Biology subjects

Juvvadi, P. R.

Publications and source records attributed to Juvvadi, P. R..

4 recordsLinked to original sources

bioRxiv2024

Structure-guided design, synthesis, and characterization of C22- and C32-modified FK520 analogs with enhanced activity against human pathogenic fungi

Invasive fungal infections are a leading cause of death worldwide. Translating molecular insights into clinical benefits is challenging because fungal pathogens and their hosts share similar eukaryotic physiology. Consequently, current antifungal treatments have limited efficacy, may be poorly fungicidal in the host, can exhibit toxicity, and are increasingly compromised by emerging resistance. We have established that the phosphatase calcineurin (CaN) is required for invasive fungal disease and an attractive target for antifungal drug development. CaN is a druggable target, and there is vast clinical experience with the CaN inhibitors FK506 and cyclosporin A (CsA). However, while FK506 and its natural analog FK520 exhibit antifungal activity, they are also immunosuppressive in the host and thus not fungal-selective. We leverage our pathogenic fungal CaN-FK506-FKBP12 complex X-ray structures and biophysical data to support structure-based ligand design as well as structure-activity relationship analyses of broad-spectrum FK506/FK520 derivatives with potent antifungal activity and reduced immunosuppressive activity. Here we apply molecular docking studies to develop antifungal C22- or C32-modified FK520 derivatives with improved therapeutic index scores. Among them, the C32-modified FK520 derivative JH-FK-44 (7) demonstrates a significantly improved therapeutic index compared to JH-FK-08, our lead compound to date. NMR binding studies with C32-derivatives are consistent with our hypothesis that C32 modifications disrupt the hydrogen bonding network in the human complex while introducing favorable electrostatic and cation-{pi} interactions with the fungal FKBP12 R86 residue. These findings further reinforce calcineurin inhibition as a promising strategy for antifungal therapy. SignificanceInvasive fungal infections cause significant mortality worldwide, and current antifungal treatments are often ineffective, toxic, or face growing resistance. This research identifies calcineurin (CaN), a critical protein for fungal survival, as a potential target for developing new antifungal drugs. Although existing CaN inhibitors such as FK506 (tacrolimus) and FK520 (ascomycin) possess antifungal properties, their immunosuppressive effects limit their clinical utility. By studying the structure of human and fungal FKBP12-FK506 or FK520 complexes with CaN, we have designed and synthesized modified FK520 derivatives with strong antifungal activity and reduced immunosuppressive effects. These new derivatives are expected to have significantly improved therapeutic profiles, offering hope for more effective and safer antifungal treatments.

microbiology↗

bioRxiv2023

Enhanced fungal specificity and in vivo therapeutic efficacy of a C-22 modified FK520 analog against C. neoformans

Fungal infections are of mounting global concern, and the current limited treatment arsenal poses challenges when treating such infections. In particular, infections by Cryptococcus neoformans are associated with high mortality, emphasizing the need for novel therapeutic options. Calcineurin is a protein phosphatase that mediates fungal stress responses, and calcineurin inhibition by the natural product FK506 blocks C. neoformans growth at 37{degrees}C. Calcineurin is also required for pathogenesis. However, because calcineurin is conserved in humans, and inhibition with FK506 results in immunosuppression, the use of FK506 as an anti-infective agent is precluded. We previously elucidated the structures of multiple fungal calcineurin-FK506-FKBP12 complexes and implicated the C-22 position on FK506 as a key point for differential modification of ligand inhibition of the mammalian versus fungal target proteins. Through in vitro antifungal and immunosuppressive testing of FK520 (a natural analog of FK506) derivatives, we identified JH-FK-08 as a lead candidate for further antifungal development. JH-FK-08 exhibited significantly reduced immunosuppressive activity and both reduced fungal burden and prolonged survival of infected animals. JH-FK-08 exhibited additive activity in combination with fluconazole in vivo. These findings further advance calcineurin inhibition as an antifungal therapeutic approach. ImportanceFungal infections cause significant morbidity and mortality globally. The therapeutic armamentarium against these infections is limited and development of antifungal drugs has been hindered by the evolutionary conservation between fungi and the human host. With rising resistance to the current antifungal arsenal and an increasing at-risk population, there is an urgent need for the development of new antifungal compounds. The FK520 analogs described in this study display potent antifungal activity as a novel class of antifungals centered on modifying an existing orally-active FDA approved therapy. This research advances the development of much needed newer antifungal treatment options with novel mechanisms of action.

microbiology↗

bioRxiv2022

Structure-guided synthesis of FK506 and FK520 analogs with increased selectivity exhibit in vivo therapeutic efficacy against C. neoformans

Calcineurin is an essential virulence factor that is conserved across human fungal pathogens including Cryptococcus neoformans, Aspergillus fumigatus, and Candida albicans. Although an excellent target for antifungal drug development, the serine-threonine phosphatase activity of calcineurin is conserved in mammals and inhibition of this activity results in immunosuppression. FK506 (tacrolimus) is a naturally produced macrocyclic compound that inhibits calcineurin by binding to the immunophilin FKBP12. Previously, our fungal calcineurin-FK506-FKBP12 structure-based approaches identified a non-conserved region of FKBP12 that can be exploited for fungal-specific targeting. These studies led to the design of an FK506 analog, APX879, modified at the C22 position that was less immunosuppressive yet maintained antifungal activity. We now report high resolution protein crystal structures of fungal FKBP12 and a human truncated calcineurin-FKBP12 bound to a natural FK506 analog, FK520 (ascomycin). Based on information from these structures and the success of APX879, we synthesized and screened a novel panel of C22-modified compounds derived from both FK506 and FK520. One compound, JH-FK-05 demonstrates broad-spectrum antifungal activity in vitro and is non-immunosuppressive in vivo. In murine models of pulmonary and disseminated C. neoformans infection, JH-FK-05 treatment significantly reduced fungal burden and extended animal survival alone and in combination with fluconazole. Furthermore, molecular dynamic simulations performed with JH-FK-05 binding to fungal and human FKBP12 identified additional residues outside of the C22 and C21 positions that could be modified to generate novel FK506 analogs with improved antifungal activity. SignificanceDue to rising rates of antifungal drug resistance and a limited armamentarium of antifungal treatments, there is a paramount need for novel antifungal drugs to treat systemic fungal infections. Calcineurin has been established as an essential and conserved virulence factor in several fungi, making it an attractive antifungal target. However, due to the immunosuppressive action of calcineurin inhibitors, they have not been successfully utilized clinically for antifungal treatment in humans. Recent availability of crystal structures of fungal calcineurin bound inhibitor complexes have enabled the structure-guided design of FK506 analogs and led to a breakthrough in the development of a compound increased for fungal specificity. The development of a calcineurin inhibitor with reduced immunosuppressive activity and therapeutic antifungal activity would add a significant tool to the treatment options for these invasive fungal infections with exceedingly high rates of mortality.

microbiology↗

bioRxiv2020

Designing Selective and Non-Immunosuppressive Antifungal FK506 Analogs: Structures, Biophysics and Dynamics of Fungal and Human Calcineurin-Inhibitor Complexes

Calcineurin is a critical enzyme in fungal pathogenesis and antifungal drug tolerance and, therefore, an attractive antifungal target. Current clinically-accessible calcineurin inhibitors, such as FK506, are immunosuppressive to humans, so exploiting calcineurin inhibition as an antifungal strategy necessitates fungal-specificity in order to avoid inhibiting the human pathway. Harnessing fungal calcineurin-inhibitor crystal structures, we recently developed a less immunosuppressive FK506 analog, APX879, with broad-spectrum antifungal activity and demonstrable efficacy in a murine model of invasive fungal infection. Our overarching goal is to better understand, at a molecular level, the interaction determinants of the human and fungal FK506-binding proteins (FKBP12) required for calcineurin inhibition in order to guide the design of fungal-selective, non-immunosuppressive FK506 analogs. To this end, we characterized high-resolution structures of the M. circinelloides FKBP12 bound to FK506, and of the A. fumigatus, M. circinelloides and human FKBP12 proteins bound to the FK506 analog, APX879, which exhibits enhanced selectivity for fungal pathogens. Combining structural, genetic and biophysical methodologies with molecular dynamics simulations, we identify critical variations in these structurally similar FKBP12-ligand complexes that will guide the rational design of inhibitors with enhanced fungal-selectivity. Significance statementInvasive fungal infections are a leading cause of death in the immunocompromised patient population. The rise in drug resistance to current antifungals highlights the urgent need to develop more efficacious and highly selective agents. Numerous investigations of major fungal pathogens have confirmed the critical role of the calcineurin pathway for fungal virulence, making it an attractive target for antifungal development. Although FK506 inhibits calcineurin, it is immunosuppressive in humans and cannot be used as an antifungal. By combining structural, genetic, biophysical, and in silico methodologies, we pinpoint regions of FK506 and a less immunosuppressive analog, APX879, that could be altered to enhance fungal selectivity. This work represents a significant advancement toward realizing calcineurin as a viable target for antifungal drug discovery.

biochemistry↗