Antimicrobial peptide GL13K
GL13K: An antimicrobial peptide with therapeutic potential
Bacterial biofilms are a key obstacle in any medical device or implant application. Once established, bacterial biofilms are virtually impossible to eradicate under biological conditions. Long term use of antibiotics or biocides are problematic due to bacterial resistance and toxicity.
To address this need, we have used a biologically based approach to discover novel antimicrobial peptides (AMPs). Rather than screening vast peptide libraries, structural analysis was used to identify human proteins that could serve as a template for AMP design. This approach is a more direct and less time-consuming screening, and is predicted to present less toxicity and bacterial resistance due to the human origin of the new AMPs (no bacteria are known to be resistant to human AMPs).
The lead peptide emerging from this approach, GL13K was used to develop a novel antimicrobial coating that prevents biofilm formation, is bactericidal, can kill drug-resistant bacteria and exhibits low mammalian toxicity. The coating is very stable and resists degradation by mechanical, hydrolytic and/or enzymatic challenges.
We invite you to browse the information below to learn more about GL13K and the applied peptide coatings or contact the investigators directly for more information and collaborative/licensing opportunities.
“Drug-resistant pathogens are a growing menace to all people, regardless of age, gender, or socioeconomic background. They endanger people in affluent, industrial societies like the United States, as well as in less-developed nations.” (Interagency-Task-Force-on-Antimicrobial-Resistance, 2011).
Antibiotic resistance is on the rise and mortality from bacterial infection and attendant sepsis is increasing. Many bacteria show a high incidence of resistance to available antibiotics and can develop antibiotic resistance during treatment with broad spectrum antibiotics. Increased resistance to common antibiotics has led to the reintroduction of polymyxin (colistin) as a last resort, although concerns over toxicity remain. To overcome antibiotic resistance, treatment with multiple antibiotics is recommended. Since few anti-bacterial agents are in development, there is a need to identify such agents either for use alone or in combination with existing antibiotics. Cationic antimicrobial peptides (AMP) have shown some promise as a new class of antibiotic, although several obstacles remain to be overcome.
Challenges facing AMPs
The identification of AMPs led to extensive research with the hope of identifying novel antibiotics for the treatment of bacterial infections. However, despite promising in vitro results the use of AMPs in vivo has met with several challenges and no new AMP has reached clinical use to date. Some of the challenges associated with AMPs include host-toxicity; lack of activity under physiological conditions; post-translational modifications; and concerns over resistance to human host-defense proteins. We have introduced a new family of AMPs that includes peptide modifications to target specific antimicrobial activities and bacterial types and address several of the challenges faced by other AMPs.
Introduction of GL13K
In recent years, we have designed new antimicrobial peptides based on the sequence of the human salivary protein Parotid Secretory Protein (PSP; BPIFA2). A recent modification of these peptides produced the peptide GL13K, which has shown promise in preliminary in vitro and in vivo experiments. A further modification by introduction of D-amino acids has improved stability and folding to decrease the effective concentration and enhance activity in PBS without an apparent increase in toxicity. Thus, preliminary data for GL13K suggests that this peptide can overcome several of the challenges encountered with other cationic AMPs. The short (13 amino acids) linear structure and absence of post-translational modifications simplifies synthesis and lowers cost.
In addition to the positive profile described above, GL13K shares several of the potential advantages with other AMPs: The peptide is active against several bacterial species; rapid onset of killing; bactericidal activity; inactivates lipopolysaccharide and exhibits anti-inflammatory activity in vivo. Importantly, GL13K is active against drug-resistant bacteria. In fact, drug-resistant Pseudomonas aeruginosa showed increased susceptibility to GL13K. In general, resistance to AMPs has been slow to develop and the mechanism of action targeting bacterial cell membranes for entry into the cell allows for activity against metabolic inactive or dormant cells, an important advantage for treatment of biofilms. Indeed, GL13K has shown promising activity against both aerobic and anaerobic biofilms. Full eradication of P. aeruginosa biofilms can be achieved by a combination of GL13K and tobramycin.
Titanium implants are used extensively in medicine and dentistry with a high success rate. However, depending on conditions, 20% of dental implants, 10% of orthopedic fixation devices, and up to 4 % of orthopedic joint replacements develop peri-implant infections over their lifetime. Bacteria can invade the inert implant surfaces where they establish biofilms that are largely out of reach for the host natural defenses and can infect the tissues surrounding the implant. Infection can occur at early and late stages after surgery. This increases the challenge for protecting the surface since continuous local and/or systemic prophylaxis is currently not clinically feasible. In fact, these bacterial biofilms are extremely difficult to eradicate with traditional antibiotics due to the low metabolism of cells in the biofilm. Dentistry has developed successful strategies to combat bacterial biofilms on tooth surfaces without the use of antibiotics. Studies in the highly accessible oral sites have shown that strategies that prevent biofilm formation allow host defenses to control the overall microbiome and maintain the health of surrounding tissues.
Using these principles, peri-implantitis can be prevented by inhibiting growth of biofilm and kill bacteria through contact with the implant surface over extended periods after surgery. To that purpose, we have developed a novel antimicrobial peptide coating, which unlike conventional antibiotics, is effective against cells that are not metabolically active.
The coating contains the peptide GL13K, which was developed from the human salivary protein Parotid Secretory Protein, and it is effective against planktonic or biofilm Gram positive and Gram negative bacteria with low host toxicity. The GL13K peptide is covalently coupled to titanium surfaces using silane chemistry.
The GL13K peptide coatings:
- exhibit sustained antimicrobial activity against key pathogens of oral infections such as Porphyromonas gingivalis and Streptococcus mutans.
- retain antimicrobial activity after sterilization by autoclaving and repeated cycles of exposure to biological fluids and bacterial challenge.
- are cytocompatible with fibroblasts and osteoblasts.
- are resistant to mechanical, hydrolytic, and enzymatic/proteolytic degradation
- uniquely disrupt the integrity of the wall of gram positive bacteria such as Streptococcus gordonii.
Research reported on this web site was supported by the National Institute of Dental and Craniofacial Research of the National Institutes of Health under award numbers R01DE017989, R01DE12205, and R90DE023058; the Office of the Vice-president for Research at the University of Minnesota through the Grant-in-Aid of Research, Artistry, and Scholarship Program; and the 3M Foundation through a 3M Non-Tenured Faculty Award. Parts of this work were carried out in the University of Minnesota I.T. Characterization Facility, which receives partial support from NSF through the MRSEC program.
Characterization of the GL13K peptide family
Hirt, H., and S.U. Gorr. 2013. Antimicrobial peptide GL13K is effective in reducing biofilms of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 57:4903-4910.
Balhara, V., R. Schmidt, S.U. Gorr, and C. Dewolf. 2013. Membrane selectivity and biophysical studies of the antimicrobial peptide GL13K. Biochimica et Biophysica Acta. 1828:2193-2203.
Abdolhosseini, M., S.R. Nandula, J. Song, H. Hirt, and S.U. Gorr. 2012. Lysine substitutions convert a bacterial-agglutinating peptide into a bactericidal peptide that retains anti-lipopolysaccharide activity and low hemolytic activity. Peptides. 35:231-238.
Abdolhosseini, M., J.B. Sotsky, A.P. Shelar, P.B. Joyce, and S.U. Gorr. 2012. Human parotid secretory protein is a lipopolysaccharide-binding protein: identification of an anti-inflammatory peptide domain. Molecular and Cellular Biochemistry. 359:1-8.
Gorr, S.U. 2012. Antimicrobial peptides in periodontal innate defense. Frontiers of Oral Biology. 15:84-98.
Gorr, S.U., and M. Abdolhosseini. 2011. Antimicrobial peptides and periodontal disease. Journal of Clinical Periodontology. 38 Suppl 11:126-141.
Gorr, S.U., M. Abdolhosseini, A. Shelar, and J. Sotsky. 2011. Dual host-defence functions of SPLUNC2/PSP and synthetic peptides derived from the protein. Biochemical Society Transactions. 39:1028-1032.
Gorr, S.U. 2009. Antimicrobial peptides of the oral cavity. Periodontology 2000. 51:152-180.
Gorr, S.U., J.B. Sotsky, A.P. Shelar, and D.R. Demuth. 2008. Design of bacteria-agglutinating peptides derived from parotid secretory protein, a member of the bactericidal/permeability increasing-like protein family. Peptides. 29:2118-2127.
Geetha, C., S.G. Venkatesh, L. Bingle, C.D. Bingle, and S.U. Gorr. 2005. Design and validation of anti-inflammatory peptides from human parotid secretory protein. J Dent Res. 84:149-153.
Bingle, C.D., and S.U. Gorr. 2004. Host defense in oral and airway epithelia: chromosome 20 contributes a new protein family. Int. J. Biochem. Cell Biol. 36:2144-2152.
Geetha, C., S.G. Venkatesh, B.H. Fasciotto Dunn, and S.U. Gorr. 2003. Expression and anti-bacterial activity of human parotid secretory protein (PSP). Biochemical Society Transactions. 31:815-818.
Biofunctional peptide coatings
X. Chen, H. Hirt, Y. Li, S.U. Gorr, C. Aparicio. 2014. Antimicrobial GL13K peptide coatings killed and ruptured the wall of Streptococcus gordonii and prevented formation and growth of biofilms. PLOS One. Accepted.
E. Salvagni, G.Y. Berguig, E. Engel, J.C. Rodriguez-Cabello, G. Coullerez, M. Textor, F.J. Gil, J.A. Planell, C. Aparicio. 2014. A bioactive elastin-like recombinamer reduces unspecific protein adsorption and enhances cell response on titanium surfaces. Colloids and Surfaces B: Biointerfaces 114: 225-233.
Y. Li, X. Chen, A. Ribeiro, E. Jensen, K.H. Holmberg. J.C. Rodriguez-Cabello, C. Aparicio. 2014. Hybrid nanotopographic surfaces obtained by biomimetic mineralization of statherin-inspired elastin-like recombinamers. Advanced Healthcare Materials 3:1638-1647.
K.V. Holmberg, M. Abdolhosseini, X. Chen, Y. Li, S. U. Gorr, C. Aparicio. 2013. Bio-inspired Antimicrobial Peptide Coating for Dental Implants. Acta Biomaterialia 9(9): 8224-31.
X. Chen, P. Sevilla, C. Aparicio. 2013. Surface biofunctionalization by covalent co-immobilization of oligopeptides. Colloids and Surfaces B: Biointerfaces. 107:189-97.
X. Chen, Y. Li, C. Aparicio. 2013. Biofunctional Coatings for Dental Implants. In Thin Films and Coatings in Biology. Biological and Medical Physics -Biomedical Engineering Series; ed. S. Nazarpour Springer-Verlag. ISBN 978-94-007-2592-8. pp 105-143.
US 8569449 B2
Synthetic peptides and peptide mimetics
Inventors: Sven-Ulrik Gorr
Assignee: University of Louisville Research Foundation, Inc.
Summary of invention :The present inventors have designed several peptides based on the sequences of human Parotid Secretory Protein (PSP, also called SPLUNC2 or C20ORF70).
WO 2011036326 A3
Novel recombinant proteinic polymers and method for bioactivating surfaces with said polymers
Inventors: Conrado Aparicio and 9 more
Assignee: Universitat Politècnica de Catalunya, Universidad de Valladolid
Summary of the invention: The invention relates to proteinic polymers or mixtures thereof comprising domains that can promote the nucleation/growth and/or adhesion of calcium phosphates of biological interest, and domains that can promote cellular adhesion by means of peptides of the proteins of the extracellular matrix. The invention also relates to methods for activating materials for osseous implants using said proteinic polymers and to the implants that can be obtained by said methods.
- University of Copenhagen, Denmark, Cand. Scient (MSc), 1977-1984 (Biochemistry)
- University of Basel, Switzerland, Post-grad, 1984-1985 (Pharmacology)
- University of Copenhagen, Denmark, PhD, 1987-1990 (Cell Biology)
- University of Louisville, Kentucky, Res. Assoc., 1985-1988 (Cell Biology)
After research training in Denmark, Switzerland and the U.S., I joined the faculty of the University of Louisville and in 2009 moved to the University of Minnesota. I have over 20 years experience as an independent investigator with additional roles as a research administrator at the National Institutes of Health and the University of Minnesota. Our research is focused on the function of salivary glands and salivary proteins and the design of antimicrobial peptides based on salivary protein structures. Thus we have studied the salivary secretory protein Parotid Secretory Protein (BPIFA2) for 10+ years. In 2001, we were the first to report the cDNA sequence of the human protein and found that the protein appeared to be related to host-defense and lipid-binding proteins. Based on this proposed similarity we designed and tested potential antimicrobial peptides. The lead peptide, GL13K, is active against drug-resistant bacteria, planktonic and biofilm bacteria, Gram negative and Gram positive bacteria and appears to act by removing lipid micelles from the cell membrane. We have built on these observations with active collaborations to further characterize these novel peptides and their innovative use as antibacterial coatings for implant surfaces.
- Technical University of Catalonia, Spain, MSEng, 1997 (Mechanical Engineering)
- Technical University of Catalonia, Spain, PhD, 2005 (Biomaterials)
- Northwestern University, Chicago, Illinois, Post-doc, 2006-2007 (Nanobiomaterials)
I am an investigator with a research focus on dental biomaterials, their design, synthesis, as well as the characterization and analysis of their physical, chemical, mechanical, and biological properties.
I am a materials engineer with 15 years experience in the field of biomaterials for dental and orthopaedic applications. I initially worked on developing new surface treatments to improve osseointegration of dental implants that resulted in a patent that was licensed to a company that produces dental implants. Our work received the European Award in Basic Research in Dentistry.
Later, I expanded my research interests to applications of peptides and recombinant polymers in tissue engineering and regenerative medicine as a result of my work at the Institute for BioNanotechnology in Medicine at Northwestern University.
I arrived to the University of Minnesota in 2008 and started a research program on biological and hybrid coatings for dental and orthopaedic applications. We have established different methodologies to multi-functionalize metallic surfaces anchoring bioactive peptides or recombinamers in collaboration with local and International research groups. Indeed, our successful collaboration with Sven-Ulrik Gorr, Ph.D. at the University of Minnesota School of Dentistry has resulted in the development of GL13K-peptide coatings that have shown sustained resistance to degradation with broad antimicrobial activity and promising applications for dental implants, dental abutments, orthodontic appliances, total joint implants, intramedular pins, orthopaedic external fixators, osseointegrated prosthesis, etc.
My background and record of scholarly publications reflects a multi- and interdisciplinary approach to biomaterials and biomechanics science and engineering, including several biomolecular, in vitro cellular, and in vivo studies.
- Massa Abdolhosseini, post-doctoral fellow
- Chitta Geetha, post-doctoral fellow
- Helmut Hirt, post-doctoral fellow
- S. Rao Nandula, Research Associate
- Anu Shelar, M.S. student
- Jonathan Song, DDS student
- Julie Sotsky, research assistant
- In memoriam: S.G. Venkatesh, post-doctoral fellow
- Xi Chen, PhD student
- Kyle Holmberg (now Kyle H. Vining), DDS
- Yuping Li, post-doctoral fellow