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Hodges Lab Research


Research is grouped into the following six topic areas:
  1. Synthetic Peptide Vaccines for Emerging and Re-emerging Viruses, and Development of Synthetic Peptide Anti-adhesion Bacterial Vaccine for Prevention of Pseudomonas aeruginosa (PA) Bacterial infections
  2. Development of Antimicrobial peptides (AMPs) as Therapeutics
  3. Transmission of Stability Information Along a Two-stranded α-helical Coiled-Coil
  4. Topic Pending
  5. Development of New HPLC and Capillary Electrophoresis Methodology​
  6. Development of New Broad Spectrum Anticancer Drugs​
Synthetic Peptide Vaccines for Emerging and Re-emerging Viruses

I have developed an extremely simple, innovative, and robust platform technology to present α-helical epitopes from native proteins to the immune system such that the resulting conformation-specific antibodies bind to the native protein target. This procedure involves a process called templating where the helical sequence of interest is inserted into a parallel two-stranded α-helical coiled-coil and disulfide-bridged template which maintains the native helical conformation of the epitope, presents all the surface-exposed residues of the α-helix in the native protein, and optimizes stability of the immunogen.

Viruses that enter cells using Class 1 Viral Fusion Proteins such as Ebola, Influenza A, Respiratory Syncytial Virus and coronaviruses such as SARS-CoV and MERS-CoV are ideal targets for this technology. The stem domains of the fusion glycoproteins of these diverse viruses share a common coiled-coil structure that must be triggered to undergo a massive conformational change in order to induce fusion of the virus envelope with host cell membranes and initiate virus infections in vitro and in vivo. Our templated, two-stranded immunogens elicited stem targeted, virus neutralizing antibodies against SARS-CoV and the hemagglutinin (HA) protein of influenza A.

We have shown that passive administration of rabbit antibodies to a selected immunogen can protect mice against death after intranasal challenge with influenza A. This antibody also recognizes purified HA proteins from different influenza subtypes, suggesting that this stem-targeted immunogen may serve as a “universal” influenza vaccine that can protect against multiple subtypes. 

This research is the result of an accumulation of an extensive body of published work on de novo design of coiled-coil proteins from my laboratory. Selected publications are listed.

Jiang, Z., L. Gera, C.T. Mant and R.S. Hodges. Antibody cross-reactivity to hemagglutinin protein antigens demonstrates feasibility for development of a “Universal” Influenza A synthetic peptide vaccine.  In Enabling Peptide Research from Basic Research to Drug Discovery, Proceedings of the 24th American Peptide Symposium, Orlando, FL (V. Srivastava, A. Yudin and M. Lebl, editors) pp. 36-39 (2015). Published by the American Peptide Society and Propt Scientific Publishing, 2015.

Yan, Z., W.J. Hartsock, Z. Qian, K.V. Holmes and R.S. Hodges.  Strategies for designing peptide immunogens to elicit α-helical conformation specific antibodies reactive with native proteins.  In “Small Wonders:  Peptides for Disease Control” (K. Rajasekaran, J.W. Cary and J. Jaynes, Eds.) ACS Symposium Series, Chapter 6, pp. 93-136 (2012).

Tripet, B., D. Kao, S. Jeffers, K. Holmes and R.S. Hodges.  Template-based Coiled-coil antigens elicit neutralizing antibodies to the SARS-Coronavirus.  J. Structural Biology 155:  176-194 (2006).

Tripet, B., M.W. Howard, M. Jobling, R.K. Holmes, K.V. Holmes, and R.S. Hodges. Structural characterization of the SARS-Coronavirus Spike Fusion Protein Core. J. Biol. Chem. 279: 20836-20849 (2004).​

Development of Synthetic Peptide Anti-adhesion Bacterial Vaccine for Prevention of Pseudomonas aeruginosa (PA) Bacterial infections

I have determined the key adhesin for the attachment of PA to the epithelial cell surface and identified the receptor binding domain as a 17-residue region of the pilin protein. Antibodies to the receptor binding domain (RBD) block adherence of the organism and provide protection against PA in animal models.

We have determined the three-dimensional structures of the receptor binding domains from multiple strains of PA and shown that though sequences vary substantially from one strain to another, there is a common structural motif.

We have shown that a synthetic peptide vaccine is superior to a pilin protein-based vaccine. Using a rational design approach, we have developed a single 17-residue peptide immunogen that generates antibodies that target the RBD of more than one strain of PA.

My present goal is to design a consensus sequence immunogen that will provide maximum antibody cross-reactivity and affinity to all known strains of PA.

Selected publications are listed.

Hartsock, W.J., C. Hackbarth and R.S. Hodges.  Antiadhesion synthetic consensus sequence peptide-based vaccines for Pseudmonas aeruginosa.  In Handbook of Biologically Active Peptides, 2nd edition, (A.J. Kastin, Editor in Chief) Chapter 77, pp. 563-570 (2013).

Hackbarth, C. and R.S. Hodges.  Synthetic Peptide Vaccine Development: Designing Dual Epitopes into a Single Pilin Peptide Immunogen Generates Antibody Cross-reactivity between Two Strains of Pseudomonas aeruginosa.  Chem. Biol. & Drug Design 76: 293-304 (2010).  (Journal Cover Figure)  PMC2949483 PMID20807222

Kao, D. and R.S. Hodges.  Advantages of a synthetic peptide immunogen over a protein immunogen in the development of an anti-pilus vaccine for Pseudomonas aeruginosa.  Chem. Biol. and Drug Design 74: 33-42 (2009).   PMC2756486 PMID19519742

Kao, D., M.E. Churchill, R.T. Irvin and R.S. Hodges. Animal protection and structural studies of a consensus sequence vaccine targeting the receptor binding domain of the type IV pilus of Pseudomonas aeruginosa.  J. Mol. Biol. 374: 426-442 (2007). PMC3493149 PMID17936788​​​​

Development of Antimicrobial peptides (AMPs) as Therapeutics

The dramatic and ever-increasing emergence of many relevant strains of bacteria resistant to traditional antibiotics (of which no radically new structural class has been introduced into medical practice over the past 30 years) is a major issue in human health. The problem is further complicated by the fact that not only has there been an explosion of resistance to antibiotics but also a rapid increase in multi-drug resistance. We have now seen the development of so called “Superbugs” that are resistant to most or all of the available antibiotics. Vertebrates and other organisms have developed a defense system to microbial infections composed of distinct groups of broad spectrum antimicrobial peptides (AMPs).

My group has been studying amphipathic α-helical AMPs for years and has made six innovative discoveries critical to the development of AMPs as therapeutics. 1) Discovery of “specificity determinants,” that is, an amino acid substitution(s) in the non-polar face of amphipathic α-helical or cyclic β-sheet AMPs that dramatically reduces toxicity to human red blood cells; 2) The development of a novel method to measure self-association of small amphipathic molecules; 3) the discovery that increasing hydrophobicity on the non-polar face beyond an optimum can lead to inactive AMPs; 4) the novel discovery that “specificity determinants” when inserted into native broad-spectrum AMPs directed selectivity to Gram-negative pathogens; 5) Methicillin-resistant S. aureus (MRSA) strains are more susceptible to AMPs than Methicillin-sensitive S. aureus (MSSA) strains. This suggested that pathogens in developing antibiotic resistance are modifying their membrane composition/structure which makes the membrane more sensitive to AMPs; 6) advances in our understanding of mechanism of action of amphipathic α-helical AMPs and the desired properties, for a clinical therapeutic; (i) the importance of lack of secondary structure in aqueous medium but inducible α-helical structure in the presence of the hydrophobic environment of the membrane; (ii) “specificity determinants” reduce or eliminate toxicity by decreasing or eliminating transmembrane penetration into eukaryotic membranes but allowing AMP access to the interface region of prokaryotic membranes; (iii) reducing self-association in aqueous environment is an important property influencing biological activity; (iv) there is an optimum hydrophobicity window to maintain high antimicrobial activity; (v) the sole target of the AMP should be the bacterial membrane and the AMP should not be involved in any stereoselective interaction with chiral enzymes or lipids or protein receptors; (vi) peptides should be prepared in the all-D conformation to provide resistance to proteolysis. 

Selected publications are listed.

Jiang, Z., L. Gera, C.T. Mant and R.S. Hodges. Design of new antimicrobial peptides (AMPs) with “specificity determinants” that encode selectivity for gram-negative pathogens and remove gram-positive and hemolytic activity from broad-spectrum AMPs.  In Enabling Peptide Research from Basic Research to Drug Discovery, Proceedings of the 24th American Peptide Symposium, Orlando, FL (V. Srivastava, A. Yudin and M. Lebl, editors) pp. 245-248 (2015). Published by the American Peptide Society and Propt Scientific Publishing, 2015.

Jiang, Z., A.J. Vasil, M.L. Vasil and R.S. Hodges.  “Specificity determinants” improve therapeutic indices of two antimicrobial peptides Piscidin 1 and Dermaseptin S4 against gram-negative pathogens Acinetobacter baumannii and Pseudomonas aeruginosa.  Pharmaceuticals 7: 366-391 (2014); DOI 10.3390/ph7040366.

Z. Jiang, A.I. Vasil, L. Gera, M.L. Vasil and R.S. Hodges.  Rational design of a-helical antimicrobial peptides to target gram-negative pathogens, Acinetobacter baumannii and Pseudomonas aeruginosa:  Utilization charge, “specificity determinants”, total hydrophobicity, hydrophobe type and location as design parameters to improve the  therapeutic ratio. Chem. Biol & Drug Design 77: 225-240 (2011). (Journal Cover Figure)  PMC3063396  PMID21219588

Hodges, R.S., Z. Jiang, J. Whitehurst, and C.T. Mant.  Devleopment of antimicrobial peptides as therapeutic agents.  In, “Development of Therapeutic Agents Handbook” (Ed. by Shayne C. Gad), John Wiley and Sons Inc. pp. 285-358 (2011).​ 

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Transmission of Stability Information Along a Two-stranded α-helical Coiled-Coil

Tropomyosin (Tm) is most widely known for its role in muscle contraction; its calcium-dependent cooperation with the troponin complex in regulating the interaction of myosin and actin required to generate the power stroke within the sarcomeres of muscle cells. Tm is expressed in all eukaryotic cell types in more than 40 isoforms that are widely distributed within individual cells. Consequently, Tm plays a critical role in numerous biologically relevant processes and is associated with several diseases, including cardiomyopathy and cancer. 

Tm is a two-stranded α-helical coiled-coil of 284-residues per polypeptide chain. How signals are transmitted along this rod-like molecule of more than 400 Ao remains a major challenge for protein chemists. We made the first identification of a stability control region (SCR) residues 97-118 in the 284-residue tropomyosin sequence that controls overall protein stability but is not required for folding. We have shown that a single mutation L110A destabilizes the entire Tm (1-131) molecule, showing that the effect of this mutation is transmitted 165 Ao along the coiled-coil in the N-terminal direction. The single mutation A109L prevents the SCR from transmitting stabilizing information and separates the coiled-coil into two domains, one that is ~9oC more stable than wild type and one that is ~16oC less stable.  Learning how stability information is transmitted along rod-like molecules is crucial to understanding function and signaling.

Selected publications are listed.

Kirwan, J.P. and R.S. Hodges.  Transmission of stability information through the N-domain of tropomyosin is interrupted by a stabilizing mutation (A109L) in the hydrophobic core of the stability control region (97-118).  J. Biol. Chem. 389: 4356-4366 (2014).

Kirwan, J.P. and R.S. Hodges.  Critical interactions in the stability control region of tropomyosin.  J. Structural Biology 170: 294-306 (2010).  PMC2856757  PMID20144718

Hodges, R.S., J. Mills, S. McReynolds, J.P. Kirwan, B. Tripet, and D. Osguthorpe.  Identification of a unique “stability control region” that controls protein stability of tropomyosin:  a two-stranded a-helical coiled-coil.  J. Mol. Biol. 392: 747-762 (2009).  PMC2756485  PMID19627992

Kwok, S.C. and R.S. Hodges.  Stabilizing and Destabilizing Clusters in the Hydrophobic Core of Long Two-stranded a-Helical Coiled-Coils.  J. Biol. Chem. 279: 21576-21588 (2004).​

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​Topic Pending​
Development of New HPLC and Capillary Electrophoresis Methodology

The development of new separation methodology is critical to research in academia, biotechnology and pharmaceutical sectors. A few examples of new methodology developed in our laboratory are listed below: a new mixed-mode hydrophilic/cation-exchange chromatographic method for the separation of peptides that rivals reversed-phase chromatography (RPC) where selectivity is completely different; a new capillary electrophoresis method called “Ion-interaction capillary zone electrophoresis” which separates not only by charge/ mass ratio but by subtle differences in hydrophobicity as well; a novel method to measure self-association of small amphipathic molecules referred to as temperature profiling in RPC; a one-step purification of recombinant proteins from a whole cell extract by RPC; and we have determined the intrinsic hydrophilicity/hydrophobicity of amino acid side-chains in peptides in the absence of nearest-neighbor or conformational affects and showed that this scale was the most accurate determination of side-chain hydrophilicity/hydrophobicity and of general value to all fields of biomedical science. 

Selected publications are listed.

Mant, C.T. and R.S. Hodges.  Separation of peptides on HALO 2-micron particles.  Current Protocols in Protein Science 85: 11.6.1-11.6.16 (2016). doi:10.1002/cpps.12 (Wileyonlinelibrary.com, John Wiley & Sons, Inc.).

Mant, C.T., Z. Jiang, B. E. Boyes and R.S. Hodges. An improved approach to hydrophilic interaction chromatography of peptides: Salt gradients in the presence of high isocratic acetonitrile concentrations.  J. Chromatography A 1277: 15-25 (2013).  PMC3639484  PMID23332786

Mant, C.T. and R.S. Hodges.  Design of peptide standards with the same composition and minimal sequence variation to monitor performance/selectivity of reversed-phase matrices.  J. Chromatography A , 1230: 30-40 (2012). PMC3294100  PMID22326185

Mant, C.T., D. Cepeniene and R.S. Hodges. Reversed-phase HPLC of Peptides: Assessing column and solvent selectivity on standard, polar embedded and polar end-capped columns.  J. Separation Science 33:3005-3021 (2010). No PMC  PMID21038458

Mant, C.T., J.M. Kovacs, H.M. Kim, D.D. Pollock and R.S. Hodges.  Intrinsic amino acid side-chain hydrophilicity/hydrophobicity coefficients determined by reversed-phase high-performance liquid chromatography of model peptides: comparison with other hydrophilicity/hydrophobicity scales.  Biopolymers (Peptide Science) 92: 573-595 (2009).  PMC2792893 PMID19795449​

Development of New Broad Spectrum Anticancer Drugs

The goal of the proposed project is to develop new broad spectrum anticancer drugs using an innovative and non-traditional approach where we create highly potent small molecules with a simple 3-component “A-B-C” structure (500-700 daltons) and nanmolar activity. ​