The study and optimization of small molecule function is often impeded by the time-intensive and specialist-dependent process that is typically used to make such compounds. In contrast, general and ...automated platforms have been developed for making peptides, oligonucleotides, and increasingly oligosaccharides, where synthesis is simplified to iterative applications of the same reactions. Inspired by the way natural products are biosynthesized via the iterative assembly of a defined set of building blocks, we developed a platform for small molecule synthesis involving the iterative coupling of haloboronic acids protected as the corresponding N-methyliminodiacetic acid (MIDA) boronates. Here we summarize our efforts thus far to develop this platform into a generalized and automated approach for small molecule synthesis. We and others have employed this approach to access many polyene-based compounds, including the polyene motifs found in >75% of all polyene natural products. This platform further allowed us to derivatize amphotericin B, the powerful and resistance-evasive but also highly toxic last line of defense in treating systemic fungal infections, and thereby understand its mechanism of action. This synthesis-enabled mechanistic understanding has led us to develop less toxic derivatives currently under evaluation as improved antifungal agents. To access more Csp3 -containing small molecules, we gained a stereocontrolled entry into chiral, non-racemic α-boryl aldehydes through the discovery of a chiral derivative of MIDA. These α-boryl aldehydes are versatile intermediates for the synthesis of many Csp3 boronate building blocks that are otherwise difficult to access. In addition, we demonstrated the utility of these types of building blocks in accessing pharmaceutically relevant targets via an iterative Csp3 cross-coupling cycle. We have further expanded the scope of the platform to include stereochemically complex macrocyclic and polycyclic molecules using a linear-to-cyclized strategy, in which Csp3 boronate building blocks are iteratively assembled into linear precursors that are then cyclized into the cyclic frameworks found in many natural products and natural product-like structures. Enabled by the serendipitous discovery of a catch-and-release protocol for generally purifying MIDA boronate intermediates, the platform has been automated. The synthesis of 14 distinct classes of small molecules, including pharmaceuticals, materials components, and polycyclic natural products, has been achieved using this new synthesis machine. It is anticipated that the scope of small molecules accessible by this platform will continue to expand via further developments in building block synthesis, Csp3 cross-coupling methodologies, and cyclization strategies. Achieving these goals will enable the more generalized synthesis of small molecules and thereby help shift the rate-limiting step in small molecule science from synthesis to function.
Multiple human diseases ensue from a hereditary or acquired deficiency of iron-transporting protein function that diminishes transmembrane iron flux in distinct sites and directions. Because other ...iron-transport proteins remain active, labile iron gradients build up across the corresponding protein-deficient membranes. Here we report that a small-molecule natural product, hinokitiol, can harness such gradients to restore iron transport into, within, and/or out of cells. The same compound promotes gut iron absorption in DMT1-deficient rats and ferroportin-deficient mice, as well as hemoglobinization in DMT1- and mitoferrin-deficient zebrafish. These findings illuminate a general mechanistic framework for small molecule–mediated site- and direction-selective restoration of iron transport. They also suggest that small molecules that partially mimic the function of missing protein transporters of iron, and possibly other ions, may have potential in treating human diseases.
Loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) compromise epithelial HCO
and Cl
secretion, reduce airway surface liquid pH, and impair respiratory host ...defences in people with cystic fibrosis
. Here we report that apical addition of amphotericin B, a small molecule that forms unselective ion channels, restored HCO
secretion and increased airway surface liquid pH in cultured airway epithelia from people with cystic fibrosis. These effects required the basolateral Na
, K
-ATPase, indicating that apical amphotericin B channels functionally interfaced with this driver of anion secretion. Amphotericin B also restored airway surface liquid pH, viscosity, and antibacterial activity in primary cultures of airway epithelia from people with cystic fibrosis caused by different mutations, including ones that do not yield CFTR, and increased airway surface liquid pH in CFTR-null pigs in vivo. Thus, unselective small-molecule ion channels can restore host defences in cystic fibrosis airway epithelia via a mechanism that is independent of CFTR and is therefore independent of genotype.
Erythropoietin (EPO) signaling is critical to many processes essential to terminal erythropoiesis. Despite the centrality of iron metabolism to erythropoiesis, the mechanisms by which EPO regulates ...iron status are not well-understood. To this end, here we profiled gene expression in EPO-treated 32D pro-B cells and developing fetal liver erythroid cells to identify additional iron regulatory genes. We determined that FAM210B, a mitochondrial inner-membrane protein, is essential for hemoglobinization, proliferation, and enucleation during terminal erythroid maturation. Fam210b deficiency led to defects in mitochondrial iron uptake, heme synthesis, and iron–sulfur cluster formation. These defects were corrected with a lipid-soluble, small-molecule iron transporter, hinokitiol, in Fam210b-deficient murine erythroid cells and zebrafish morphants. Genetic complementation experiments revealed that FAM210B is not a mitochondrial iron transporter but is required for adequate mitochondrial iron import to sustain heme synthesis and iron–sulfur cluster formation during erythroid differentiation. FAM210B was also required for maximal ferrochelatase activity in differentiating erythroid cells. We propose that FAM210B functions as an adaptor protein that facilitates the formation of an oligomeric mitochondrial iron transport complex, required for the increase in iron acquisition for heme synthesis during terminal erythropoiesis. Collectively, our results reveal a critical mechanism by which EPO signaling regulates terminal erythropoiesis and iron metabolism.
Leigh syndrome is a fatal neurometabolic disorder caused by defects in mitochondrial function. Mechanistic target of rapamycin (mTOR) inhibition with rapamycin attenuates disease progression in a ...mouse model of Leigh syndrome (Ndufs4 knock-out (KO) mouse); however, the mechanism of rescue is unknown. Here we identify protein kinase C (PKC) downregulation as a key event mediating the beneficial effects of rapamycin treatment of Ndufs4 KO mice. Assessing the impact of rapamycin on the brain proteome and phosphoproteome of Ndufs4 KO mice, we find that rapamycin restores mitochondrial protein levels, inhibits signalling through both mTOR complexes and reduces the abundance and activity of multiple PKC isoforms. Administration of PKC inhibitors increases survival, delays neurological deficits, prevents hair loss and decreases inflammation in Ndufs4 KO mice. Thus, PKC may be a viable therapeutic target for treating severe mitochondrial disease.
Deficiencies of protein ion channels underlie many currently incurable human diseases. Robust networks of pumps and channels are usually responsible for the directional movement of specific ions in ...organisms ranging from microbes to humans. We thus questioned whether minimally selective small molecule mimics of missing protein channels might be capable of collaborating with the corresponding protein ion pumps to restore physiology. Here we report vigorous and sustainable restoration of yeast cell growth by replacing missing protein ion transporters with imperfect small molecule mimics. We further provide evidence that this tolerance for imperfect mimicry is attributable to collaboration between the channel-forming small molecule and protein ion pumps. These results illuminate a mechanistic framework for pursuing small molecule replacements for deficient protein ion channels that underlie a range of challenging human diseases.
Mitochondrial diseases represent a spectrum of disorders caused by impaired mitochondrial function, ranging in severity from mortality during infancy to progressive adult-onset disease. Mitochondrial ...dysfunction is also recognized as a molecular hallmark of the biological ageing process. Rapamycin, a drug that increases lifespan and health during normative ageing, also increases survival and reduces neurological symptoms in a mouse model of the severe mitochondrial disease Leigh syndrome. The Ndufs4 knockout (Ndufs4
) mouse lacks the complex I subunit NDUFS4 and shows rapid onset and progression of neurodegeneration mimicking patients with Leigh syndrome. Here we show that another drug that extends lifespan and delays normative ageing in mice, acarbose, also suppresses symptoms of disease and improves survival of Ndufs4
mice. Unlike rapamycin, acarbose rescues disease phenotypes independently of inhibition of the mechanistic target of rapamycin. Furthermore, rapamycin and acarbose have additive effects in delaying neurological symptoms and increasing maximum lifespan in Ndufs4
mice. We find that acarbose remodels the intestinal microbiome and alters the production of short-chain fatty acids. Supplementation with tributyrin, a source of butyric acid, recapitulates some effects of acarbose on lifespan and disease progression, while depletion of the endogenous microbiome in Ndufs4
mice appears to fully recapitulate the effects of acarbose on healthspan and lifespan in these animals. To our knowledge, this study provides the first evidence that alteration of the gut microbiome plays a significant role in severe mitochondrial disease and provides further support for the model that biological ageing and severe mitochondrial disorders share underlying common mechanisms.
Small molecules can powerfully benefit society, but the study and optimization of their function is too often impeded by the time-intensive and specialist-dependent process that is typically used to ...make them. In contrast, general and automated platforms have been developed for peptide, oligonucleotide, and increasingly oligosaccharide synthesis, resulting in on-demand access to these molecules, even for non-specialists. A more generalized and automated approach for making small molecules could similarly help shift the rate limiting step in small molecule science from synthesis to function. Targeting this goal, we have developed a fully automated and increasingly general platform for iterative coupling of boronate building blocks. Analogous to peptide synthesis, the process involves iterative coupling of haloboronic acids protected as the corresponding
N
-methyliminodiacetic acid (MIDA) boronates. This platform has enabled us and other groups to access many polyene natural products, including the polyene motifs in >75% of all polyene natural products. It further allowed us to derivatize and thereby understand the powerful but also highly toxic antifungal natural product amphotericin B, which has led to the development of less toxic derivatives currently under evaluation as drug candidates. We also discovered a stereocontrolled entry into chiral, non-racemic α-boryl aldehydes, which are versatile intermediates for the synthesis of many Csp
3
boronate building blocks that are otherwise difficult to access. We have also expanded the scope of the platform to include Csp
3
-rich, polycyclic molecules using a linear-to-cyclized strategy, in which Csp
3
boronate building blocks are iteratively assembled into linear precursors that are then cyclized into the cyclic frameworks found in many natural products and natural product-like structures. Enabled by the serendipitous discovery of a catch-and-release protocol for generally purifying MIDA boronate intermediates, the platform has been automated. The synthesis of 14 distinct classes of small molecules, including pharmaceuticals, materials, and polycyclic natural products has been achieved using this new synthesis machine. It is anticipated that the scope of small molecules accessible by this platform will continue to expand via further developments in building block synthesis, Csp
3
cross-coupling methodologies, and cyclization strategies.
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