Amphotericin B (AmB) is a prototypical small molecule natural product that can form ion channels in living eukaryotic cells and has remained refractory to microbial resistance despite extensive clinical utilization in the treatment of life-threatening fungal infections for more than half a century. It is now widely accepted that AmB kills yeast primarily via channel-mediated membrane permeabilization. Enabled by the iterative cross-coupling-based synthesis of a functional group deficient derivative of this natural product, we have discovered that channel formation is not required for potent fungicidal activity. Alternatively, AmB primarily kills yeast by simply binding ergosterol, a lipid that is vital for many aspects of yeast cell physiology. Membrane permeabilization via channel formation represents a second complementary mechanism that further increases drug potency and the rate of yeast killing. Collectively, these findings (i) reveal that the binding of a physiologically important microbial lipid is a powerful and clinically validated antimicrobial strategy that may be inherently refractory to resistance, (ii) illuminate a more straightforward path to an improved therapeutic index for this clinically vital but also highly toxic antifungal agent, and (iii) suggest that the capacity for AmB to form proteinlike ion channels might be separable from its cytocidal effects.small molecules | protein-like functions | N-methyliminodiacetic acid boronates

Amphotericin B is the archetype for small molecules that form transmembrane ion channels. However, despite extensive study for more than five decades, even the most basic features of this channel structure and its contributions to the antifungal activities of this natural product have remained unclear. We herein report that a powerful series of functional group-deficient probes have revealed many key underpinnings of the ion channel and antifungal activities of amphotericin B. Specifically, in stark contrast to two leading models, polar interactions between mycosamine and carboxylic acid appendages on neighboring amphotericin B molecules are not required for ion channel formation, nor are these functional groups required for binding to phospholipid bilayers. Alternatively, consistent with a previously unconfirmed third hypothesis, the mycosamine sugar is strictly required for promoting a direct binding interaction between amphotericin B and ergosterol. The same is true for cholesterol. Synthetically deleting this appendage also completely abolishes ion channel and antifungal activities. All of these results are consistent with the conclusion that a mycosamine-mediated direct binding interaction between amphotericin B and ergosterol is required for both forming ion channels and killing yeast cells. The enhanced understanding of amphotericin B function derived from these synthesis-enabled studies has helped set the stage for the more effective harnessing of the remarkable ion channel-forming capacity of this prototypical small molecule natural product.

The clinically vital antimycotic agent amphotericin B represents the archetypal example of a channelforming small molecule. The leading model for self-assembly of the amphotericin B channel predicts that C(41) carboxylate and the C(3′) ammonium ions form intermolecular salt bridges/hydrogen bonds that are critical for stability. We herein report a flexible degradative synthesis pathway that enables the removal of either or both of these groups from amphotericin B. We further demonstrate with extensive NMR experiments that deleting these groups does not alter the conformation of the polyene macrolide skeleton. As predicted by the leading model, amphotericin B derivatives lacking the mycosamine sugar that contains the C(3′) ammonium ion are completely inactive against Saccharomyces cerevisiae. However, strikingly -and in strong contradiction with the current model -the amphotericin B derivative lacking the C(41) carboxylate is at least equipotent to the natural product. Collectively, these findings demonstrate that the leading model for the mechanism of action of amphotericin B must be significantly revised -either the C(41) carboxylate is not required for channel formation, or channel formation is not required for antifungal activity.The leading model for the antifungal action of amphotericin B (AmB, 1) involves its selfassembly into a membrane-spanning ion channel. 1 This natural product thus represents a potential prototype for small molecules with the capacity to perform ion channel-like functions in living systems. Efforts to harness this potential and/or improve the notoriously poor therapeutic index of this clinically vital antimycotic 2 would benefit from a molecular-level understanding of this channel activity.Although the evidence that AmB can self-assemble in lipid membranes to form discrete ion conducting channels is strong, 1,3 the molecular architecture of this channel assemblage and its role in antifungal activity remain poorly understood. 4 Despite this, the leading "barrelstave" model 5 is an often cited textbook classic (Fig. 1A). 6 Extensive computer modeling studies predict that this complex is stabilized by a ring of salt bridges 7a and/or hydrogen bonds 7b-c at the channel periphery between oppositely-charged C(41)-carboxylate and C(3′)-

Chemogenetic libraries, collections of well-defined chemical probes, provide tremendous value to biomedical research but require substantial effort to ensure diversity as well as quality of the contents. We have assembled a chemogenetic library by data mining and crowdsourcing institutional expertise. We are sharing our approach, lessons learned, and disclosing our current collection of 4,185 compounds with their primary annotated gene targets (https://github.com/Novartis/MoaBox). This physical collection is regularly updated and used broadly both within Novartis and in collaboration with external partners. ll

The assembly of chemogenetic libraries composed of chemical probes provides tremendous value to biomedical research, but requires substantial effort to ensure diversity as well as quality of the contents. We are assembling a chemogenetic library by data mining and crowdsourcing institutional expertise. We are sharing our methodology, lessons learned, and disclosing our current collection of 4186 compounds with their primary annotated gene targets.

Antibody–drug conjugates (ADCs) are a novel modality that allows targeted delivery of potent therapeutic agents to the desired site. Herein we report our discovery of NAMPT inhibitors as a novel nonantimitotic payload for ADCs. The resulting anti-c-Kit conjugates (ADC-3 and ADC-4) demonstrated in vivo efficacy in the c-Kit positive gastrointestinal stromal tumor GIST-T1 xenograft model in a target-dependent manner.

Small molecules that inhibit the metabolic enzyme NAMPT have emerged as potential therapeutics in oncology. As part of our effort in this area, we took a scaffold morphing approach and identified 3-pyridyl azetidine ureas as a potent NAMPT inhibiting motif. We explored the SAR of this series, including 5 and 6 amino pyridines, using a convergent synthetic strategy. This lead optimization campaign yielded multiple compounds with excellent in vitro potency and good ADME properties that culminated in compound 27.

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