Browsing by Subject "Tacrolimus"
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Item Open Access Characterization of the FKBP12-Encoding Genes in Aspergillus fumigatus.(PLoS One, 2015) Falloon, Katie; Juvvadi, Praveen R; Richards, Amber D; Vargas-Muñiz, José M; Renshaw, Hilary; Steinbach, William JInvasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the Δfkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn't localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don't play an important role in virulence. While the Δfkbp12-2 and Δfkbp12-3 strains didn't show any discernable phenotype, the Δfkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated "paradoxical growth effect" at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A. fumigatus FKBP12-1 and human FKBP12 will be critical for the generation of fungal-specific FK506 analogs to inhibit fungal calcineurin and treat invasive fungal disease.Item Open Access Costimulation blockade alters germinal center responses and prevents antibody-mediated rejection.(Am J Transplant, 2014-01) Kim, EJ; Kwun, J; Gibby, AC; Hong, JJ; Farris III, AB; Iwakoshi, NN; Villinger, F; Kirk, AD; Knechtle, SJDe novo donor-specific antibody (DSA) after organ transplantation promotes antibody-mediated rejection (AMR) and causes late graft loss. Previously, we demonstrated that depletion using anti-CD3 immunotoxin combined with tacrolimus and alefacept (AMR regimen) reliably induced early DSA production with AMR in a nonhuman primate kidney transplant model. Five animals were assigned as positive AMR controls, four received additional belatacept and four received additional anti-CD40 mAb (2C10R4). Notably, production of early de novo DSA was completely attenuated with additional belatacept or 2C10R4 treatment. In accordance with this, while positive controls experienced a decrease in peripheral IgM(+) B cells, bela- and 2C10R4-added groups maintained a predominant population of IgM(+) B cells, potentially indicating decreased isotype switching. Central memory T cells (CD4(+) CD28(+) CD95(+)) as well as PD-1(hi) CD4(+) T cells were decreased in both bela-added and 2C10R4-added groups. In analyzing germinal center (GC) reactions in situ, lymph nodes further revealed a reduction of B cell clonal expansion, GC-follicular helper T (Tfh) cells, and IL-21 production inside GCs with additional belatacept or 2C10R4 treatment. Here we provide evidence that belatacept and 2C10R4 selectively suppresses the humoral response via regulating Tfh cells and prevents AMR in this nonhuman primate model.Item Open Access FKBP12 dimerization mutations effect FK506 binding and differentially alter calcineurin inhibition in the human pathogen Aspergillus fumigatus.(Biochemical and biophysical research communications, 2020-05) Juvvadi, Praveen R; Bobay, Benjamin G; Gobeil, Sophie MC; Cole, D Christopher; Venters, Ronald A; Heitman, Joseph; Spicer, Leonard D; Steinbach, William JThe 12-kDa FK506-binding protein (FKBP12) is the target of the commonly used immunosuppressive drug FK506. The FKBP12-FK506 complex binds to calcineurin and inhibits its activity, leading to immunosuppression and preventing organ transplant rejection. Our recent characterization of crystal structures of FKBP12 proteins in pathogenic fungi revealed the involvement of the 80's loop residue (Pro90) in the active site pocket in self-substrate interaction providing novel evidence on FKBP12 dimerization in vivo. The 40's loop residues have also been shown to be involved in reversible dimerization of FKBP12 in the mammalian and yeast systems. To understand how FKBP12 dimerization affects FK506 binding and influences calcineurin function, we generated Aspergillus fumigatus FKBP12 mutations in the 40's and 50's loop (F37 M/L; W60V). Interestingly, the mutants exhibited variable FK506 susceptibility in vivo indicating differing dimer strengths. In comparison to the 80's loop P90G and V91C mutants, the F37 M/L and W60V mutants exhibited greater FK506 resistance, with the F37M mutation showing complete loss in calcineurin binding in vivo. Molecular dynamics and pulling simulations for each dimeric FKBP12 protein revealed a two-fold increase in dimer strength and significantly higher number of contacts for the F37M, F37L, and W60V mutations, further confirming their varying degree of impact on FK506 binding and calcineurin inhibition in vivo.Item Open Access Harnessing calcineurin-FK506-FKBP12 crystal structures from invasive fungal pathogens to develop antifungal agents.(Nature communications, 2019-09) Juvvadi, Praveen R; Fox, David; Bobay, Benjamin G; Hoy, Michael J; Gobeil, Sophie MC; Venters, Ronald A; Chang, Zanetta; Lin, Jackie J; Averette, Anna Floyd; Cole, D Christopher; Barrington, Blake C; Wheaton, Joshua D; Ciofani, Maria; Trzoss, Michael; Li, Xiaoming; Lee, Soo Chan; Chen, Ying-Lien; Mutz, Mitchell; Spicer, Leonard D; Schumacher, Maria A; Heitman, Joseph; Steinbach, William JCalcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection.Item Open Access Leveraging Fungal and Human Calcineurin-Inhibitor Structures, Biophysical Data, and Dynamics To Design Selective and Nonimmunosuppressive FK506 Analogs.(mBio, 2021-12) Gobeil, Sophie M-C; Bobay, Benjamin G; Juvvadi, Praveen R; Cole, D Christopher; Heitman, Joseph; Steinbach, William J; Venters, Ronald A; Spicer, Leonard DCalcineurin 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 fungus-selective, nonimmunosuppressive FK506 analogs. To this end, we characterized high-resolution structures of the Mucor circinelloides FKBP12 bound to FK506 and of the Aspergillus 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. The work presented here, aimed at the rational design of more effective calcineurin inhibitors, indeed suggests that modifications to the APX879 scaffold centered around the C15, C16, C18, C36, and C37 positions provide the potential to significantly enhance fungal selectivity. IMPORTANCE Invasive 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 the FK506 scaffold and a less immunosuppressive analog, APX879, centered around the C15 to C18 and C36 to C37 positions that could be altered with selective extensions and/or deletions to enhance fungal selectivity. This work represents a significant advancement toward realizing calcineurin as a viable target for antifungal drug discovery.Item Open Access Outcome of transplantation for acute lymphoblastic leukemia in children with Down syndrome.(Pediatric blood & cancer, 2014-06) Hitzler, Johann K; He, Wensheng; Doyle, John; Cairo, Mitchell; Camitta, Bruce M; Chan, Ka Wah; Diaz Perez, Miguel A; Fraser, Christopher; Gross, Thomas G; Horan, John T; Kennedy-Nasser, Alana A; Kitko, Carrie; Kurtzberg, Joanne; Lehmann, Leslie; O'Brien, Tracey; Pulsipher, Michael A; Smith, Franklin O; Zhang, Mei-Jie; Eapen, Mary; Carpenter, Paul A; CIBMTR Pediatric Cancer Working CommitteeWe report on 27 patients with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) who received allogeneic hematopoietic cell transplantation (HCT) between 2000 and 2009. Seventy-eight percent of patients received myeloablative conditioning and 52% underwent transplantation in second remission. Disease-free survival (DFS) was 24% at a median of 3 years. Post-transplant leukemic relapse was more frequent than expected for children with DS-ALL (54%) than for non-DS ALL. These data suggest leukemic relapse rather than transplant toxicity is the most important cause of treatment failure. Advancements in leukemia control are especially needed for improvement in HCT outcomes for DS-ALL.Item Open Access Pre-engraftment syndrome after myeloablative dual umbilical cord blood transplantation: risk factors and response to treatment.(Bone marrow transplantation, 2013-07) Kanda, J; Kaynar, L; Kanda, Y; Prasad, VK; Parikh, SH; Lan, L; Shen, T; Rizzieri, DA; Long, GD; Sullivan, KM; Gasparetto, C; Chute, JP; Morris, A; Winkel, S; McPherson, J; Kurtzberg, J; Chao, NJ; Horwitz, MEHigh fevers and/or rashes prior to neutrophil engraftment are frequently observed after umbilical cord blood (UCB) transplantation, and the condition is referred to as pre-engraftment syndrome (PES). Few studies have evaluated the risk factors for and treatment response to PES. Therefore, we retrospectively characterized PES in 57 consecutive engrafted patients (≥ 12 years old) who received myeloablative dual UCB transplantation. All patients received TBI (≥ 13.2 Gy)-based myeloablative conditioning. Tacrolimus (n=35) or CYA (n=22) combined with mycophenolate mofetil was used as GVHD prophylaxis. PES was defined as the presence of non-infectious fever (≥ 38.5 °C) and/or rash prior to or on the day of neutrophil engraftment. The incidence (95% confidence interval) of PES was 77% (66-88%). The incidence of PES was significantly higher in patients who received CYA as a GVHD prophylaxis than those who received tacrolimus (P<0.001), and this association was confirmed in the multivariate analysis. The occurrence of PES did not impact OS or tumor relapse, although it may have increased non-relapse mortality (P=0.071). The incidence of acute GHVD or treatment-related mortality was not influenced by the choice to use corticosteroids to treat PES. This study suggests that use of CYA for GVHD prophylaxis increases the risk of PES following dual UCB transplantation.Item Open Access Structure-Guided Synthesis of FK506 and FK520 Analogs with Increased Selectivity Exhibit In Vivo Therapeutic Efficacy against Cryptococcus.(mBio, 2022-06) Hoy, Michael J; Park, Eunchong; Lee, Hyunji; Lim, Won Young; Cole, D Christopher; DeBouver, Nicholas D; Bobay, Benjamin G; Pierce, Phillip G; Fox, David; Ciofani, Maria; Juvvadi, Praveen R; Steinbach, William; Hong, Jiyong; Heitman, JosephCalcineurin 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 nonconserved region of FKBP12 that can be exploited for fungus-specific targeting. These studies led to the design of an FK506 analog, APX879, modified at the C-22 position, which 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 C-22-modified compounds derived from both FK506 and FK520. One compound, JH-FK-05, demonstrates broad-spectrum antifungal activity in vitro and is nonimmunosuppressive 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 the C-22 and C-21 positions that could be modified to generate novel FK506 analogs with improved antifungal activity. IMPORTANCE Due 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 has enabled the structure-guided design of FK506 analogs and led to a breakthrough in the development of a compound with increased fungal specificity. The development of a calcineurin inhibitor with reduced immunosuppressive activity and maintained therapeutic antifungal activity would add a significant tool to the treatment options for these invasive fungal infections with exceedingly high rates of mortality.