recombinant subunit proteins enabling increase soluble expression from heterologous host systems and higher production yields during manufacturing while maintaining antigenic properties and eliminating undesirable features.
Rapid intervention: the role of antivirals in containing or controlling pandemic influenza SMITH JR International Medical Leader: Tamiflu, F. Hoffmann-La Roche Ltd, Basel, Switzerland [Roche: EHRLICH II character count (not including spaces): 2,200; current character count: 2,198] The continuing spread of the influenza A(H5N1) virus among poultry in Southeast Asia, Europe, the Middle East and Africa represents the most serious risk of a human influenza pandemic in decades. As no matched vaccines, and only limited amounts of pre-pandemic vaccines, will be available at the start of a pandemic, the principal interventions are likely to be antivirals and social distancing. Modelling data suggest that antiviral treatment of infected individuals and post-exposure prophylaxis (PEP) of household contacts, combined with containment, could halve infection rates during a pandemic. The oral antiviral agent oseltamivir (Tamiflu^®) is active against multiple influenza strains and is the only neuraminidase inhibitor to have shown activity in the clinical management of patients infected with H5N1. As such, oseltamivir is the only antiviral that is strongly recommended by WHO for the treatment of individuals with confirmed or strongly suspected H5N1 infections and PEP of high-risk exposure groups. WHO has recently highlighted that ‘early treatment with oseltamivir is recommended, and data from uncontrolled clinical trials suggest that it improves survival’, underscoring the need for rapid medical intervention. WHO advises stockpiling of oseltamivir to ensure that sufficient supplies are available for pandemic use, as timelines for ‘surge production’ will not meet the requirements for a rapid response. Roche, the manufacturer of oseltamivir, has scaled up production capacity and can now supply up to 400 million treatment courses annually. At present, 85 governments worldwide have oseltamivir stockpiles. Cumulatively, this is sufficient to treat <5% of the world’s population. To maximise efficacy, individuals infected with the pandemic virus should receive treatment as soon as possible (ideally within 2 days) after symptom onset. If this cannot be achieved, stockpiles may not be used to maximum benefit. Roche is holding a global stockpile of 3 million treatment courses as a ‘rapid response stockpile’ to be donated for use by WHO exclusively at the site of outbreak of a pandemic in an attempt to contain or slow its spread. Roche has also donated a regional stockpile consisting of a further 2 million treatment courses to WHO to serve the needs of developing countries. Once a pandemic is declared, Roche will fill oseltamivir orders as follows: 1) delivery of rapid response stockpile to WHO; 2) fulfilment of existing pandemic orders from governments and other groups; 3) increase in containment effort with WHO and other international agencies.	Activation of Ancient Stress-resistance Pathways by Molecular Triggers, Age and Disease Intervention SMITH SONNEBORN J University of Wyoming, Laramie, WY USA Background: Not only the genetic blueprint, but also the ability to access survival pathways by epigenetic signaling determine species survival. The pathways, though present and thought lost to man, only require an activation trigger of latent protective mechanisms. Molecular mimics of the environmental cues have emerged as triggers of the protective and rejuvenating molecular pathways from the studies from this laboratory and from numerous other investigators. Hibernation is the environmental response to stress. Delta opioids are used to tolerate hemorragic stress in our pubished studies. Objectives:To assess the effect of one such agent to tolerate ischemic stress of hemorrhage, deltophin D, (DeltDvar) a mimic of hibernation factor, our following experiments were preformed. Methods: Rats were fitted with femoral arterial and venous catheters for measurements of mean arterial pressure (MAP), heart rate (HR), and intravenous (i.v.) injections of isotonic saline, 1 mg/kg Delt-Dvar, or 2 mg/kg Delt-Dvar. During hemorrhaging, 30% (5 mL) of total blood volume was collected from the arterial catheter. MAP-HR was fitted to a logistic equation to determine baroreceptor reflex properties. Results: Saline and 1 mg/kg Delt-Dvar rats treated posthemorrhage had similar MAP and HR after hemorrhage. In contrast, 2 mg/kg Delt-Dvar administered after hemorrhaging led to a faster and more complete recovery of MAP than compared with the other groups. In hemorrhaged rats, the average HR gain (bpm/mmHg) after 2 mg/kg Delt-Dvar treatment was greater and the BP50 (BP at one-half the HR range) was significantly lower than after saline treatment. Conclusion: After hemorrhage, 1) stimulation of Delt Dvar opioid receptors leads to improved MAP, and this recovery may involve a change in baroreflex sensitivity. 2) In our other studies, using, DeltE, another peptide, significant extension of survival without fluid resuscitation was found. Since deltorphins preserve function during stress, and strength muscles during hibernation, agonists of the delta opioid receptor may stimulate type 2 muscles in the absence of exercise. The longevity pathways to be activated by pharmaceutical triggers are identified by their conservation through evolution to withstand stress. The magic bullet(s) would include all of the mimics of environmental cues or selected mimics when the activated pathway would intervene in specific diseases.
Novel Modular Nanotransporters which Significantly Enhance Efficacy of Transported Drugs and Impart Cell Specificity to Them SOBOLEV AS^1,2 ^1Institute of Gene Biology, Moscow Russia; ^2Moscow State University, Moscow, Russia Background: A major challenge in the development of specific and effective cancer treatments is the fact that exploiting a molecular target that is accessible (e.g. cell membrane or extracellular matrix) is critical for achieving tumor selectivity while delivery of the therapeutic to the cell nucleus is generally required for maximizing the therapeutic effect. An intriguing approach to this conundrum is to utilize a hybrid molecule to achieve both goals by linking together modules with different functionalities. Methods: We have employed recombinant technology to develop targeted therapeutics that include modules for addressed delivery both to tumor cells and into compartments within these cells that are the most sensitive to the drug. Our modular nanotransporters (NT) are polypeptides possessing a) an internalizable ligand module providing for target cell recognition and subsequent receptor-mediated endocytocis of the NT by the cell; b) an endosomolytic module ensuring escape of the NT from the endosomes; c) a module containing a nuclear localization sequence, thereby enabling translocation of the NT into the cell nucleus; and d) a carrier molecule for attachment of a drug. Results: We produced different NTs containing different ligand modules enabling recognition and internalization of the NTs by the following target cancer cells: melanoma, glioma, epidermoid carcinoma, acute myeloid leukemia and neuroblastoma. Cytotoxic efficacy of either -emitting radionuclides or photosensitizers transported by the NTs turned out to be 10-3000 times higher than that of free drugs. Moreover, the NTs impart cell specificity to the drugs: e.g., free photosensitizers are equally photocytotoxic for target and non-target cells, whereas if they are attached to the NT they become not photocytotoxic for non-target cells at the concentrations that were photocytotoxic for target cancer cells. Conclusions: Cell specificity and high efficacy of many therapeutics can be achieved with the use of modular NTs with preset properties, which would ensure recognition of the desired target cell and subsequent directed transport to the subcellular compartment of choice.	Predatory Bdellovibrio Bacteria as Living Antibiotics-Nature’s Magic Bullets to Treat Antibiotic-Resistant Gram-Negative Infections SOCKETT RE, LAMBERT C, WOODS R, CAPENESS M, HOBLEY L, CHANG C Institute of Genetics, School of Biology, University of Nottingham Medical School, QMC Nottingham NG7 2UH UK Background: Bdellovibrio bacteriovorus is a naturally predatory bacterium that attaches to other Gram-negative bacteria invading the periplasm. Bdellovibrio cells are small and highly motile due to a single sheathed flagellum and efficiently collide with their prey in liquid media and in biofilms. Once they have entered the prey bacterial periplasm they become sessile and attach to the inner membrane of the prey, killing it and then they release organized waves of enzymes which hydrolyse the prey cytoplasm, allowing uptake of prey nutrients into the Bdellovibrio which is living in the periplasm. The Bdellovibrio grow as a long sausage-shaped cell until the prey nutrients are exhausted, then they septate into small cells, regain flagellar motility and burst from the husk of the exhausted prey to invade and kill more bacteria. This is a fascinating life-cycle but what is more important is that prey bacteria for Bdellovibrio include Proteus, Escherichia, Salmonella, Serratia, Acinetobacter, Pseudomonas, Burkholderia and many other important Gram-negative pathogens of man and animals. We are working to investigate the natural predatory properties of Bdellovibrio and to apply them to treat infections where antibiotic resistance is an emerging problem. Methods and Results:. We collaborated in the sequencing of the first, and to date, only published Bdellovibrio genome in 2004 and since then have been examining, using chip arrays the transcription from that genome when the Bdellovibrio attack prey bacteria. We have verified our results using QPCR and have then inactivated key genes that we found to be upregulated to see the effect upon predation. We see the genes and their products that we have studied so far as the “first bite” of the Bdellovibrio into prey and are beginning to understand the order in which prey are degraded. We have also established that Bdellovibrio preys efficiently upon bacteria in serum and that typeIV pili are important for its entry into prey Conclusions: Bdellovibrio is now being converted from a charming microbiological curiosity, to a potent curative for the next wave of human and animal infections, using our combination of genetic and microscopic approaches.
LC-MS/MS Shotgun Proteomics of Lung Cancer Pleural Effusions Identifies the Prognostically Relevant Epithelial-to-Mesenchymal Transition Protein Periostin SOLTERMANN A^1, OSSOLA R^2, TISCHLER V^1, AEBERSOLD R^2, MOCH H^1 ^1University Hospital Zürich, Zürich, Switzerland; ^2Swiss Federal Institute of Technology, Zürich, Switzerland Background: Malignant pleural effusion of advanced lung cancer is a valid source for detection of secreted N-glycosylated biomarker proteins (N-GP), because tumor cells grow during weeks in this liquid. Therefore, such N-GP effusion profiles are likely to contain tumor progression factors. Among them, epithelial-to-mesenchymal transition (EMT) proteins are of particular interest, since EMT is a key program facilitating invasive tumor growth into its surrounding desmoplastic stroma. Methods: Malignant pleural effusions of 5 patients with lung adenocarcinoma and 5 non-malignant controls were used for triplicate N-GP capturing by solid-phase extraction. After trypsin digest and PNGase F release, liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) was performed. For biomarker protein validation, a tumor tissue microarray of 533 patients with surgically resected non-small cell lung cancer (NSCLC) was analysed by immunohistochemistry. Results: In the total of 10 effusion samples, 170 non-redundant proteins were detected with probability 0.9 to 1. The specificity for the N-glycomotif was 88%. Mass spectrometric penetration into the moderate to low protein concentration range (mikro-nanogram/mL) occurred. The EMT protein periostin was confidently identified in several malignant effusions. Of the 533 NSCLC patients, 48% had squamous cell carcinoma, 47% adenocarcinoma and 5% adeno-squamous carcinoma. High protein expression of periostin in either peritumoral desmoplastic stroma or tumor epithelia, independently scored by two pathologists, correlated with male gender, higher stage, higher pT category and larger tumor size; and in only stroma with tumor relapse (all p-values <0.05). Further, high stromal expression was found to be a prognostic factor for decreased progression-free survival on univariate analysis (p-value 0.007). Conclusions: Pleural effusion is a useful biomarker source for lung cancer. Reduction of sample complexity by N-GP capturing allows detection of low abundance biomarker proteins. The EMT protein periostin is closely associated with advanced disease and may thus be integrated in progression models of NSCLC.	Treatment of Agitation in Dementia: Polypharmacy for symptom relief as the Magic Bullet SOMMER, BR^1, FENN^2, HH, KETTER TA^1 ^1Stanford University School of Medicine, Stanford, CA, USA ^2Acute Geropsychiatry Unit, Menlo Park Division, Palo Alto VA Health Care System, Palo Alto CA, USA Background: Few controlled studies are available to guide the clinician in treating potentially assaultive elderly individuals with psychiatric disorders. This is an important concern, with up to 90% of dementia patients suffering from agitation during its course. Safety concerns limit the use of benzodiazepines and antipsychotic medications, making anticonvulsants an attractive alternative. With no one medication as a magic bullet, the side effects profile of a given medication may be used to its advantage in treating a patient’s unique target symptoms. In other cases, targeted dosing of several medications concomitantly for specific symptom relief may yield the most effective results. Each medication has side effects that may be contraindicated in this population. Methods: We reviewed the current research on the efficacy, safety and tolerability of anticonvulsant medications used for individuals over 60 with agitation due to dementia. Recommendations for use under specific circumstances are made, depending on the specific symptoms of the patient. Results: Gabapentin, useful in patients with co-existing pain, may cause ataxia. Oxcarbazepine, with few efficacy studies, is associated with severe hyponatremia. Carbamazepine, important in the treatment of bipolar disorder and pain disorders, may cause hyponatremia and pancytopenia. Topiramate may be helpful in the weight gain commonly induced by other medications such as the commonly prescribed antipsychotic drugs, but is associated with significant cognitive impairment. Valproate, also used for pain syndromes may cause transaminase elevation, pancreatitis, and hyponatremia. Conclusions: With no medication approved for the treatment of agitation associated with dementia, the clinician has a dilemma in nonetheless needing to treat the out of control yet frail patient. There is no one class of medication that is most effective or best tolerated for this vulnerable population. Better outcomes often are achieved by combining different medications to optimize efficacy while limiting adverse effects. Anticonvulsants are an example of a medication class that may be used to advantage in this way; either to augment the effect of other drugs for therapeutic effects, or to diminish side effects.
Alpha 1 Antitrypsin (AAT) for the Treatment of Autoimmune Diseases SONG S Department of Pharmaceutics, University of Florida Background: Autoimmune diseases including type 1 diabetes (T1D) and rheumatoid arthritis (AR) result in self destructions and tissue injuries. Although multiple factors contribute to these diseases, an imbalance of the immune regulatory pathways plays an important role in the development. Therefore, immunoregulatory and anti-inflammatory strategies hold great potential for the prevention of these autoimmune diseases. Alpha 1 antitrypsin (AAT) is a serine proteinase inhibitor and exhibits various anti-inflammatory effects and protects against tissue damage or injury. The goal of our studies is to develop AAT thereapies for the treatment of autoimmune diseases. Methods: We have tested the feasibility of AAT protein and gene therapy for the prevention of type 1 diabetes in none-obese diabetic (NOD) mouse model and the prevention of RA in collagen induced arthritis (CIA) mouse model. For AAT gene delivery, we used recombinant adeno-associated virus vectors (rAAV). Results: AAT gene therapy, contrary to control vector (expressing elafin) and saline, decreased insulitis, reduced the levels of autoantibody and prevented type 1 diabetes efficiently and in dose-dependent manner. T cell receptor spectratyping indicated that AAT gene therapy altered T cell repertoire diversity in splenocytes from NOD mice. Adoptive transfer experiments demonstrated that AAT gene therapy attenuated cellular immunity associated with beta cell destruction. Our results showed that AAT protected islet cells against apoptosis and block cytotoxicity of NK cells. Our recent studies also showed that AAT protein and gene therapies significantly delayed arthritis development in CIA mouse model. Conclusion: Alpha 1 antitrypsin (AAT) has great potential for the treatment of type 1 diabetes and rheumatoid arthritis. This study was supported by grants from NIDDK and Juvenile Diabetes research Foundation	Drug Resistance in Surgical Infection SONNENFELD G^1, AVILES H^1, FUJII H^2, SUN BX^2, CHEADLE WG^3 ^1Department of Biological Sciences, Binghamton University, State University of New York, Binghamton, NY USA; ^2Amino Up Chemical Company, Sapporo, Japan; ^3Department of Surgery, University of Louisville, Louisville, KY, USA Despite development of new antibiotics and protective surgical techniques, infection remains a serious problem in severely injured surgical trauma patients. Infection is the leading cause of late death in severely injured patients. Although the bacteria responsible for the infections, usually gram negative enterobacteriacae, may be sensitive to antibiotics in vitro, the severely injured patients often do not respond to antibiotic therapy. Evidence suggests that trauma-induced deficiencies in the immune response, particularly involving antigen presentation and cytokine production, interfere with the ability of the patient to clear the infection even after antibiotic treatment. Treatments to enhance the immune response, such as interleukin-2 and interferon-gamma therapy, have been used to enhance the immune response of severely injured trauma patients. Although these treatments have been very effective in animal models, the fact that they are regulatory cytokines and have other effects beyond restoring immune response has made their use in human systems problematic. Active Hexose Correlated Compound (AHCC) is an extract from the mycelia of the Basidiomycete mushroom that is non-toxic and has been shown to enhance immune responses and resistance to gram-negative enterobacteriacae. The use of AHCC in the intramuscular thigh infection of acutely food –deprived mice with Klebsiella pneumoniae, a model of trauma and infection, resulted in reduced bacterial load and total clearance of bacteria over time, which did not occur in controls. Cytokine production was also enhanced in these mice. These results suggest that AHCC treatment may be a novel approach for overcoming “drug resistance” related to an impaired immune response in severe trauma victims. Funded in part by a grant from the Amino Up Chemical Company, Sapporo, Japan.