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Rapamycin and FK506 are macrocyclic natural products with an extraordinary mode of action—they form binary complexes with FKBP through a shared FKBP-binding domain before forming ternary complexes with their respective targets, 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 that are 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 ischemia 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., Graphical abstract

Rapadocin is a novel rapamycin-inspired polyketide–tetrapeptide hybrid macrocycle that possesses highly potent and isoform-specific inhibitory activity against the human equilibrative nucleoside transporter 1 (hENT1). Rapadocin contains an epimerizable chiral center in phenylglycine and an olefin group, and can thus exist as a mixture of four stereoisomers. Herein, we report the first total synthesis of the four stereoisomers of rapadocin using two different synthetic strategies and the assignment of their structures. The inhibitory activity of each of the four synthetic isomers on both hENT1 and hENT2 was determined. It was found that the stereochemistry of phenylglycine played a more dominant role than the configuration of the olefin in the activity of rapadocin. These findings will guide the future design and development of rapadocin analogs as new modulators of adenosine signaling., Rapadocin is a novel rapamycin-inspired polyketide–tetrapeptide hybrid macrocycle that possesses highly potent and isoform-specific inhibitory activity against the human equilibrative nucleoside transporter 1 (hENT1).

Glucose transporters play an essential role in cancer cell proliferation and survival and have been pursued as promising cancer drug targets. Using microarrays of a library of new macrocycles known as rapafucins, which were inspired by the natural product rapamycin, we screened for new inhibitors of GLUT1. We identified multiple hits from the rapafucin 3D microarray and confirmed one hit as a bona fide GLUT1 ligand, which we named rapaglutin A (RgA). We demonstrate that RgA is a potent inhibitor of GLUT1 as well as GLUT3 and GLUT4, with an IC50 value of low nanomolar for GLUT1. RgA was found to inhibit glucose uptake, leading to a decrease in cellular ATP synthesis, activation of AMP-dependent kinase, inhibition of mTOR signaling, and induction of cell-cycle arrest and apoptosis in cancer cells. Moreover, RgA was capable of inhibiting tumor xenografts in vivo without obvious side effects. RgA could thus be a new chemical tool to study GLUT function and a promising lead for developing anticancer drugs.

The antifungal drug itraconazole was recently found to exhibit potent antiangiogenic activity and has since been repurposed as an investigational anticancer agent. Itraconazole has been shown to exert its antiangiogenic activity through inhibition of the mTOR signaling pathway, but the molecular mechanism of action was unknown. We recently identified the mitochondrial protein VDAC1 as a target of itraconazole and a mediator of its activation of AMPK, an upstream regulator of mTOR. However, VDAC1 could not account for the previously reported inhibition of cholesterol trafficking by itraconazole, which was also demonstrated to lead to mTOR inhibition. In this study, we demonstrate that cholesterol trafficking inhibition by itraconazole is due to direct inhibition of the lysosomal protein NPC1. We further map the binding site of itraconazole to the sterol-sensing domain of NPC1 using mutagenesis, competition with U18666A, and molecular docking. Finally, we demonstrate that simultaneous AMPK activation and cholesterol trafficking inhibition leads to synergistic inhibition of mTOR, endothelial cell proliferation, and angiogenesis.

Arsenic is a cancer chemotherapeutic but hepatotoxicity can be a limiting side effect. O(2)-vinyl 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (V-PROLI/NO) is a nitric oxide (NO) donor prodrug and metabolized by liver cytochromes P450 (CYP450) to release NO. The effects of V-PROLI/NO pretreatment on the toxicity of arsenic (as NaAsO(2)) were studied in a rat liver cell line (TRL 1215). The cells acted upon the prodrug to release NO, as assessed by nitrite levels, in a time-dependent fashion to maximal levels of 8-fold above basal levels. Pretreatment with V-PROLI/NO markedly reduced arsenic cytolethality which was directly related to the level of NO produced by V-PROLI/NO treatment. Cyp1a1 expression was directly related to the level of NO production and to reduced arsenic cytotoxicity. V-PROLI/NO pretreatment markedly reduced arsenic-induced apoptosis and suppressed phosphorylation of JNK1/2. V-PROLI/NO pretreatment facilitated additional increases in arsenic-induced metallothionein, a metal-binding protein important in arsenic tolerance. Thus, V-PROLI/NO protects against arsenic toxicity in rat liver cells, reducing cytolethality, apoptosis and dysregulation of MAPKs, through generation of NO formed after metabolism by liver cell enzymes, possibly including Cyp1a1. CYP450 required for NO production from V-PROLI/NO treatment in the rat and human appears to differ as we have previously studied the ability of V-PROLI/NO to prevent arsenic toxicity in human liver cells where it reduced toxicity apparently through a CYP2E1-mediated metabolic mechanism. None-the-less, it appears that both rat and human liver cells act upon V-PROLI/NO via a CYP450-related mechanism to produce NO and subsequently reduce arsenic toxicity.

V-PYRRO/NO is a well studied nitric oxide (NO) prodrug which has been shown to protect human liver cells from arsenic, acetaminophen, and other toxic assaults in vivo. Its proline-based analogue, V-PROLI/NO, was designed to be a more biocompatible form that decomposes to the naturally occurring metabolites of proline, NO, and glycolaldehyde. Like V-PYRRO/NO, this cytochrome P450-activated prodrug was previously assumed to passively diffuse through the cellular membrane. Using (14)C-labeled proline in a competition assay, we show that V-PROLI/NO is transported through proline transporters into multiple cell lines. A fluorescent NO-sensitive dye (DAF-FM diacetate) and nitrite excretion indicated elevated intracellular NO release after metabolism over V-PYRRO/NO. These results also allowed us to predict and design a more permeable analogue, V-SARCO/NO. We report a proline transporter-based strategy for the selective transport of NO prodrugs that may have enhanced efficacy and aid in development of further NO prodrugs with increased permeability.

We report the stabilization of the nitric oxide (NO) prodrugs and anticancer lead compounds, PABA/NO (O(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate) and "Double JS-K" 1,5-bis-{1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diol-2-ato}-2,4-dinitrobenzene, through their incorporation into polymer-protected nanoparticles. The prodrugs were formulated in block copolymer-stabilized nanoparticles with sizes from 220 to 450 nm by a novel rapid precipitation process. The block copolymers, with polyethylene glycol (PEG) soluble blocks, provide a steric barrier against NO prodrug activation by glutathione. Too rapid activation and NO release has been a major barrier to effective administration of this class of compounds. The nanoparticle stabilized PABA/NO are protected from attack by glutathione as evidenced by a significant increase in time taken for 50% decomposition from 15 min (unformulated) to 5 h (formulated); in the case of Double JS-K, the 50% decomposition time was extended from 4.5 min (unformulated) to 40 min (formulated). The more hydrophobic PABA/NO produced more stable nanoparticles and correspondingly more extended release times in comparison with Double JS-K. The hydrophobic blocks of the polymer were either polystyrene or polylactide. Both blocks produced nanoparticles of approximately the same size and release kinetics. This combination of PEG-protected nanoparticles with sizes appropriate for cancer targeting by enhanced permeation and retention (EPR) and delayed release of NO may afford enhanced therapeutic benefit.

An improved synthesis of V-PROLI/NO, a cytochrome P450-activated nitric oxide (NO) prodrug, in an overall yield of 26% in four steps from prolinol is reported; the previously published yield of this transformation was 1%. Using this revised strategy, the sarcosine analogue (14) of V-PROLI/NO was prepared. Finally, the methyl ester of V-PROLI/NO (15) was found to be an esterase-activated prodrug form of V-PROLI/NO.

The reported synthesis of O 2 -vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), a hepatoprotective agent, was cumbersome and limited by a poor overall yield of 4% from sodium 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (PYRRO/NO). We report an improved synthesis of V-PYRRO/NO in two steps with a significantly higher overall yield of 40% from PYRRO/NO. Using this protocol, a number of structural analogues of V-PYRRO/NO were prepared in good yields.

Improved therapies are needed for non-small cell lung cancer. Diazeniumdiolate-based nitric oxide (NO)–releasing prodrugs are a growing class of promising NO-based therapeutics. Recently, we have shown that O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, 1) is effective against non-small cell lung cancer (NSCLC) cells in culture and in vivo. Here we report mechanistic studies with compound 1 and its homopiperazine analogue, and structural modification of these into more stable prodrugs. Compound 1 and its homopiperazine analogue were potent cytotoxic agents against NSCLC cells in vitro and in vivo, concomitant with activation of the SAPK/JNK stress pathway and upregulation of its downstream effector ATF3. Apoptosis followed these events. An aryl-substituted analogue, despite extended half-life in the presence of glutathione, did not activate JNK or have anti-tumor activity. The data suggest that rate of reactivity with glutathione and activation of JNK/ATF3 are determinants of cancer cell killing by these prodrugs.

GlcNAc-PROLI/NO prodrugs that are activated by N-acetylglucosaminidase to release nitric oxide (NO) are described. A classical acid−amine coupling is used to bifunctionalize these PROLI/NO prodrugs, which on activation generate up to 4 mol of NO, a peptide residue, and an N-acetylglucosamine residue. Many of the prodrugs synthesized are efficient sources of intracellular NO.