Rapamycin-inspired macrocycles with new target specificity

Abstract

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.

Discovery of a Potent GLUT Inhibitor from a Library of Rapafucins by Using 3D Microarrays

“…To further determine the dependence of RgA on endogenous FKBP, we knocked out three major isoforms of FKBP ( FKBP12 , 51 , and 52 ) using CRISPR‐Cas9 in Jurkat T‐cells . Unlike the ENT1 inhibitor rapadocin, knockout of the three FKBP isoforms showed negligible effect on the sensitivity of cells to RgA (Figure S7). Taken together, these results strongly suggest that the inhibitory activity of RgA is independent of endogenous FKBP.…”

Discovery of a Potent GLUT Inhibitor from a Library of Rapafucins by Using 3D Microarrays

“…The ability of rapafucins to bind FKBP proteins to form a tight complex confers a number of advantages for drug leads, including greater stability, higher intracellular accumulation, larger size and better pharmacokinetic and pharmacodynamic properties than smaller molecules lacking the ability to bind FKBP. 6] We have designed and synthesized a 45 000 compound rapafucin library and identified promising hits against several targets, including a potent and isoform‐specific inhibitor of the human equilibrative nucleoside transporter (hENT)1 that showed in vivo efficacy in an animal model of ischemic kidney reperfusion injury . Given that hENT1 and GLUT belong to the same superfamily of solute carrier transporters, we were prompted to screen the rapafucin library for new GLUT1 inhibitors.…”

“…The highest signal‐to‐background ratio (SBR) was obtained at a PEGMA/DMAEMA ratio of 8:2 (Figure b, Figure S2). Using this optimized diazirine‐containing 3D surface grafted onto a glass slide, we robotically arrayed a rapafucin library containing 3918 individual compounds to the glass. As a comparison, we also arrayed the same 3918 individual rapafucins onto a slide grafted onto a 2D surface displaying the same diazirine as previously described .…”

“…High concentrations of FK506 and rapamycin had negligible effect on the inhibitory activity of RgA in the [ 3 H]‐2DG uptake assay (Figure d). To further determine the dependence of RgA on endogenous FKBP, we knocked out three major isoforms of FKBP ( FKBP12 , 51 , and 52 ) using CRISPR‐Cas9 in Jurkat T‐cells . Unlike the ENT1 inhibitor rapadocin, knockout of the three FKBP isoforms showed negligible effect on the sensitivity of cells to RgA (Figure S7).…”

“…Theh ighest signal-to-background ratio (SBR) was obtained at aPEGMA/DMAEMA ratio of 8:2(Figure 1b,F igure S2). Using this optimized diazirine-containing 3D surface grafted onto aglass slide,werobotically arrayed arapafucin library [7] containing 3918 individual compounds to the glass.A s ac omparison, we also arrayed the same 3918 individual rapafucins onto as lide grafted onto a2 Ds urface displaying the same diazirine as previously described. [9] Once stock solutions of the rapafucin library were arrayed on the 2D or 3D surface and most of solvent carrier was evaporated, the crosslinking reaction was initiated by irradiating the surfaces with 365 nm UV light.…”