Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742
Performing Department
Veterinary Medicine
Non Technical Summary
Bacteria within the genus Rickettsia are the causative agents of many significant animal and human infections. These bacteria are transmitted to mammals by blood-feeding arthropods, whereby the bacteria parasitize the mammalian circulatory system. Without prompt and appropriate therapeutic intervention these bacteria destroy mammalian blood vessels with extremely high risk of mortality. Yet, antibiotics are the only therapeutic strategy to defend against these infections.Rickettsia species are obligate intracellular pathogens that can only grow within a host cell. The bacteria hijack to the host cell to create a hospitable environment for growth. Therefore, we believe that modulation of specific host cell activities could make the intracellular environment inhospitable to the bacteria. We hypothesize that chemical manipulation of the host functions that contribute to bacterial growth will block Rickettsia infection. We refer to this strategy as "host-targeted" therapeutics, as we will target the host to prevent bacterial infection. Accordingly, we have examined 640 FDA approved drugs for the ability to inhibit Rickettsia growth. 30 compounds were effective at reducing Rickettsia growth without host toxicity. The leading class of host-targeted anti-Rickettsia therapeutics are Calcium channel blockers that are commonly used to treat high blood pressure. Herein, we describe experiments to assess the ability of Calcium channel blockers to act as antibiotics to fight Rickettsia infections in animal and human hosts.
Animal Health Component
(N/A)
Research Effort Categories
Basic
60%
Applied
(N/A)
Developmental
40%
Goals / Objectives
The goal of this proposal is to perform in vitro and in vivo analysis of potential host-targeted chemical inhibitors of Rickettsia infections, with the long-term goal of repurposing these drugs to supplement current treatment strategies and/or serve as the first line of defense against pathogenic Rickettsia species. Candidate drugs have previously been identified through a screen of available FDA-approved drugs12 and will be first be assessed for efficacy in tissue culture models of Rickettsia infection. The promising compounds will then be applied to small animal models of infection to assess in vivo efficacy in preventing and treating infection. As many different animal species are susceptible to Rickettsia infection these drugs can be used for animal health. Therefore, the objectives will support Animal health and production and animal products AFRI research priority, and a goal of the National Institutes of Health to re-purpose existing drugs for other uses. We will perform a comprehensive assessment of the translational potential of previously identified FDA-approved Calcium channel blockers (CCBs) for anti-bacterial therapeutics by completing the two following objectives:Objective 1: Characterization of antibacterial activity of CCBs against pathogenic Rickettsia speciesA. Assess CCB efficacy in multiple cell culture models of Rickettsia infection. Each candidate compound will be analyzed for efficacy in inhibition of Rickettsia proliferation in cell culture systems available in the PI's laboratory. Determination of tissue culture inhibitory and toxic concentrations will be valuable for validation of the therapeutic potential of previously identified candidate therapeutics and will also eliminate any "false-positive" screen hits. The CCBs will be assessed for 1) prophylactic (pre-exposure) and therapeutic (post-exposure) efficacy, 2) usefulness against multiple different Rickettsia pathogens, and 3) activity in multiple host species.B. Determine efficacy of combinatorial Doxycycline and Calcium channel inhibitor treatment. A major obstacle to developing new therapeutics for lethal diseases is that is unethical to withhold a proven therapeutic for the candidate drug. However, we believe the current therapeutic regimen can be improved because the case fatality rate remains persistently high and Doxycycline is ineffective once the patient has demonstrated clinical signs of infection for three days. Therefore, we propose to assess the effectiveness of the candidate anti-Rickettsia compounds in combination with the current therapeutic regimen.C. Identify the host/pathogen interaction that is targeted by anti-Rickettsia drugs. While each of the FDA-approved compounds being examined herein has a defined molecular target for normal use, it is imperative that we confirm the biological mechanism of anti-bacterial activity. This type of analysis is especially pertinent to increase understanding of the basic biology of the Rickettsia/host interaction and to identify bona fide host processes that contribute to virulence of these bacteria. Therefore, we will employ chemical and genetic manipulation of host pathways to determine if the previously identified molecular target of each candidate drug is also responsible for anti-bacterial activity against Rickettsia species.Objective 2: Assess effectiveness of host-targeted therapeutics in murine models of Rickettsia infection.A. Evaluate prophylactic capacity of CCBs in preventing Rickettsia infections. As an initial assessment of the translational capacity of each candidate compound for use mammals, we will pre-treat mice with each in vitro-validated anti-infective drug before challenge with different Rickettsia species. This treatment strategy models the regimen that may be required in places with high frequency of Rickettsia infection where prophylactic treatment is supported.B. Appraise therapeutic efficacy of compounds in treating ongoing Rickettsia infections. The drugs identified herein would be most frequently used as a therapeutic after emergence of the clinical signs and symptoms of Rickettsia infection. We therefore propose to assess the capability of each candidate compound to impede Rickettsia infection after emergence of the first clinical signs of infection.
Project Methods
Objective 1: Characterization of antibacterial activity of screen hits against pathogenic Rickettsia speciesObjective 1a. Assess CCB efficacy in multiple cell culture models of Rickettsia infection. We first propose to identify the "therapeutic window" where each of the six CCBs inhibits Rickettsia growth while not affecting host cell viability. Initially, we will initially analyze the only pathogenic Rickettsia that can be manipulated under BSL-2 conditions, R. parkeri. Toxicity will be assessed by the common MTT viability assay.Objective 1b. Determine efficacy of combinatorial doxycycline and calcium channel inhibitors treatment. A major challenge in developing infection blocking drugs for fatal diseases is that withholding an existing therapeutic for an experimental regimen is both unethical and dangerous. While it is vital to establish efficacy of each candidate anti-infective, new therapeutics will likely by combined with canonical anti-infectives for clinical applications. This concept correlates with our proposed use of CCBs to supplement antibiotic therapy, because antibiotic therapy can be ineffective after 3 days of illness. We will first calculate the prophylactic TCIC50, toxic concentration, and 10% tissue culture inhibitory concentration (TCIC10) of Doxycycline. We will then assess Rickettsia inhibition using the TCIC10 of Doxycycline supplemented with multiple concentrations of each CCB. We anticipate that combinatorial treatment will result in greater anti-Rickettsia activity than each drug alone, thus establishing the concept of combining Doxycycline with the CCBs.Objective 1c. Identify the host/pathogen interaction that is targeted by anti-Rickettsia drugs. While each of the FDA-approved compounds being examined herein has a defined molecular target, it is imperative that we confirm the biological mechanism of anti-bacterial activity. This type of analysis is especially pertinent to increase understanding of the basic biology of the Rickettsia/host interaction and to identify host processes that contribute to virulence of these bacteria.All cells continuously pump Ca2+ out of the cytoplasm into the extracellular space and organelles. Various cell signaling events can lead to opening of calcium channels to allow flow of Ca2+ back into the cytoplasm. Increased cytoplasmic The increase in cytoplasmic Ca2+ is detected by intracellular calcium-receptors (e.g. calmodulin) which mediate changes to cellular physiology. Since multiple types of CCBs with variant channel specificity were identified in our original screen, we hypothesize that properly regulated calcium signaling promotes intracellular Rickettsia growth. Therefore, we will employ non-CCB methods to disrupt intracellular calcium signaling to assess if the calcium gradient contributes to Rickettsia proliferation. We propose to employ Ca2+-ATPase inhibitors (Thapsigargin and 2,5-Ditertbutylhydro-quinone) and calcium ionophores to disrupt formation of the calcium gradient and will subsequently analyze their growth inhibitory effects as described in Objective 1a.Objective 2: Assess in vivo effectiveness of chemicals in animal models of Rickettsia infection.Objective 2a: Evaluate prophylactic capacity of CCBs in preventing Rickettsia infection Mice will be subjected to intravenous Rickettsia infection while treated with the candidate anti-infective. Each experiment will contain five groups: 1) protected (daily 10 mg/kg Doxycycline), 2) mock-treated, and 3-5) each of the three candidate anti-bacterial drugs. We will use the minimum lethal dose (MLD) of each Rickettsia species so that we have the greatest chance of identifying a significant increase in survival associated with any drug treatment. Each group will consist of 22 total animals, with 10 animals used to assess survival rate and monitor clinical signs of infection, including: hunched posture, ruffled fur, and weight loss. 3 pre-identified animals will be removed at specific time-points after infection for temporal analysis of pathologic lesions and for qPCR-based quantification of Rickettsia loads. As the data from animal models of infection may be used for future FDA new drug use applications, all animal studies will be conducted according to Title 21, Chapter 1, Part 58 "Good Laboratory Practice for Nonclinical Laboratory Studies." By combining analysis of clinical signs of infection with temporal ex vivo analysis, we have significant potential to assess the efficacy of each drug.As an initial assessment of the translational capacity of each candidate compound, we will pre-treat animals with each CCB before challenge with each Rickettsia species. Animals will be treated one hour before and throughout the infection. We will subsequently allow the infection to proceed to recovery or until animals are deemed to be fatally infected. 3 pre-identified mice will be removed at 1, 3, 5, and 7 days post infection for analysis of bacterial burden and pathology. This objective will involve 330 mice (110 mice/experiment x 3 Rickettsia species). We will also isolate DNA from portions of major organs to assess bacterial load by quantitative PCR using primers directed against chromosomal Rickettsia and Mus musculus genes, whereby bacterial load is ratio of Rickettsia to murine DNA.To further analyze rickettsial load and to identify the signs of parasitism in target organs, we will perform immunohistopathology analyses. Rickettsia will be stained using a specific anti-Rickettsia antibody followed by standard immunohistochemical staining. By analyzing the clinical signs of infection, quantifying bacterial load in distal organs, and examining pathological lesions we have a significant chance of identifying changes to infection associated with each prophylactic regimen.Objective 2b. Appraise therapeutic efficacy of compounds in treating Rickettsia infection. The drugs identified herein would be most frequently used as a therapeutic after emergence of the clinical signs and symptoms of Rickettsia infection. We propose to assess the capability of each candidate compound to impede Rickettsia infection after emergence of the first clinical signs of infection. The R. rickettsii, R. conorii, and R. australis mouse models of infection employed in Objective 2 all follow a similar time course of disease. 3-4 days after infection, the mice begin to show the first signs of infection, including weight loss and ruffled fur. Subsequently, the clinical signs become more ominous with reduced mobility, hunched posture, and >15% weight loss before typically succumbing to infection at day 5-7. We propose to treat the infected mice after emergence of the first clinical signs of infection at day 3 post-infection. Mice will be treated starting at D=3 with the drug regimen until recovery or until deemed to be fatally infected. 3 pre-identified mice will be removed at D=4, 5, 6, and 7 after infection for analysis of bacterial load in distal organs and pathological lesions as described above. In total, this objective will involve 330 mice (110 mice/experiment x 3 Rickettsia species). This experimental design allows testing of each drug in a manner that closely resembles the anticipated clinical setting as an anti-bacterial therapeutic.