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Rapamycin and FK506 are macrocyclic natural products with an extraordinary mode of action, in which they form binary complexes with FK506-binding protein (FKBP) through a shared FKBP-binding domain before forming ternary complexes with their respective targets, mechanistic target of rapamycin (mTOR) and calcineurin, respectively. Inspired by this, we sought to build a rapamycin-like macromolecule library to target new cellular proteins by replacing the effector domain of rapamycin with a combinatorial library of oligopeptides. We developed a robust macrocyclization method using ring-closing metathesis and synthesized a 45,000-compound library of hybrid macrocycles (named rapafucins) using optimized FKBP-binding domains. Screening of the rapafucin library in human cells led to the discovery of rapadocin, an inhibitor of nucleoside uptake. Rapadocin is a potent, isoform-specific and FKBP-dependent inhibitor of the equilibrative nucleoside transporter 1 and is efficacious in an animal model of kidney ischaemia reperfusion injury. Together, these results demonstrate that rapafucins are a new class of chemical probes and drug leads that can expand the repertoire of protein targets well beyond mTOR and calcineurin. Rapamycin and FK506 are macrocycles that contain an FKBP-binding domain and an effector domain responsible for interacting with their respective targets, mTOR and calcineurin. Now, a 45,000-compound macrocycle library has been synthesized by fusing oligopeptides with synthetic FKBP-binding domains. Screening and subsequent optimization yielded a highly potent FKBP-dependent inhibitor of hENT1.

Calcineurin 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. FK506 is a potential antifungal compound that inhibits calcineurin, but it also has immunosuppressive activity. Here, Juvvadi et al. report the structure of FK506 in complex with the FK506-binding protein FKPB12 and calcineurin, and design a less immunosuppresive FK506 analog with antifungal activity in mice.

The human fungal pathogen Mucor circinelloides develops spontaneous resistance to an antifungal drug both through mutation and through a newly identified epigenetic RNA-mediated pathway; RNA interference is spontaneously triggered to silence the fkbA gene, giving rise to drug-resistant epimutants that revert to being drug-sensitive once again when grown in the absence of drug. Epimutants confer drug resistance RNA interference (RNAi) is a mechanism conserved across eukaryotes that controls multiple cellular functions. This study reports that the opportunistic human pathogen Mucor circinelloides can develop spontaneous resistance to the antifungal drug FK506 (tacrolimus) via two distinct mechanisms. One is through conventional Mendelian mutation, whereas the other, surprisingly, is via a newly identified epigenetic RNAi-mediated pathway. Joseph Heitman and colleagues show that RNAi is spontaneously triggered to silence a gene, fkbA , that encodes the peptidylprolyl isomerase FKBP12. This enzyme interacts with the drug to form a complex that inhibits calcineurin, blocking the transition to hyphae. The resulting drug-resistant 'epimutants' revert to drug sensitivity when grown in the absence of drug. Microorganisms evolve via a range of mechanisms that may include or involve sexual/parasexual reproduction, mutators, aneuploidy, Hsp90 and even prions. Mechanisms that may seem detrimental can be repurposed to generate diversity. Here we show that the human fungal pathogen Mucor circinelloides develops spontaneous resistance to the antifungal drug FK506 (tacrolimus) via two distinct mechanisms. One involves Mendelian mutations that confer stable drug resistance; the other occurs via an epigenetic RNA interference (RNAi)-mediated pathway resulting in unstable drug resistance. The peptidylprolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin 1 . Calcineurin inhibition by FK506 blocks M. circinelloides transition to hyphae and enforces yeast growth 2 . Mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance and restore hyphal growth. In parallel, RNAi is spontaneously triggered to silence the fkbA gene, giving rise to drug-resistant epimutants. FK506-resistant epimutants readily reverted to the drug-sensitive wild-type phenotype when grown without exposure to the drug. The establishment of these epimutants is accompanied by generation of abundant fkbA small RNAs and requires the RNAi pathway as well as other factors that constrain or reverse the epimutant state. Silencing involves the generation of a double-stranded RNA trigger intermediate using the fkbA mature mRNA as a template to produce antisense fkbA RNA. This study uncovers a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes.

Background：Orthotopic liver transplantation （OLT） improves the prognosis of patients with hepatocellular carcinoma （HCC）.Moreover,the complement system is a powerful immune effector that can affect liver function and process of liver cirrhosis.However,studies correlating the complement system with tacrolimus metabolism after OLT are scarce.In this study,the role of single nucleotide polymorphisms （SNPs） associated with the sixth complement component （C6） in tacrolimus metabolism was investigated during the early stages of liver transplantation.Methods：The study enrolled 135 adult patients treated with OLT for HCC between August 2011 and October 2013.Ten SNPs in C6 gene and rs776746 in cytochrome P450 3A5 （CYP3A5） gene were investigated.The tacrolilnus levels were monitored daily during 4 weeks after transplantation.Results：Both donor and recipient CYP3A5 rs776746 allele A were correlated with decreased concentration/dose （C/D） ratios.Recipient C6 rs9200 allele G and donor C6 rs10052999 homozygotes were correlated with lower C/D ratios.Recipient CYP3A5 rs776746 allele A （yielded median tacrolimus C/D ratios of 225.90 at week 1 and 123.61 at week 2）,C6 rs9200 allele G （exhibited median tacrolimus C/D ratios of 211.31 at week l,110.23 at week 2,and 99.88 at week 3）,and donor CYP3A5 rs776746 allele A （exhibited median C/D ratios of 210.82 at week l,111.06 at week 2,77.49 at week 3,and 85.60 at week 4） and C6 rs 10052999 homozygote （exhibited median C/D ratios of 167.59 at week 2,157.99 at week 3,and 155.36 at week 4） were associated with rapid tacrolimus metabolism.With increasing number of these alleles,patients were found to have lower tacrolimus C/D ratios at various time points during the 4 weeks after transplantation.In multiple linear regression analysis,recipient C6 rs9200 group （AA vs.GG/GA） was found to be related to tacrolimus metabolism at weeks 1,2,and 3 （P =0.005,P =0.045,and P =0.033,respectively）,whereas donor C6 rs10052999 group （CC/TT vs.TC） was demonstrated to be correlated with tacrolimus metabolism only at week 4 （P =0.001）.Conclusions：Recipient C6 gene rs9200 polymorphism and donor C6 gene rs10052999 polymorphism are new genetic loci that affect tacrolimus metabolism in patients with HCC after OLT.

Detection of protein–protein interactions (PPIs) is important for understanding numerous processes in mammalian cells; however, existing PPI detection methods often give significant background signals. Here, we propose a novel PPI-detection method based on kinase-mediated growth induction of mammalian cells. In this method, target proteins are fused to the intracellular domain of c-kit (c-kit ICD) and expressed in interleukin-3-dependent mammalian cells. The PPI induces dimerization and activation of c-kit ICDs, which leads to cell growth in the absence of interleukin-3. Using this system, we successfully detected the ligand-dependent homo-interaction of FKBP F36V and hetero-interaction of FKBP and FRB T2098L , as well as the constitutive interaction between MDM2 and a known peptide inhibitor. Intriguingly, cells expressing high-affinity peptide chimeras are selected from the mixture of the cell populations dominantly expressing low-affinity peptide chimeras. These results indicate that this method can detect PPIs with low background levels and is suitable for peptide inhibitor screening.

Arteriolar hyalinosis is a common histological finding in renal transplant recipients treated with the calcineurin inhibitor tacrolimus; however, the pathophysiologic mechanisms remain unknown. In addition to increasing transforming growth factor (TGF)-β levels, tacrolimus inhibits calcineurin by binding to FK506-binding protein 12 (FKBP12). FKBP12 alone also inhibits TGF-β receptor activation. Here we tested whether tacrolimus binding to FKBP12 removes an inhibition of the TGF-β receptor, allowing ligand binding, ultimately leading to receptor activation and arteriolar hyalinosis. We found that specific deletion of FKBP12 from endothelial cells was sufficient to activate endothelial TGF-β receptors and induce renal arteriolar hyalinosis in these knockout mice, similar to that induced by tacrolimus. Tacrolimus-treated and knockout mice exhibited significantly increased levels of aortic TGF-β receptor activation as evidenced by SMAD2/3 phosphorylation, along with increased collagen and fibronectin expression compared to controls. Treatment of isolated mouse aortas with tacrolimus increased TGF-β receptor activation and collagen and fibronectin expression. These effects were independent of calcineurin, absent in endothelial denuded aortic rings, and could be prevented by the small molecule TGF-β receptor inhibitor SB-505124. Thus, endothelial cell TGF-β receptor activation is sufficient to cause vascular remodeling and renal arteriolar hyalinosis.

Invasive 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.

Immunophilins comprise two protein families, cyclophilins (CYPs) and FK506-binding proteins (FKBPs), and are the major receptors for the immunosuppressive drugs cyclosporin A (CsA) and FK506 (tacrolimus), respectively. Most eukaryotic species have at least one immunophilin and some of them have been associated with pathogenesis of infectious or parasitic diseases or the action of antiparasitic drugs. The human malarial parasite Plasmodium falciparum has 13 immunophilin or immunophilin-like genes but the functions of their products are unknown. We set out to identify the parasite proteins that interact with the major CYPs, PfCYP19A and PfCYP19B, and the FKBP, PfFKBP35, using a combination of co-immunoprecipitation and yeast two-hybrid screening. We identified a cohort of putative interacting partners and further investigation of some of these revealed potentially novel roles in parasite biology. We demonstrated that (i) P. falciparum CYPs interacted with the heat shock protein 70, (ii) treatment of parasites with CYP ligands disrupted transport of the rhoptry-associated protein 1, and (iii) PfFKBP35 interacted with parasite histones in a way that might modulate gene expression. These findings begin to elucidate the functions of immunophilins in malaria. Furthermore, the known antimalarial effects of CsA, FK506 and non-immunosuppressive derivatives of these immunophilin ligands could be mediated through these partner proteins.

Low tacrolimus trough concentration-to-dose ratio (CDR) is recognized as an indicator of high tacrolimus metabolism. However, its impact on long-term transplant outcomes and potential for clinical intervention remains unclear. In the largest study to date, we analyzed the impact of a low CDR at post-transplant year 1 on graft loss and patient mortality in 21,865 kidney transplants. We also performed a longitudinal analysis of CDR dynamics and conducted a genetic correlation in a subset of 1,257 patients. Low CDR at year 1 was significantly associated with increased hazards of graft failure (HR up to 2.80) and infection-related mortality (HR = 1.63), even in patients with therapeutic trough levels and good graft function. In the longitudinal analysis, normalizing initially low CDR by year 2 significantly improves graft survival. Low CDR was identified in a substantial proportion of the cohort (25.2%). Black, female, and younger recipients (<50 years) had higher odds of having a low CDR. The CYP3A5*1A genotype was also strongly associated with low CDR (approximately 8-fold higher odds). Patients with a low tacrolimus CDR represent a large high-risk population. The normalization of tacrolimus CDR through co-medication with diltiazem and reductions in steroid dosing may improve graft survival. Our findings support personalized tacrolimus management based on metabolic profiling and genetic testing.

Analysis of the human repertoire of the FK506-binding protein (FKBP) family of peptidyl-prolyl cis/trans isomerases has identified an expansion of genes that code for human FKBPs in the secretory pathway. There are distinct differences in tissue distribution and expression levels of each variant. In this article we describe the characterization of human FKBP19 (Entrez Gene ID: FKBP11), an FK506-binding protein predominantly expressed in vertebrate secretory tissues. The FKBP19 sequence comprises a cleavable N-terminal signal sequence followed by a putative peptidyl-prolyl cis/trans isomerase domain with homology to FKBP12. This domain binds FK506 weakly in vitro. FKBP19 mRNA is abundant in human pancreas and other secretory tissues and high levels of FKBP19 protein are detected in the acinar cells of mouse pancreas.