AZITHROMYCIN ALCHEMY: A JOURNEY FROM PRECISION IMMUNOMODULATION TO TARGET EXPLORATION AND BEYOND

Azithromycin (AZM) is a macrolide antibiotic that is commonly used in respiratory infections. In addition to this, AZM exhibits immunomodulatory properties and has shown utility in a spectrum of inflammatory conditions, including cystic fibrosis and chronic obstructive pulmonary disease. However, the emergence of antibiotic resistance with increased use of AZM and other macrolides for their secondary anti-inflammatory effects is of major concern. To address this, our studies aimed to pinpoint AZM’s mechanism of action while addressing antibiotic resistance issues through generation of nonantibiotic immunomodulatory AZM derivatives. We strategically designed these derivatives by modifying sugar moieties known to be responsible for antibiotic activity while also improving its anti-inflammatory efficacy through targeted synthesis. Minimum inhibitory concentration assays showed a significant loss of antibiotic potency against Staphylococcus aureus in 10 out of 11 analogs. Immunomodulatory potential was assessed through both assays testing NF-κB translocation in monocytes as well as interleukin- 12 secretion from macrophages. These assays both indicated that the majority of the nonantibiotic AZM derivatives exerted superior anti-inflammatory effects compared to their parent compound. Together, these data suggest that the anti-inflammatory mechanisms of AZM can be uncoupled from its antibiotic effects using a targeted approach and offer a path forward for rational design of further immunomodulatory agents.Chapter 4 delves into the nuanced anti-inflammatory mechanisms of AZM, seeking to address the unresolved structure-activity relationship (SAR) and the dearth of identified drug targets. Employing a meticulously designed series of novel AZM analogs and probes, our research aimed to elucidate the intricacies of AZM's immunomodulatory mechanism. Two noteworthy biotinylated AZM derivatives, namely compounds 11 and 12, demonstrated anti-inflammatory potential. Exhibiting the capacity to diminish NF-κB activity, suppress IL-12/23 (p40) secretion, and maintain cytotoxicity below 5%, these compounds emerge as promising candidates for sophisticated in vitro assays aimed at target identification. A groundbreaking enhancement to our toolkit includes the biorthogonal clickable AZM derivative, HS-4-58 (P), and the fluorescent AZM probe, HS-4-86. These probes are meticulously crafted to unravel the intricate network of AZM's binding partners and enable real-time tracking in a zebrafish model of inflammation.Chapter 5 embarks on the transformative potential of AZM in concert with synergistic combinations. Recognizing the multifaceted nature of inflammation, we strategically explored synergies with agents targeting diverse inflammatory pathways, presenting a holistic paradigm for optimal control and resolution of inflammatory responses. Our investigation unveils three strategic dimensions: augmenting AZM's potency through covalent warheads, synergizing with Pitavastatin (PIT), and pioneering HDAC3 inhibition for enhanced epigenetic modulation. Navigating the domain of irreversible covalent inhibitors, exemplified by Compound 10, our inquiry delved into acrolein-based derivatives. Experimental validation highlights the superior efficacy of Compound 10. Beyond unveiling heightened pharmacological potency, our findings dissect the intricate dynamics of acryloyl moieties' strategic placement, unveiling a delicate interplay between positioning, toxicity modulation, and anti-inflammatory efficacy. Experimental validation also showcases the increased potency of Compound M, a PIT-conjugated AZM derivative, in comparison to AZM. Our findings illuminate sophisticated strategies—covalent precision, synergistic augmentation, and selective HDAC3 modulation to enhance AZM's anti-inflammatory fitness.In Chapter 6, we delve into the realm of capuramycin-type nucleoside antibiotics as an independent endeavor, specifically focusing on glycorandomization of nucleosides using the OleD Loki variant glycosyl transferase enzyme. This deliberate approach serves as a promising strategy to enhance and broaden our nucleoside antibiotics platform. Moreover, by leveraging the OleD Loki variant glycosyl transferase enzyme, we aim to achieve glycorandomization of AZM, enabling the semisynthetic synthesis of a diverse library comprising aminosugar-conjugated AZM derivatives. This diverse library holds significance in our exploration of immunomodulation and proton trapping within macrophage lysosomes.In addition to the utility of the compounds developed herein, a useful framework is provided to further modification of macrolide antibiotic. Our findings contribute nuanced insights into AZM's anti-inflammatory properties, presenting a diverse toolkit for further exploration. This research, with its methodological rigor and innovative probes, holds potential for impactful contributions to the understanding of AZM's immunomodulatory potential and its promising role as a therapeutic agent in the realm of inflammatory diseases.