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Using Molecular Biology to Develop a Novel Drug Targeting Antibiotic-Tolerant Lyme Persisters

This study focuses on developing a novel, targeted anti-persister drug for Lyme disease by utilizing molecular biology techniques. It addresses the growing problem of antibiotic-tolerant Lyme persisters and aims to improve treatment options.

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Using Molecular Biology to Develop a Novel Drug Targeting Antibiotic-Tolerant Lyme Persisters

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  1. Jonathan Locke BIOL-546 Using Molecular Biologyto Develop a Novel Drug Targeting Antibiotic-TolerantLyme Persisters

  2. Ixodes scapularis (deer tick)Eastern North America Ixodes pacificus Pacific North America Ixodes ricinusEurope Ticks 1 • Anaplasmosis • Babesiosis • Bartonellosis • Borreliosis (Lyme Disease) - Borreliaburgdorferi • Bourbon virus infection • Colorado tick fever • Erlichiosis • Heartland virus infection • Mycoplasmosis • Powassan virus infection • Rickettsia • Rocky Mountain Spotted Fever • Southern tick-associated rash illness • Tick-borne relapsing fever • Tularemia • CDC. Life cycle of Hard Ticks that Spread Diseasehttps://www.cdc.gov/ticks/life_cycle_and_hosts.html

  3. Lyme is Spreading 3 More than 300,000 cases/year 2 1 4 5 6 7 Lyme, CT • https://blogs.scientificamerican.com/observations/in-the-battle-against-lyme-disease-the-ticks-are-winning/, Mary Beth Pfeiffer, June 2018 • https://www.nytimes.com/2018/08/14/health/lyme-disease-vaccine.html, Karen Zraick, 2018 • Bay Area Lyme Foundation / CDC. Lyme Disease Statistics and Facts. CDC. • https://www.nrdc.org/experts/kim-knowlton/half-us-counties-talking-ticks-must-health, Kim Knowlton, April 2018 • Lyme Disease: An Even Bigger Threat Than You Think. Wall Street Journal, Jun 2018 • Lyme Disease Maps. https://www.cdc.gov/lyme/stats/maps.html • Ibid.

  4. Symptoms 1 1. Early localized Lyme - flu-like symptoms, skin rash, joint pain, stiff neck, swollen lymph nodes Lyme spirochete’s endoflagella allow dissemination into viscous tissues, affecting all organ systems 2. Early disseminated Lyme- neurological symptoms, fatigue, pain, weakness, vision and heart problems, facial paralysis 3. Late disseminated Lyme - arthritis, migraines, light sensitivity, sound sensitivity, skin pain, vertigo, severe pain, severe fatigue, migrating pain, joint pain, neck pain, insomnia, somnolence, mental fogginess, concentration problems, memory issues, numbness, ataxia, paralysis, seizures, disability, death Erythema Migrans (EM) rash 2 Facial Paralysis (Bell’s Palsy) 3 • CDC. Signs and Symptoms of Untreated Lyme Diseasehttps://www.cdc.gov/lyme/signs_symptoms/index.html • Ibid. • Ibid. Swollen Knee

  5. 10-36% of patients that receive early diagnosis and treatment develop a disabling chronic form of the illness Prognosis 1, 2 3 • Ibid., CDC • Ibid., Cameron, 2008 • https://www.lymedisease.org/lyme-basics/lyme-disease/chronic-lyme/, Aucott (John Hopkins) 2013

  6. Persistent Lyme Infection Persistent Borrelia Infection in Patients with Ongoing Symptoms of Lyme Disease Eva Sapi, et al. MDPI. Apr 2018 • Reason for ongoing symptoms has been debated for decades • In April 2018, 12 chronic Lyme patients and 10 controls were accurately identified from tissue and fluid samples by PCR and culture of viable spirochetes in a double-blind study • Odds of randomly guessing the binary disease state of 22 unknown samples is 1 in 2.4 million 1 (A) (Top left): Darkfield microscopy of blood culture showing live spirochete and spherules. Magnification 400×. (B) (Bottom left): Dieterle silver stain of culture fluid from Case 10 showing live spirochetes. Magnification 1000×. (C) (Top right): Borrelia immunostain of culture fluid from Case 9 showing live spirochetes. Magnification 1000×. (D) (Bottom right). Typical dermal filaments from patient with Morgellons disease. Magnification 100×. • Eva Sapi et al, Peristent Borrelia Infection in Patients with Ongoing Symptoms of Lyme Disease, Apr 2018

  7. Borrelia burgdorferi Bacteria PleomorphicFastidiousParasiticHeterotroph 2 Planktonic Spiral Spirochetes 3 Leptospira Treponema Biofilm Aggregation Leptospirosis Syphilis, Yaws T. pallidum Borrelia Periodontitis 4 T. denticola Round Body Relapsing Fever Lyme Disease B. burgdorferi B. miyamotoi B. hermsii B. afzelii B. parkeri B. gariniii • CDC. Public Health Image Library. ID #6631 • E. Sapi. et al. Evidence of In Vivo Existence of Borrelia Biofilm in Borrelial Lymphocytomas. European Journal of Microbiology and Immunology, Mar 2016. • Øystein Brorson, et al. Destruction of spirochete Borrelia burgdorferi round-body propagules (RBs) by the antibiotic Tigecycline. PNAS, Nov 2009.

  8. Lyme Persisters Borrelia burgdorferi, the Causative Agent of Lyme Disease, Forms Drug-Tolerant Persister Cells 1 Kim Lewis et al, Antimicrobial Agents and Chemotherapy, May 2015 Kim Lewis, Northeastern Eva Sapi, University of New Haven Ying Zhang, John Hopkins Jay Rajadas, Stanford University Neil Spector, Duke University Dr. Richard Horowitz • Kim Lewis et al., Borrelia burgdorferi, the Causative Agent of Lyme Disease, Forms Drug-Tolerant Persister Cells. AAC, May 2015

  9. Molecular Biology Developing a novel, targeted anti-persister drug using molecular biology techniques 1 Drug Discovery • Target Selection - Find a gene that makes a protein that might be critical to viability or virulence • 2. Target Validation - Determine if that gene is truly critical • 3. Lead Finding - Find a reasonable small molecule that binds to the target protein • 4. Lead Optimization - Improve binding affinity without reducing efficacy or safety Molecular biology can be used to increase precisionand decrease risk in the drug discovery process • Raymond G Hill, et al. Drug Discovery and Development. Elsevier, 2013

  10. Developing an Anti-persister Drug 1. Target Selection We want to find a plausible drug target to investigate. If our small molecule drug interferes with this target protein, we want B. burgdorferi to lose viability or virulence. Examples of known targets: Ouyang Z, He M, Oman T, Yang XF, Norgard MV. A manganese transporter, BB0219 (BmtA), is required for virulence by the Lyme disease spirochete, Borrelia burgdorferi. PNAS. 2009 Ignas Bunikis. An RND-Type Efflux System in Borrelia burgdorferi Is Involved in Virulence and Resistance to Antimicrobial Compounds. PLoS, Feb 2008 A (potentially) unknown target: Felipe C. Cabello. Sleeper cells: the stringent response and persistence in the Borreliella (Borrelia) burgdorferi enzootic cycle. Environmental Microbiology, Aug 2017 The stringent response mediated by the alarmone (p)ppGpp […] may also be involved in the recently described non‐genetic phenotypic phenomenon of tolerance to otherwise lethal doses of antimicrobials […]

  11. Developing an Anti-persister Drug 1. Target Selection Investigating a potential target: Felipe C. Cabello. Sleeper cells: the stringent response and persistence in the Borreliella (Borrelia) burgdorferi enzootic cycle. Environmental Microbiology, Aug 2017 • Look for bacteria that have known alarmone proteins - E. coli has these! • Locate the gene for the synthase enzyme that makes them - E. coli has RelA! • Get the amino acid sequence for RelA • Do a Blast database search to find all bacteria with a sequence similar to RelA • Use a tool like MEME to generate a “motif” from these sequences (a search pattern that indicates what parts of the sequence are most conserved) • Use a tool like FIMO to search the B. burgdorferi genome for homologies with the sequence motif. • Call our new gene RelB for now. Bioinformatics Highly conserved Not very conserved 1 A sequence motif “logo” • Wikipedia

  12. Developing an Anti-persister Drug 2. Target Validation We want to see if RelB is truly critical to viability or if the bacterium has some other pathway or workaround it can use to stay alive without it. 1 Transformation RelB • Locate restriction recognition sites for RelB • Obtain a sticky-ended restriction enzyme for those sites • Cut out an antibiotic resistance gene using the sticky-ended restriction enzyme • Use the same restriction enzyme to remove RelB and in its place, add the resistance gene, transforming the bacteria into resistant mutants • Kill off the non-mutants with the antibiotic corresponding to the resistance gene • Expose the (recombinant) mutants to antibiotics that should trigger the alarmone pathway (which no longer exists in the mutants) and see whether they survive as persisters or if they die. ABX resist • Google Image Search. Pearson, 2010.

  13. Developing an Anti-persister Drug 3. Lead Finding We want to find a small molecule that’s safe and binds with high affinity to our validated target protein, producing the same effect on viability that was observed when knocking out the gene during target validation. 1 • Use recombinant DNA techniques to add RelB to yeast or E. coli to produce biosynthetic RelBenzyme for use in determining structure and in a protein binding assay Genetic Recombination • Determine the structure of RelB • Use x-ray crystallography on RelB to determine the precise structure • Use I-Tasser protein folding software to predict the structure of RelB • Use Autodock Vina docking software to screen a large library of small moleculesto find existing molecules with good binding affinity. A molecule with good bindingaffinity is a “hit”. If it tests well, it can become a lead compound. • Test the compound against the biosynthetic enzyme in a protein binding assay, then in vitro and finally in an animal model to make sure it’s a viable lead ready for optimization. • Borreliacidal activity of Borrelia metal transporter A (BmtA) binding small molecules by manganese transport inhibition, Dhananjay Wagh, et al, Drug Design, Development and Therapy, Feb 2015

  14. Developing an Anti-persister Drug 4. Lead Optimization We want to improve the binding affinity without affecting drug safety or efficacy. • Use drug design tools to derive a novel compound from the hit with improved binding affinity without compromising safety (ADME/Tox) Absorption Distribution Metabolism Excretion In Silico Drug Design • Test the compound in an assay to see if it has the desired effect in vitro andthen in an animal model. If it does, we enter pre-clinical development.

  15. Questions?

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