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Scholarly Works (49 results)

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UC Irvine Electronic Theses and Dissertations (2020)

Antibiotic resistant bacteria are an increasing threat to global human health. These drug resistant infections are challenging to treat and contain, and the antibiotic discovery pipeline does not match the pace of the growing number of antibiotic resistant bacteria. In Chapter 1, I summarize the outlook of antibiotic resistance and how the new antibiotic, teixobactin, serves as a promising template to combat drug resistant infections. Teixobactin is a peptide antibiotic that kills Gram-positive bacteria without detectable resistance and is able to kill pathogens that are considered as urgent and serious threats by the CDC. I provide a summary of the mechanism of action of teixobactin, structure-relationship studies of its pharmacophore, and various synthentic methods to make the antibiotic. In the last section of this chapter, I review how fluorescent antibiotics are versatile probes to understand the mode of action of antibiotics, elucidate off-target effects (toxicity studies), detect antibiotic resistance, and serve as diagnostics for detecting infections in-vivo or ex-vivo.

Chapter 2 describes the first synthesis and application of a fluorescent teixobactin analogue that exhibits antibiotic activity and binds to the cell walls of Gram-positive bacteria. The teixobactin analogue, Lys(Rhod)9,Arg10-teixobactin, has a fluorescent tag at position 9 and an arginine in place of the natural allo-enduracididine residue at position 10. The fluorescent teixobactin analogue retains partial antibiotic activity, with minimum inhibitory concentrations of 4–8 µg/mL across a panel of Gram-positive bacteria, as compared to 1–4 µg/mL for the unlabeled Arg10-teixobactin analogue. Lys(Rhod)9,Arg10-teixobactin is prepared by a regioselective labeling strategy that labels Lys9 with an amine-reactive rhodamine fluorophore during solid-phase peptide synthesis, with the resulting conjugate tolerating subsequent solid-phase peptide synthesis reactions. Treatment of Gram-positive bacteria with Lys(Rhod)9,Arg10-teixobactin results in septal and lateral staining, which is consistent with an antibiotic targeting cell wall precursors. Concurrent treatment of Lys(Rhod)9,Arg10-teixobactin and BODIPY FL vancomycin results in septal co-localization, providing further evidence that Lys(Rhod)9,Arg10-teixobactin binds to cell wall precursors. Controls with either Gram-negative bacteria, or an inactive fluorescent homologue with Gram-positive bacteria, showed little or no staining in fluorescence micrographic studies. Lys(Rhod)9,Arg10-teixobactin can thus serve as a functional probe to study Gram-positive bacteria and their interactions with teixobactin.

Chapter 3 describes a new approach to selectively label Lys9,Arg10-teixobactin and Lys10-teixobactin with a variety of N-Hydroxysuccinimide (NHS) ester fluorophores. The reaction affords regioselective labeling of the lysine sidechain amines of either Lys10- or Lys9,Arg10-teixobactin. Using this labeling method, we were able to generate four fluorescent teixobactin analogues, bearing different fluorophores, that retain antibiotic activity and stain the cell walls of Gram-positive bacteria. This approach also enabled us to determine that position 10 tolerates fluorophores better than position 9, using MIC assays and fluorescence microscopy as readouts. Structured illumination microscopy of the fluorescent teixobactin analogues in live B. subtilis cells enabled further study of the aggregation of teixobactin in bacterial membranes, with observation of both clusters and aggregates of the antibiotic in bacterial membranes. To further understand the aggregation of teixobactin in live bacteria, we used fluorescence lifetime imaging Förster resonance energy transfer (FLIM-FRET) microscopy, which demonstrated that fluorescent teixobactin analogues are in intimately interacting with each other in live cells. Lastly, we treated NRK-52E rat kidney cells with our fluorescent teixobactin analogues to determine if teixobactin had any nephrotoxic effects due to its unfavorable aggregation propensity. Taken together, we demonstrate useful applications of fluorescent teixobactin analogues that enabled elucidation of its on-target and off-target effects in bacterial and kidney cells, respectively.

Chapter 4 reports the biological activities of a toxic, fluorescent peptide derived from Aβ15-36 (peptide 1*) using the LDH release assay as well as confocal laser scanning microscopy (CLSM). The fluorescent L (7-hydroxycoumarin-4-yl) ethyl glycine (7-HC) amino acid was synthesized and incorporated into coumarin-QK15-36 (peptide 1*), thereby making it suitable for localization studies. Peptide 1* was shown to be toxic to human neuroblastoma SH-SY5Y cells over a range of concentrations using the LDH release assay. Furthermore, peptide 1* was uniformly internalized in the cytosol of SH-SY5Y cells, suggesting that it mediates toxicity by disrupting intracellular homeostasis. This is the first time the Nowick lab has characterized the cellular localization of a toxic peptide and the results from this study may further our understanding of Aβ neurotoxicity.

Chapter 5 summarizes my teaching and chemical education research experiences at UC Irvine. I provide reflections of my experiences in two pedagogical training programs offered at UC Irvine and summarize my experiences as an instructor of record for Chemistry 51LB and Chemistry 101W. This chapter concludes with a summary of my involvement in two chemical education research studies, where I learned about research survey design and data analysis.

    Cover page: Fluorescent Teixobactin Probes
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2022)
    Teixobactin is a promising new antibiotic that kills a spectrum of Gram-positive pathogens that are considered to be urgent threats by the CDC and the WHO. Better understanding of the novel mechanism of action of teixobactin may assist in developing new antibiotics and furthering our understanding of antibiotic resistance. This chapter describes the synthesis and application of fluorescent teixobactin analogs in fluorescence microscopy to study the mode of action of teixobactin. The first part of this chapter describes the synthesis and purification of fluorescent teixobactin analogs using two synthetic approaches. The second part of this chapter describes the application of the fluorescent teixobactin analogs to visualize their interactions with molecular targets in B. subtilis using fluorescence microscopy. The methods described herein provide synthetic access to chemical probes that may help further the understanding of antibiotic resistance.
      Cover page: Synthesis and application of fluorescent teixobactin analogs
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2021)
      This paper describes the structure-based design of a preliminary drug candidate against COVID-19 using free software and publicly available X-ray crystallographic structures. The goal of this tutorial is to disseminate skills in structure-based drug design and to allow others to unleash their own creativity to design new drugs to fight the current pandemic. The tutorial begins with the X-ray crystallographic structure of the main protease (Mpro) of the SARS coronavirus (SARS-CoV) bound to a peptide substrate and then uses the UCSF Chimera software to modify the substrate to create a cyclic peptide inhibitor within the Mpro active site. Finally, the tutorial uses the molecular docking software AutoDock Vina to show the interaction of the cyclic peptide inhibitor with both SARS-CoV Mpro and the highly homologous SARS-CoV-2 Mpro. The supporting information provides an illustrated step-by-step protocol, as well as a video showing the inhibitor design process, to help readers design their own drug candidates for COVID-19 and the coronaviruses that will cause future pandemics. An accompanying preprint in bioRxiv [https://doi.org/10.1101/2020.08.03.234872] describes the synthesis of the cyclic peptide and the experimental validation as an inhibitor of SARS-CoV-2 Mpro.
        Cover page: Structure-based drug design of an inhibitor of the SARS-CoV-2 (COVID-19) main protease using free software: A tutorial for students and scientists
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2021)
        The antibiotic teixobactin targets bacterial cell walls. Previous research has proposed that the active form of teixobactin is a nano-/micron-sized supramolecular assembly. Here, we use cryogenic transmission electron microscopy to show that at 1 mg/mL, teixobactin forms sheet-like assemblies that selectively act upon the cell wall. At 4 μg/mL, teixobactin is active, and aggregates are formed either transiently or sparingly at the cell surface.
          Cover page: Visualizing Teixobactin Supramolecular Assemblies and Cell Wall Damage in B. Subtilis Using CryoEM
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2016)
          We previously showed that expression of the anti-HIV innate proteins human beta-defensin 2 (hBD2) and hBD3 in adult oral epithelial cells reduces HIV transepithelial transmission by inactivation of virus. However, fetal/infant oral epithelia lack beta-defensin expression, leading to transmission of HIV. The mechanisms of hBD2- and hBD3-mediated HIV inactivation in adult oral epithelial cells are poorly understood. Here we found that heparan sulfate proteoglycans (HSPGs) on the apical surfaces of epithelial cells facilitate simultaneous binding of hBDs and HIV gp120 to the cell surface. HSPG-facilitated binding of hBDs and HIV gp120 to the cell surface did not affect viral attachment. HBD2 or -3 cointernalized with virions in endosomes, formed oligomers, and reduced infectivity of HIV. The anti-HIV effect of combining hBD2 and hBD3 was substantially higher than that of the individual peptides. These findings advance our understanding of the mechanisms of anti-HIV resistance in adult oral epithelium.
            Cover page: Human beta-defensins 2 and -3 cointernalize with human immunodeficiency virus via heparan sulfate proteoglycans and reduce infectivity of intracellular virions in tonsil epithelial cells.
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2024)
            Optimal patient management protocols for metastatic castration-resistant prostate cancer (mCRPC) are poorly defined and even further complexified with new therapy approvals, such as radiopharmaceuticals. The prostate-specific membrane antigen (PSMA)-targeted agent 177Lu vipivotide tetraxetan ([177Lu]Lu-PSMA-617), approved after the phase III VISION study, presents physicians with additional aspects of patient management, including specific adverse event (AE) monitoring and management, as well as radiation safety. Drawing on our experience as VISION study investigators, here we provide guidance on best practices for delivering PSMA-targeted radiopharmaceutical therapy (RPT) to patients with mCRPC. After a comprehensive review of published evidence and guidelines on RPT management in prostate cancer, we identified educational gaps in managing the radiation safety and AEs associated with [177Lu]Lu-PSMA-617. Our results showed that providing sufficient education on AEs (e.g., fatigue and dry mouth) and radiation safety principles is key to effective delivery and management of patient expectations. Patient counseling by health care professionals, across disciplines, is a cornerstone of optimal patient management during PSMA-targeted RPT. Multidisciplinary collaboration is crucial, and physicians must adhere to radiation safety protocols and counsel patients on radiation safety considerations. Treatment with [177Lu]Lu-PSMA-617 is generally well tolerated; however, additional interventions may be required, such as dosing modification, medications, or transfusions. Urinary incontinence can be challenging in the context of radiation safety. Multidisciplinary collaboration between medical oncologists and nuclear medicine teams ensures that patients are monitored and managed safely and efficiently. In clinical practice, the benefit-to-risk ratio should always be evaluated on a case-by-case basis.
              Cover page: Best Patient Care Practices for Administering PSMA-Targeted Radiopharmaceutical Therapy.
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2020)
              This report describes the first synthesis and application of a fluorescent teixobactin analogue that exhibits antibiotic activity and binds to the cell walls of Gram-positive bacteria. The teixobactin analogue, Lys(Rhod)9,Arg10-teixobactin, has a fluorescent tag at position 9 and an arginine in place of the natural allo-enduracididine residue at position 10. The fluorescent teixobactin analogue retains partial antibiotic activity, with minimum inhibitory concentrations of 4-8 μg/mL across a panel of Gram-positive bacteria, as compared to 1-4 μg/mL for the unlabeled Arg10-teixobactin analogue. Lys(Rhod)9,Arg10-teixobactin is prepared by a regioselective labeling strategy that labels Lys9 with an amine-reactive rhodamine fluorophore during solid-phase peptide synthesis, with the resulting conjugate tolerating subsequent solid-phase peptide synthesis reactions. Treatment of Gram-positive bacteria with Lys(Rhod)9,Arg10-teixobactin results in septal and lateral staining, which is consistent with an antibiotic targeting cell wall precursors. Concurrent treatment of Lys(Rhod)9,Arg10-teixobactin and BODIPY FL vancomycin results in septal colocalization, providing further evidence that Lys(Rhod)9,Arg10-teixobactin binds to cell wall precursors. Controls with either Gram-negative bacteria, or an inactive fluorescent homologue with Gram-positive bacteria, showed little or no staining in fluorescence micrographic studies. Lys(Rhod)9,Arg10-teixobactin can thus serve as a functional probe to study Gram-positive bacteria and their interactions with teixobactin.
                Cover page: A Fluorescent Teixobactin Analogue
                • Article
                • Peer Reviewed
                LBL Publications (2022)
                Interferometric microscopes are used to measure surface roughness, from which the computed power spectral density function can be used to extract bandwidth-limited values of the various surface properties, such as root-mean-square (rms) height and slope errors. Measurements with a microscope equipped with different objectives that have an overlapping spatial frequency range usually give different rms results over the common frequency bandwidth. This is a result of different instrument transfer functions (ITFs) that attenuate spatial frequencies by different amounts over the overlapping range. We report on the use of binary pseudo-random array (BPRA) standards to characterize the ITF of an interferometric microscope with the various objectives. We use a simple model of a 1D binary Results show that the spectrum for an undersampled array is a cosine function, rather than a straight line, constant white noise spectrum. We have an analytical model of the ITF that includes the effects of an obscured aperture and defocus, in addition to the usual parameters of numerical aperture, wavelength, and sampling period. In addition, the model includes the effect of aliasing of spatial frequency components beyond the Nyquist back into the sub-Nyquist region. We compare the model PSD predictions to the measurements performed with different objectives. Departures of the measured PSDs from the model predictions indicate that there are higher-order ITF corrections yet to be identified and included in the model.
                  Cover page: Calibration, modeling, parameterization, and verification of the instrument transfer function of an interferometric microscope
                  • Article
                  • Peer Reviewed
                  UC Irvine Previously Published Works (2024)
                  PURPOSE: 18F-Flotufolastat (18F-rhPSMA-7.3) is a newly approved prostate-specific membrane antigen targeting radiopharmaceutical for diagnostic imaging of prostate cancer (PCa). SPOTLIGHT (National Clinical Trials 04186845) evaluated 18F-flotufolastat in men with suspected PCa recurrence. Here, we present results of predefined exploratory endpoints from SPOTLIGHT to evaluate the impact of clinical factors on 18F-flotufolastat detection rates (DR). METHODS AND MATERIALS: The impact of baseline prostate-specific antigen (PSA), PSA doubling time (PSAdt), and International Society of Urologic Pathology Grade Group (GG) on 18F-flotufolastat DR was evaluated among all SPOTLIGHT patients with an evaluable scan, with DR stratified according to the patients prior treatment (radical prostatectomy ± radiation therapy [RP] or radiation therapy only [RT]). The patients underwent positron emission tomography 50 to 70 minutes after receiving 18F-flotufolastat (296 MBq IV), and scans were read by 3 blinded central readers, with the majority read representing agreement between ≥2 readers. RESULTS: In total, 389 men (median PSA: 1.10 ng/mL) were evaluable. By majority read, 18F-flotufolastat identified distant lesions in 39% and 43% of patients treated with prior RP or RT, respectively. The overall DR broadly increased with increasing PSA (<0.2 ng/mL: 33%; ≥10 ng/mL: 100%). Among patients with PSA <1 ng/mL, 68% had positive scans, and 27% had extrapelvic findings. PSAdt was available for 145/389 (37%) patients. PSAdt did not appear to influence 18F-flotufolastat DR (77%-90% across all PSAdt categories). Among patients with prior RP, DR ranged from 70% to 83% across PSAdt categories, and 100% DR was reported for all post-RT patients. In total, 362/389 (93%) patients had baseline GG data. Overall DRs were uniformly high (75%‒95%) across all GG. When stratified by prior treatment, DRs across all GG were 69% to 89% in patients with prior RP and ≥96% in patients with prior RT. CONCLUSIONS: 18F-Flotufolastat-positron emission tomography enabled the accurate detection of recurrent PCa lesions across a wide range of PSA, PSAdt, and International Society of Urologic Pathology GG, thus supporting its clinical utility for a broad range of patients with recurrent PCa.
                    Cover page: Impact of Clinical Factors on 18F-Flotufolastat Detection Rates in Men With Recurrent Prostate Cancer: Exploratory Analysis of the Phase 3 SPOTLIGHT Study.
                    • Article
                    • Peer Reviewed
                    UC San Diego Previously Published Works (2018)

                    Objective

                    To assess the feasibility of performing national, randomized trials of dietary interventions for localized prostate cancer.

                    Methods

                    The Men's Eating and Living (MEAL) study (CALGB 70807 [Alliance]) is a phase III clinical trial testing the efficacy of a high-vegetable diet to prevent progression in patients with prostate cancer on active surveillance (AS). Participants were randomized to a validated diet counselling intervention or to a control condition. Chi-squared and Kruskal-Wallis analyses were used to assess between-group differences at baseline.

                    Results

                    Between 2011 and 2015, 478 (103%) of a targeted 464 patients were randomized at 91 study sites. At baseline, the mean (sd) age was 64 (6) years and mean (sd) PSA concentration was 4.9 (2.1) ng/mL. Fifty-six (12%) participants were African-American, 17 (4%) were Hispanic/Latino, and 16 (3%) were Asian-American. There were no significant between-group differences for age (P = 0.98), race/ethnicity (P = 0.52), geographic region (P = 0.60), time since prostate cancer diagnosis (P = 0.85), PSA concentration (P = 0.96), clinical stage (T1c or T2a; P = 0.27), or Gleason sum (Gleason 6 or 3+4 = 7; P = 0.76). In a pre-planned analysis, the baseline prostate biopsy samples of the first 50 participants underwent central pathology review to confirm eligibility, with an expectation that <10% would become ineligible. One of 50 participants (2%) became ineligible.

                    Conclusion

                    The MEAL study shows the feasibility of implementing national, multi-institutional phase III clinical trials of diet for prostate cancer and of testing interventions to prevent disease progression in AS.
                      Cover page: Men's Eating and Living (MEAL) study (CALGB 70807 [Alliance]): recruitment feasibility and baseline demographics of a randomized trial of diet in men on active surveillance for prostate cancer.
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