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Paralleling the activation of dorsal horn microglia after peripheral nerve injury is a significant expansion and proliferation of macrophages around injured sensory neurons in dorsal root ganglia (DRG). Here we demonstrate a critical contribution of DRG macrophages, but not those at the nerve injury site, to both the initiation and maintenance of the mechanical hypersensitivity that characterizes the neuropathic pain phenotype. In contrast to the reported sexual dimorphism in the microglial contribution to neuropathic pain, depletion of DRG macrophages reduces nerve injury-induced mechanical hypersensitivity and expansion of DRG macrophages in both male and female mice. However, fewer macrophages are induced in the female mice and deletion of colony-stimulating factor 1 from sensory neurons, which prevents nerve injury-induced microglial activation and proliferation, only reduces macrophage expansion in male mice. Finally, we demonstrate molecular cross-talk between axotomized sensory neurons and macrophages, revealing potential peripheral DRG targets for neuropathic pain management. Interactions among spinal dorsal horn neurons and microglia contribute to the induction and maintenance of neuropathic pain after peripheral nerve injury. The authors show that depletion of macrophages in the dorsal root ganglia prevents and reverses ongoing nerve injury-induced hypersensitivity.

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.

Cystathionine-β-synthase (CBS), the first (and rate-limiting) enzyme in the transsulfuration pathway, is an important mammalian enzyme in health and disease. Its biochemical functions under physiological conditions include the metabolism of homocysteine (a cytotoxic molecule and cardiovascular risk factor) and the generation of hydrogen sulfide (H2S), a gaseous biological mediator with multiple regulatory roles in the vascular, nervous, and immune system. CBS is up-regulated in several diseases, including Down syndrome and many forms of cancer; in these conditions, the preclinical data indicate that inhibition or inactivation of CBS exerts beneficial effects. This article overviews the current information on the expression, tissue distribution, physiological roles, and biochemistry of CBS, followed by a comprehensive overview of direct and indirect approaches to inhibit the enzyme. Among the small-molecule CBS inhibitors, the review highlights the specificity and selectivity problems related to many of the commonly used “CBS inhibitors” (e.g., aminooxyacetic acid) and provides a comprehensive review of their pharmacological actions under physiological conditions and in various disease models.

Mitochondrial and lysosomal dysfunction have been implicated in substantia nigra dopaminergic neurodegeneration in Parkinson’s disease (PD), but how these pathways are linked in human neurons remains unclear. Here we studied dopaminergic neurons derived from patients with idiopathic and familial PD. We identified a time-dependent pathological cascade beginning with mitochondrial oxidant stress leading to oxidized dopamine accumulation and ultimately resulting in reduced glucocerebrosidase enzymatic activity, lysosomal dysfunction, and α-synuclein accumulation. This toxic cascade was observed in human, but not in mouse, PD neurons at least in part because of species-specific differences in dopamine metabolism. Increasing dopamine synthesis or α-synuclein amounts in mouse midbrain neurons recapitulated pathological phenotypes observed in human neurons. Thus, dopamine oxidation represents an important link between mitochondrial and lysosomal dysfunction in PD pathogenesis.

The calcineurin inhibitor tacrolimus is the backbone of immunosuppressive drug therapy after solid organ transplantation. Tacrolimus is effective in preventing acute rejection but has considerable toxicity and displays marked inter-individual variability in its pharmacokinetics and pharmacodynamics. The genetic basis of these phenomena is reviewed here. With regard to its pharmacokinetic variability, a single nucleotide polymorphism (SNP) in cytochrome P450 (CYP) 3A5 (6986A>G) has been consistently associated with tacrolimus dose requirement. Patients expressing CYP3A5 (those carrying the A nucleotide, defined as the *1 allele) have a dose requirement that is around 50 % higher than non-expressers (those homozygous for the G nucleotide, defined as the *3 allele). A randomised controlled study in kidney transplant recipients has demonstrated that a CYP3A5 genotype-based approach to tacrolimus dosing leads to more patients reaching the target concentration early after transplantation. However, no improvement of clinical outcomes (rejection incidence, toxicity) was observed, which may have been the result of the design of this particular study. In addition to CYP3A5 genotype, other genetic variants may also contribute to the variability in tacrolimus pharmacokinetics. Among these, the CYP3A4 *22 and POR *28 SNPs are the most promising. Individuals carrying the CYP3A4 *22 T-variant allele have a lower tacrolimus dose requirement than individuals with the CYP3A4* 22 CC genotype and this effect appears to be independent of CYP3A5 genotype status. Individuals carrying the POR *28 T-variant allele have a higher tacrolimus dose requirement than POR *28 CC homozygotes but this association was only found in CYP3A5-expressing individuals. Other, less well-defined SNPs have been inconsistently associated with tacrolimus dose requirement. It is envisaged that in the future, algorithms incorporating clinical, demographic and genetic variables will be developed that will aid clinicians with the determination of the tacrolimus starting dose for an individual transplant recipient. Such an approach may limit early tacrolimus under-exposure and toxicity. With regard to tacrolimus pharmacodynamics, no strong genotype–phenotype relationships have been identified. Certain SNPs associate with rejection risk but these observations await replication. Likewise, the genetic basis of tacrolimus-induced toxicity remains unclarified. SNPs in the genes encoding for the drug transporter ABCB1 and the CYP3A enzymes may relate to chronic nephrotoxicity but findings have been inconsistent. No genetic markers reliably predict new-onset diabetes mellitus after transplantation, hypertension or neurotoxicity. The CYP3A5 *1 SNP is currently the most promising biomarker for tailoring tacrolimus treatment. However, before CYP3A5 genotyping is incorporated into the routine clinical care of transplant recipients, prospective clinical trials are needed to determine whether such a strategy improves patient outcomes. The role of pharmacogenetics in tacrolimus pharmacodynamics should be explored further by the study of intra-lymphocyte and tissue tacrolimus concentrations.

ObjectiveIntestinal barrier loss is a Crohn’s disease (CD) risk factor. This may be related to increased expression and enzymatic activation of myosin light chain kinase 1 (MLCK1), which increases intestinal paracellular permeability and correlates with CD severity. Moreover, preclinical studies have shown that MLCK1 recruitment to cell junctions is required for tumour necrosis factor (TNF)-induced barrier loss as well as experimental inflammatory bowel disease progression. We sought to define mechanisms of MLCK1 recruitment and to target this process pharmacologically.DesignProtein interactions between FK506 binding protein 8 (FKBP8) and MLCK1 were assessed in vitro. Transgenic and knockout intestinal epithelial cell lines, human intestinal organoids, and mice were used as preclinical models. Discoveries were validated in biopsies from patients with CD and control subjects.ResultsMLCK1 interacted specifically with the tacrolimus-binding FKBP8 PPI domain. Knockout or dominant negative FKBP8 expression prevented TNF-induced MLCK1 recruitment and barrier loss in vitro. MLCK1-FKBP8 binding was blocked by tacrolimus, which reversed TNF-induced MLCK1-FKBP8 interactions, MLCK1 recruitment and barrier loss in vitro and in vivo. Biopsies of patient with CD demonstrated increased numbers of MLCK1-FKBP8 interactions at intercellular junctions relative to control subjects.ConclusionBinding to FKBP8, which can be blocked by tacrolimus, is required for MLCK1 recruitment to intercellular junctions and downstream events leading to immune-mediated barrier loss. The observed increases in MLCK1 activity, MLCK1 localisation at cell junctions and perijunctional MLCK1-FKBP8 interactions in CD suggest that targeting this process may be therapeutic in human disease. These new insights into mechanisms of disease-associated barrier loss provide a critical foundation for therapeutic exploitation of FKBP8-MLCK1 interactions.

The physical distance, or proximity, between molecules often directs biological events. The development of membrane-permeable small molecules that reversibly regulate proximity has enabled advances in fields such as synthetic biology, signal transduction, transcription, protein degradation, epigenetic memory, and chromatin dynamics. This “induced proximity” can also be applied to the development of new therapeutics. Stanton et al. review the wide range of advances and speculate on future applications of this fundamental approach. Science , this issue p. eaao5902 Proximity, or the physical closeness of molecules, is a pervasive regulatory mechanism in biology. For example, most posttranslational modifications such as phosphorylation, methylation, and acetylation promote proximity of molecules to play deterministic roles in cellular processes. To understand the role of proximity in biologic mechanisms, chemical inducers of proximity (CIPs) were developed to synthetically model biologically regulated recruitment. Chemically induced proximity allows for precise temporal control of transcription, signaling cascades, chromatin regulation, protein folding, localization, and degradation, as well as a host of other biologic processes. A systematic analysis of CIPs in basic research, coupled with recent technological advances utilizing CRISPR, distinguishes roles of causality from coincidence and allows for mathematical modeling in synthetic biology. Recently, induced proximity has provided new avenues of gene therapy and emerging advances in cancer treatment.

Drug synergy allows a therapeutic effect to be achieved with lower doses of component drugs. Drug synergy can result when drugs target the products of genes that act in parallel pathways (‘specific synergy’). Such cases of drug synergy should tend to correspond to synergistic genetic interaction between the corresponding target genes. Alternatively, ‘promiscuous synergy’ can arise when one drug non‐specifically increases the effects of many other drugs, for example, by increased bioavailability. To assess the relative abundance of these drug synergy types, we examined 200 pairs of antifungal drugs in S. cerevisiae . We found 38 antifungal synergies, 37 of which were novel. While 14 cases of drug synergy corresponded to genetic interaction, 92% of the synergies we discovered involved only six frequently synergistic drugs. Although promiscuity of four drugs can be explained under the bioavailability model, the promiscuity of Tacrolimus and Pentamidine was completely unexpected. While many drug synergies correspond to genetic interactions, the majority of drug synergies appear to result from non‐specific promiscuous synergy. Two types of drug synergy, genetic and promiscuous, are explored in S. cerevisiae . The results suggest that promiscuous synergy predominates, and that propensity to synergize is an intrinsic drug property with the potential to accelerate the search for synergistic drug combinations. Synopsis Two types of drug synergy, genetic and promiscuous, are explored in S. cerevisiae . The results suggest that promiscuous synergy predominates, and that propensity to synergize is an intrinsic drug property with the potential to accelerate the search for synergistic drug combinations. Discovered 37 synergistic interactions among antifungal chemicals Promiscuous synergy is the predominant form of drug synergy Rate of synergy is an intrinsic property of drugs that can guide searches for drug synergy

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.