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Published in final edited form as: J Am Chem Soc. 2025 Oct 23;147(44):40652–40663. doi: 10.1021/jacs.5c13262

Kupyaphores—Self-Assembling Diisocyanolipopeptide ZnII Ionophores in Mycobacterium tuberculosis ZnII/CuI/II Homeostasis and Antibacterial Effects

Tsung-Yun Wong 1,‖, Sachin Sharma 2,‖, Kritee Mehdiratta 3, Rashmi S Bhosale 4, Kaavya Nimmakayala 5, Randall K Wilharm 6, Arnab Chakraborty 7, Moyosore Orimoloye 8, Qiang Liu 9, Siddhesh S Kamat 10, Valérie C Pierre 11, Rajesh S Gokhale 12, Courtney C Aldrich 13
PMCID: PMC12713621  NIHMSID: NIHMS2118941  PMID: 41128264

Abstract

Mycobacterium tuberculosis (Mtb), the leading cause of infectious disease mortality from a single pathogen, requires essential metal ions to establish infection and persist in the host. Kupyaphores, a suite of recently identified amphiphilic diisocyanolipopeptides, were reported to assist with ZnII acquisition to support a multitude of ZnII-dependent metalloenzymes critical for Mtb’s survival and pathogenicity. However, compared to well-studied FeIII acquisition systems in Mtb, the mechanisms for ZnII acquisition and homeostasis remain virtually unexplored. Herein, we reveal them as novel metal ionophores in Mtb’s metal-fluctuating lipidic niche. A concise modular scalable synthesis was developed to assess the critical features required for activity. Synthetic kupyaphores were structurally and functionally validated, respectively, via LCMS and chemical complementation of kupyaphore-deficient (Δrv0101) Mtb. MS, NMR, and IR evidence demonstrated that kupyaphores complex ZnII as a bidentate ligand. Fluorescence competition data indicated ZnII/CuI/II binding capabilities, by which Mtb entraps excessive metals within o/w-type micelles against host-induced metal intoxication. The inhibition against Gram-positive Staphylococcus aureus and the low human toxicity imply the potential as a novel antibacterial scaffold. Collectively, this work provides insight into the ZnII/CuI/II homeostasis of Mtb and a chemical basis for the development of mechanistic tools, therapeutic conjugates against Mtb, and antibiotics.

Graphical Abstract

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INTRODUCTION

Ionophores, coined in 1968 by Breton Pressman,1 are molecules that possess a hydrophilic moiety to substitute the solvation shell of ions and a hydrophobic exterior to escort ions through a lipidic phase. Ionophores feature relatively comparable ion association and dissociation, a necessity for shuttling ions recurrently across lipid bilayers. In this regard, they differ significantly from metallophores, which chelate their respective metal ions tightly (low KD, kon ≫ koff) and thus require transporters for cell uptake and enzymes to release the metal ion.2 Ionophores discriminate between ions via their hydration energies and coordination preferences, but the flexibility in conformation can further vary the spectrum of ion selectivity among alkali, alkaline earth, and transition metals.3 Mechanically, ionophores can be categorized into carrier type and channel type: the former shields ions and migrates in lipid surroundings, whereas the latter creates a hydrophilic passage for ions to pass through hydrophobic barriers. Most naturally occurring ionophores are metabolites from bacteria, fungi, or plants, instances by scaffolds (Figure 1): depsipeptides/polypeptides (e.g., valinomycin,4 enniatins,5 and gramicidins6), polyketide derivatives (e.g., nonactin,7,8 lasalocid,9,10 calcimycin,11 monensin,12 narasin13 zincophorin,14,15 quercetin,16,17 and amphotericin B18), and terpenoids (e.g., hinokitol19,20 and ferutinin21). The ability of ionophores to alter the transmembrane ion gradient has rendered broad biological and electrochemical applications such as antibiotics22 and ion-selective electrodes.23

Figure 1.

Figure 1.

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Ionophore examples by the mechanism and scaffolds along with transported metals. Reported coordinating atoms are labeled in red. Only cation ionophores are listed.

Many transition metals (e.g., Fe, Zn, Cu, Mn, Ni, and Co) are essential trace elements for bacteria, engaging in the constitution and function of proteins, regulation of oxidative stress, and metabolic processes. The innate immune response to invading bacterial pathogens exploits this vulnerability by limiting the availability of metals to prevent bacteria from establishing infection or conversely intoxicates bacteria through exposure to excessive metals, which is termed “nutritional immunity”.24 To acquire metals, bacteria evolve strategies adapting to metal speciation resulting from the physicochemical properties of the metal and the host manipulations. Mycobacterium tuberculosis (Mtb), the pathogen of tuberculosis (TB) that reclaimed the leading worldwide cause of infectious disease mortality from COVID-19 in 2024, provides a prime instance of iron acquisition from a human host. In a physiological (pH 7.4) aerobic environment, iron predominantly exists as insoluble FeIII (Ksp of Fe(OH)3 ≈ 10−38), while ferric metalloproteins (e.g., transferrin, lactoferrin, and ferritin) further restrict the physiological free FeIII concentration to less than 10−24 M.25 To overcome this limitation, Mtb deploys the iron metallophore mycobactins (log β110 > 30)26 to sequestrate FeIII from the host. Siderophores (sidero-, “iron” in Greek) have also been discovered in other pathogens such as yersiniabactin27 (Yersinia spp., pathogens for plague), petrobactin28 and bacillibactin29 (Bacillus anthracis, anthrax), vibriobactin30 (Vibrio spp., cholera), salmochelin31 (Salmonella spp., typhoid), and enterobactin32,33 (Escherichia coli, gastrointestinal infections). ZnII, in contrast, is relatively soluble at a magnitude where the host can withhold it by ZnII-binding proteins or elevate its concentration to poison Mtb in macrophages.34 To date, no isolated structural evidence of the zinc complex has suggested a metallophore paradigm for ZnII acquisition in Mtb, but a series of recently revealed lipopeptides, kupyaphores (kupya-, Sanskrit prefix for metal), has been shown indispensable for Mtb survival in ZnII-deficient milieu.35 While the association of kupyaphores with ZnII awaits investigation, their analogs in other species have been observed bound to CuI/II, which is another relatively soluble metal utilized by the host to intoxicate Mtb via catalyzing the generation of reactive oxygen species and displacing other d-block metals in metalloproteins.36–39

The kupyaphores are structurally fascinating secondary metabolites featuring two β-isocyanoacyl C13-19 chains appended on the two amino groups of an L-ornithyl-L-phenylalaninol core assembled through a hybrid fatty acid–nonribosomal peptide synthetase (NRPS) pathway. The biosynthetic gene cluster encoded by the nrp operon (rv0097–rv0101) is highly conserved among pathogenic mycobacteria and other genera within the Actinobacteria phylum, including Streptomyces, Nocardia, and Rhodococcus (Figure 2). Biosynthesis proceeds in two distinct stages. The first stage mediated by the gene products of rv0097–rv0100 constructs the β-isocyano fatty acid building block and begins with loading of a long-chain α,β-unsaturated fatty acid onto a stand-alone acyl carrier protein (ACP, Rv0100) catalyzed by a fatty acyl AMP ligase (FAAL10, Rv0099). Next, a bifunctional thioesterase (TE, Rv0098) promotes the Michael addition of glycine to an α,β-unsaturated fatty-acyl-ACP. The defining isocyanide is forged through oxidative decarboxylation of the N-carboxymethyl-β-amino fatty acid catalyzed by the FeII α-ketoglutarate-dependent dioxygenase (Rv0097).40,41 While the final stage of biosynthesis awaits biochemical validation, the seven-domain NRPS (encoded by nrp/rv0101) is predicted to build the lipopeptide in the N- to C-terminal direction through the iterative condensation of the β-isocyano fatty acyl-ACP starter units with the first extender unit L-ornithine; elongate the peptide with L-phenylalanine; and catalyze the reductive release of the nascent lipopeptide thioester as the corresponding alcohol. The kupyaphore structure has been assigned through an elegant combination of retrobiosynthetic analysis, accurate MS/MS fragmentation data, and in vitro reconstitution of the first stage of biosynthesis. The configuration at C-3 of the β-isocyanide in kupyaphores is tentatively assigned as 3R based on the diisocyanide SF2768’s lysine congener encoded by homologous BGCs from Streptomyces spp.42

Figure 2.

Figure 2.

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Biosynthesis, purported role of kupyaphores in M. tuberculosis, and related diisocyanides in Actionobacteria. The chain length of incorporated fatty acids confers different extents “lipo-” to these NRPS products. Phagocytosed M. tuberculoss replicates in host macrophages. Protein engagement in transport across M. tuberculosis membrane remains unclear.

The nrp-knockout Mtb strain, shown by the Gokhale group, can grow under both conditions of zinc limitation and intoxication after being genetically complemented, suggesting that the NRPS product kupyaphores may contribute to Mtb’s dynamic adaptability to survive in different environments encountered in vivo. On the other hand, Glickman and co-workers demonstrated that the nrp-knockout Mtb strain can grow under copper limitation by chemically complementing with the lysine congener of diisocyanide SF2768.43,44 Their abortive attempt to rescue nrp-knockout Mtb under zinc starvation was possibly because TPEN (log Kf of ZnII-TPEN, 15.4; CuII-TPEN, 20.2)45 cannot selectively create zinc-deficient media, nor can ctpV induction quantitatively verify zinc drainage. Although these results collectively hinted that kupyaphores could involve both metals in early Mtb growth, none provided chemical complementation evidence of authentic kupyaphores. Such lipopeptides, however, are isolated from pellicle biofilms of biosafety-level-3 pathogen Mtb grown at the air–water interface in a mixture form and a scarce amount, which impedes characterization of metal binding and key mechanistic experiments to confirm their role in metal transport.

Herein, we describe a modular scalable synthesis of kupyaphores, confirm the structural assignment, measure metal binding and selectivity, and investigate the impact of the acyl chain length and stereochemistry of the two β-isocyanides on binding behaviors. Complementation of a kupyaphore-deficient Mtb strain with a synthetic standard provides functional validation while confirming kupyaphores as virulence factors required for growth under metal-limited conditions.46 Then, we demonstrate their ability to promote ZnII transport across liposomes, suggesting an ionophore role for the kupyaphores in metal acquisition and transport. Moreover, kupyaphores can retain excess ZnII and CuI/II in the oil phase of lipid droplets, helping Mtb evade the metal intoxication by host immunity. Lastly, we exhibit their potential application as antibacterial agents against Gram-positive bacteria.

RESULTS AND DISCUSSION

Based on the anticipated higher solubility and the simpler lipid metabolites from an even-numbered acyl chain that would ease prospective assays, we initially elected to prepare (3R,3′R)-C12-kupyaphore 1a. Retrosynthetically, we envisioned that kupyaphore 1a could be derived from β-isocyano fatty acid N-hydroxysuccinimide (NHS) ester 2a and L-ornithyl-L-phenylalaninol 3 through chemoselective acylation of the primary amines (Figure 3A). Due to the propensity of β-elimination during nucleophilic isocyanation,47 we opted to prepare secondary isocyanide 2a via classical dehydration of an N-formamide derived from an amine precursor. The requisite β-amino fatty acid 4a can be prepared with nearly complete enantiocontrol using Ellman’s tert-butanesulfinyl auxiliary.48

Figure 3.

Figure 3.

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(A) Retrosynthesis of kupyaphores. (B) Synthesis of C12 kupyaphore 1a. (C) Telescoping synthesis from 7a to 10a and 10a to 2a sharing intermediates shown in (B).

The synthesis commenced with condensation of decanal 5a with Ellman’s (R)-tert-butanesulfinamide auxiliary to yield (R)-tert-butanesulfinyl aldimine 6a in 89% yield exclusively as the E-isomer (Figures 3B and S1). The asymmetric Mannich-type reaction of 6a with the titanium enolate of methyl acetate afforded methyl ester 7a in 85% yield with a diastereomeric ratio of 99:1. Removal of the tert-butylsulfinimide auxiliary was accomplished with methanolic HCl to give β-aminododecanoic methyl ester 8a quantitatively. Next, formylation of 8a with acetic formic anhydride furnished β-formylaminododecanoic methyl ester 9a in 86% yield, which was quantitatively saponified with aqueous LiOH to acid 10a.49 Subsequent esterification of acid 10a and dehydration of formamide with POCl3 provided NHS ester 2a in 65% yield over two steps (Table S1). The dipeptide core of the kupyaphores was prepared by sequential EDCI-mediated coupling of bis-Cbz-L-ornithine with L-phenylalaninol and Pd-mediated hydrogenolysis of the Cbz groups to give 3 in 63% yield. Diacylation of L-ornithyl-L-phenylalaninol 3 with NHS ester 2a afforded (3R,3′R)-C12-kupyaphore 1a in 70% yield, with the remaining mass balance resulting from competitive β-elimination of the isocyanide. Noteworthily, two novel one-pot conversions were further developed in this work: (1) transamidation–saponification from sulfinamide 7a to formamidyl acid 10a and (2) POCl3-mediated esterification–formamide dehydration from 10a to isocyanoester 2a (Figure 3C). Various esters were also one-pot synthesized in comparable yields (Table S2), but we selected the only solid form NHS ester for ease of handling. In summary, this modular and efficient synthesis proceeded in five steps for the longest linear sequence from decanal 5a in a 36% overall yield. We similarly prepared diastereomeric (3S,3′S)–C12-kupyaphore 1b, the most abundant natural congener (3R,3′R)-C18-kupyaphore 1c, and truncated (3R,3′R)-C4-kupyaphore 1d (Schemes S1–2).

We first assessed the ability of the synthetic kupyaphores to rescue the growth of a M. tuberculosis Δrv0101 deletion mutant,50 which was incapable of producing kupyaphores and deficient in biofilms (Figure 4A). The addition of synthetic (3R,3′R)-C12–Kupyaphore 1a fully rescued the growth (Figure 4B) of Δrv0101 M. tuberculosis by day 10 in Sauton’s medium depleted of divalent metals with Chelex-100 resin, while natural long-chain (3R,3′R)-C18-kupyaphore 1c only partially rescued growth, potentially caused by its limited solubility. To provide more direct structural confirmation, we compared synthetic (3R,3′R)-C18-kupyaphore 1c with a freshly isolated extract of natural kupyaphores obtained from wild-type M. tuberculosis pellicle biofilms by reversed-phase LC–MS and both synthetic and natural kupyaphores eluted at 19.1 min (Figure 4C). The chemical complementation studies and analytical characterization of the synthetic kupyaphores are consistent with their structural assignment by Mehdiratta and co-workers.35

Figure 4.

Figure 4.

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(A) 5-week biofilm cultures of wild-type Mtb and Δrv0101 Mtb. The latter biofilm was fragile, thin, and absent in the reticular pattern. (B) Chemical complementation of M. tuberculosis Δrv0101 strain with kupyaphores. Growth curves for WT and Δrv0101 deletion mutants complemented with or without 200 nM 1a or 1c measured by the optical density at 600 nm (OD600) in Chelex-processed Sauton’s medium supplemented with 0.1 μM ZnII. (C) The extracted ion chromatograms of the natural and synthetic C18 kupyaphore 1c at m/z 848.6987 (ΔM ± 5 ppm) on LC–MS (ESI+).

To investigate the metal binding properties of kupyaphores, we initially employed electrospray ionization-mass spectrometry (ESI-MS). Incubation of ZnBr2 with kupyaphore 1a afforded an m/z corresponding to [Zn·1a·(H2O)4]2+ (Figure S2A). However, an attempted Job’s plot of the titration curve rendered a blunt curvature with an inexplicit stoichiometry. To complement these studies, we examined the NMR spectrum of kupyaphore 1a titrated with ZnBr2. The α-H of isocyanide (δ 4.0 ppm) shifted downfield over the addition of 1.0 equiv of ZnBr2 (Figures 5 and S3). Additionally, the diagnostic peak of the isocyanide at δ 156 ppm (13C NMR) and 2143 cm−1 (IR) shifted as 1 equiv of ZnBr2 was added (Figures S4 and S5). As for copper, Chen et al. observed both isocyanides of the C14 kupyaphore coordinating to CuI by NMR and two stoichiometries ML1 and ML2 by MS.41 Interestingly, we noticed reduction of CuII upon incubating kupyaphore 1a with CuSO4 (Figure S2B), similar to Wang et al.’s finding on diisocyanide SF2768.51

Figure 5.

Figure 5.

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Stacked plot of 1H NMR spectra showing isocyanide titrated with 1 equiv of ZnBr2.

We next sought to determine metal affinities of the kupyaphores; however, the susceptibility of the isocyanide to elimination and hydrolysis under basic and acidic conditions precluded the use of standard potentiometric methods. Instead, we performed fluorescent competition experiments using metal-selective fluorescent probes (Table S3). C12 and C18 kupyaphores significantly decreased fluorescence by liberating FluoZin-3 (Figure 6A), yet the resulting dissociation constants (KD) varied as the kupyaphores increased. We suspected their surfactant-like structure introduced additional interligand interaction that renders KD dependent on the concentration of kupyaphores. Indeed, it was observed that C12 and C18 kupyaphores 1a and 1b aggregated across the corresponding concentration span using the pyrene I1/I3 method52,53 and formed spherical o/w micelles approximately 102 nm (Figure 6B,C). Cylindrical micelles were also observed (Figure S9). Considering the biofilm likely resulting from kupyaphore micelles losing surface charge over weeks of culturing (Figures 4A and 6C), we finally measured the metal binding affinities above critical micelle concentration (CMC) through a competitive binding experiment by titration of the fluorescent probes into a fixed concentration of 500 μM kupyaphore and 500 nM zinc or other biological relevant metals (Figure 6D,E). Overall, the results suggest that the kupyaphores with long alkyl chains can self-assemble into micelles and that, unique among ionophores, this supramolecular self-assembly is key to the kupyaphore’s affinity for metals. Organization of the lipid tails in the center of the micelles preorganizes the two isocyanides into an efficient metal chelator with the correct bite angle for coordination, thereby entropically favoring metal binding above the CMC. C12 and C18 kupyaphore micelles exhibited modest binding toward ZnII and CuI/II as compared to mycobactins toward FeIII. The configuration of the C-3 isocyanide slightly affects the zinc binding (KD of diastereomer 1b = (1.2 ± 0.1) × 10−4 M, Table S4). The higher copper binding affinities are predictable by the Irving-Williams series but not necessarily reflective of a higher copper association in host cells. Prior to immune response elevating metal concentration in Mtb-containing phagosomes, the metal speciation shows a more restricted availability of CuI/II than ZnII due to other tight-binding ligands (Table S5). No iron binding was observed under the given conditions, which further distinguishes kupyaphores from the mycobactins that Mtb evolved as high-affinity hexadentate iron-chelators to obtain iron from the host (plasma [FeIIIfree] ≈ 10−24 M).54 For CaII and MgII, neither 1a nor 1c showed the displacement of the respective probes in the fluorescent competition binding assay.

Figure 6.

Figure 6.

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(A) Fluorescent competition of kupyaphores against the turn-on type ZnII probe Fluozin-3. (B) Ratiometric pyrene 1:3 method. The fluorescent intensity ratio of vibrionic bands I1/I3 (372/386 nm) decreases if pyrene partitions into a relatively nonpolar microenvironment (e.g., micelle). The concentrations at inflection points were considered as CMCs, 410 μM and 176 μM, respectively, for C12 and C18 kupyaphores. (C) Size distribution, transmission electron microscopy images, and suspensions of the kupyaphore micelle at 0 (top) and 48 h (bottom). The scale bars are 200 nm. (D) Representative curve of Fluozin-3 titrated into a mixture of kupyaphores (500 μM) and 500 nM ZnII or ZnII only (blank). (E) Conditional dissociation constants KD (M) of metal–kupyaphore complexes (n = 3, mean ± s.d.).

We next explored the transport mechanism of the kupyaphores. Metallophores universally cooperate with transporter proteins to facilitate import or export across the inner membrane of the bacterial cell envelope. Gram-negative pathogens and Mtb possess an additional set of transporters for their outer membrane and adaptor proteins to assist with transport through the periplasm. While rv0096, the first gene in the nrp operon, encodes a PPE1 protein belonging to a family of PE/PPE proteins, some of which are associated with nutrient uptake across the outer membrane,55 the role of PPE1 has not been functionally characterized. Additionally, the prominent zinc importer ZnuABC that has been proven to be a virulence factor in many bacteria (e.g., E. coli, Salmonella enterica, Campylobacter jejuni, and Y. Pestis), it is functionally unclear in Mtb for missing some conserved transmembrane domains in ZnuB and lacking the entire ATPase ZnuC.56–61 The resemblance between kupyaphores and membrane lipids led us to hypothesize that kupyaphores could self-serve as a transporter. To evaluate this hypothesis, kupyaphores were added to Fluozin-3-encapsulating 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes in zinc solution (Figure 7A).16,62 The fluorescence significantly increased after the addition of kupyaphores (in mol % of DOPC), indicating the transport of the external ZnII into the liposome (Figure 7B). Most ZnII concentrations reached plateaus despite a minor background ZnII influx, suggesting a bidirectional transport with a ZnII efflux (Figure 7C).

Figure 7.

Figure 7.

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(A) Schematic of liposome experiments. High-affinity membrane impermeant Fluozin-3 salt surrogates ZnII deposit (e.g., ZnII demanding proteins in nascent Mtb). Extravesicular zinc was 20 μM. (B) Time-course change of normalized fluorescence intensity. Sodium lauryl sulfate and additional ZnBr2 were added at 90 min to lyse liposome and to secure Fmax. (C) Intravesicular zinc concentrations vs time. (D) Epifluorescence images showing the significant turn-on of intravesicular Fluozin-3 after adding kupyaphores and the integrity of liposome before lysis. Scale bars are 50 μm. (E) Apparent initial rates of zinc transport vs kupyaphore concentrations. (F) Proposed machinery of kupyaphores facilitating ZnII traversal across outer and inner Mtb membranes.

Kupyaphores (0.1–5 mol %) enhanced the responsiveness of lipid bilayers to extravesicular ZnII, thus enhancing influx more than efflux (Δkin > Δkout) and rendering higher intravesicular ZnII concentrations at equilibrium. With higher kupyaphore concentrations, the competition against Fluozin-3 for ZnII became noticeable and led to a decline in fluorescence after 40 min (5 and 10 mol %) and a reduction in ZnII concentration at 90 min (5 > 10 mol %). Fluorescent images showed that Fluozin-3 was intravesically turned on after the addition of kupyaphores and that the liposome remained integral before lysis (Figure 7D). The initial ZnII transport rates were positively correlated to kupyaphore concentrations (Figure 7E). We supposed that kupyaphores behave as an ionophore, facilitating ZnII transport according to its gradient (Figure 7F). Initially, extravesicular ZnII are engaged by kupyaphores that are embedded or partially inserted (as mycobactins63) in the outer leaflet (Figure 7F, I). Both modes proceed to coordination in a U-shaped conformation stabilized by attractive van der Waals interactions in the lipid surroundings (Figure 7F, II). By subsequent lipid flip-flop,64,65 the complex transverses to the bilayer’s inner leaflet and unloads ZnII to a low zinc environment (Figure 7F, III and IV). This bilayer model could account for the longitudinal diffusion of ZnII–kupyaphore across the inner phospholipid membrane, obviating the need for a second set of transporters and could even promote transport across the periplasmic space. The attempt to encapsulate CuI/II indicator Phen Green SK was, however, relatively challenging since it can penetrate the lipid bilayer, but kupyaphores may also act as a CuI/II ionophore albeit likely less efficiently at the same metal concentration (lower KD, thus lower koff/kon). In short, kupyaphores embody a new class of ZnII (and perhaps CuI/II) carrier-type ionophores, by which Mtb procures essential metals to proliferate in macrophages, especially in the incipient TB infection.

As tuberculosis progresses, Mtb can program macrophages to generate a variety of amphiphilic and neutral lipids that provoke the formation of lipid droplets66–68 (LDs)—energy-storing micellar organelles in the macrophage phagosome and cytoplasm (Figure 8A). Meanwhile, such pathogenesis elicits host immune responses including elevating intraphagosomal copper and zinc to poison Mtb.34,69,70 We speculated that secreted kupyaphores are accommodated by LDs and endow them with metal binding capacity. To test this assumption, we mixed the above-CMC kupyaphores with LDs of DOPC and triglyceride oleate (TG). Consequently, we observed the dominant particle diameter shifting from 102 to 104 nm, suggesting that kupyaphore micelles merged into LDs (Figure S9). The exclusive deposition of lipophilic dyes throughout the interior of vesicles further confirmed the o/w micelle system (Figure 8B). Subsequently, we examined the ability of kupyaphore-containing LDs to retain metals (Figure 8C). Both ZnII and CuII were largely withheld by the mixed LDs, whereas no significant difference was shown between the C12 and C18 kupyaphores (Figure 8D). Intriguingly, high-triacylglycerol LDs exhibited a lower metal concentration in the dialysate (the aqueous continuous phase), inferring that the metal–kupyaphore complex partitions into not only amphiphilic lipids but also neutral lipids of LDs, thereby further reducing the metal concentrations at equilibrium (Figure 8E). Zinc and copper resistance of Mtb has been considered a critical factor to successfully infect and persist in hosts, yet contemporary counter mechanisms surround mycobacterial metallothionien (MymT71) and exporters (e.g., ZnII: CtpC;34 CuII: CtpV,72 MctB38) that regulate metal ions in the aqueous phase. Our results, nonetheless, suggested the entrapment of excessive phagosomal metals in the oil phase of kupyaphore LDs, which is a spatially and thermodynamically distinctive strategy of Mtb to evade metal intoxication. Conceivably, highly chemoselective isocyanide-based bioorthogonal reactions (e.g., [4 + 1] cycloaddition with 1,2,4,5-tetrazines73,74) may enable biolabeling of kupyaphores for further in vivo investigation or even novel antitubercular agents by interfering with kupyaphore-mediated metal homeostasis.

Figure 8.

Figure 8.

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(A) Lipid droplet distribution and involved lipids. The figure also includes a characteristic later-stage structure, granuloma, the lipid-rich center of which, caseum, contains debris and Mtb released from necrotized foamy macrophages. (B) Epifluorescence images of the kupyaphore-containing lipid droplet stained with Oil Red O (left) and Nile Red (right). The scale bars are 150 μm. (C) Metal entrapment by incubating the lipid droplet with ZnII or CuII (2.5 μM) on a rotator. (D) Determination of metal concentrations in droplet-free dialysates (molecular weight cutoff: 10000) with fluorescent probes. Molar ratios with respect to DOPC, 2.5 mM, were indicated below (n = 3, mean ± s.d.; nonsignificant (ns), p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001). (E) Proposed mechanism of kupyaphore LDs buffering excessive ZnII and CuII by acting as a phase transfer agent.

Of as much interest as the metal regulation in Mtb unique lipidic dwelling, kupyaphores bear functional features which may exert bioactivity in non-Mtb context. Ever since the first antibiotic isocyanide xanthocillin reported in 1950s, nearly 100 isocyanides borne by myriad scaffolds have been proven antimicrobial.75 Besides, ionophores can potentially disturb the ion equilibrium of microbes, causing futile energy expenditure to maintain ion homeostasis and hence their eventual collapse, e.g., monensin (Coban), lasalocid (Bovatec), narasin (Monteban), and calcimycin (A23187). Additionally, surfactants may cause vesicle rupture by micellizing membrane lipids.76,77 Due to the accessibility to the cytoplasmic membrane, Gram-positive bacteria are generally more susceptible to the three mechanisms as compared to Gram-negative bacteria, fungi, and Mtb. We thus probed the inhibitory activity of kupyahores against methicillin-resistant Staphylococcus aureus (MRSA) as a representative of Gram-positive pathogenic bacteria. C12 kupyaphore 1a showed higher inhibition than C18 kupyaphore 1c, which could be ascribed to a higher monomer concentration (as reflected by a higher CMC77) and partition events into the cytoplasmic membrane (Figure 9A). Higher zinc concentration seemed to enhance the inhibition, while C4 kupyaphore 1d remained effective, suggesting inhibition via hybrid mechanisms (Figure S11). Since the turbidity (OD600) significantly rebounded because of micellization above 100 μM, an aliquot of each suspension culture was streaked onto agar plates to further confirm the inhibition. MRSA grown with kupyaphores perceivably reduced colony formation (Figure 9B). To assess the toxicity of inhibitory concentrations, we performed a colorimetric cell viability assay based on measuring the reductive orange formazan product of yellow tetrazolium salt XTT from metabolically active cells. The LC50 of kupyaphores appeared to be over 100 μM, suggesting the selectivity toward bacteria and the safety in mammalian cells (Figure 9C). In summary, our preliminary data indicate that kupyaphores may serve as a novel antibiotic scaffold.

Figure 9.

Figure 9.

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(A) Inhibition of kupyaphores and known ZnII ionophores against MRSA after 16 h of incubation in Mueller–Hinton broth. Pyrithione and PBT2 serve as positive controls (n = 3). (B) S.aureus-containing culture suspension streaked onto agar plates and allowed additional 16 h growth for colony observation. (C) Cell viability assay with the H460 cell line (n = 2 biological replicates, mean ± s.d.).

CONCLUSION

We developed a concise five-step modular scalable total synthesis of diisocyanide kupyaphores to confirm the stereochemical assignment and impact of key structural features for the function of the critical virulence factors. The lability of the isocyanides coupled with the self-assembly of the lipopeptide proved especially challenging, but we showed that kupyaphores bind zinc and copper in the micellar form that progresses to biofilm morphology observed in Mtb suspension culture. Kupyaphores demonstrate not only a novel carrier-type ZnII ionophore self-transporting ion across the lipid bilayer but also a new paradigm of metal transport besides the long-standing FeIII metallophore in Mtb. Such ionophores, due to Mtb’s unique pathogenesis and lipid-rich niche, can also conversely store excessive zinc and copper in lipid droplets, thereby contributing Mtb’s metal resistance to host-induced metal intoxication. The dual roles of kupyaphores reveal the vulnerability in Mtb’s metal homeostasis and thus a potential therapeutic target in tuberculosis, which remains the globally leading cause of death from a single infectious pathogen. The modular synthesis and functional validation of the kupyaphores provide a cornerstone for further exploration of the ionophores. It highlights the importance of carrier-type ionophores in metal acquisition and homeostasis in bacteria. Transition metal acquisition in bacteria stems from two complementary and distinct systems. Metallophores, so far the prevalent mechanism for transition metal acquisition in the literature, are highly water-soluble ligands that form metal complexes that are also highly soluble. They are characterized by high affinity for their targeted metal ion. The metal complex is subsequently taken up via dedicated transporter proteins as well as enzymes that trigger the release of the metal from the metallophore. This mechanism is favored for weakly soluble metal ions or those tightly chelated in the media. In contrast, ionophores bind metals weakly. Both the ionophore and its metal complex are very poorly soluble in water. The hydrophobicity of the ionophore and its metal complex are key to integration within the lipid bilayer of the membrane and, therefore, to cell uptake. They do not require any dedicated transporter proteins because they are directly inserted in the lipid bilayer. Moreover, because they bind their metal weakly, they do not need any enzyme to release their metal ion. This second mechanism is more appropriate to highly soluble metal ions that are present at relatively high concentrations in the media. We postulate that transition-metal ionophores are likely more common and encourage further reevaluation of certain natural products. This study should inspire the design and synthesis of diagnostic probes,78–80 nanocarriers,81,82 or drug conjugates,83–86 and their inhibition against Gram-positive bacteria additionally exhibits a new framework for antibiotics.

Supplementary Material

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Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.5c13262.

Experimental section: methods, images, tables, NMR spectra for all compounds, characterization data (micelle, liposome, and lipid droplet), and equations for conditional KD calculation (PDF)

Compiled raw data (XLSX)

ACKNOWLEDGMENTS

This research was supported by the National Institutes of Allergy and Infectious Disease (grant: R01AI143784 and R01AI136445) and Swarna Jayanti Fellowship (to SSK) by SERB, Govt. of India (Grant: SB/SJF/2021–22/01). A.C. was supported by a Senior Research Fellowship from CSIR, India. R.S.B. was supported by funding from Department of Biotechnology, India (BT/PR40267/BTIS/137/67/2023). Illustrations were created with BioRender.com. K.N., R.K.W., and V.C.P. acknowledge the support from the National Science Foundation from grant 2203624. The TEM experiments were performed in the Characterization Facility, University of Minnesota, which receives partial support from the NSF through the MRSEC (#Award DMR-2011401) and the NNCI (#Award ECCS-2025124) programs. The widefield fluorescence microscopy experiments were supported by the resources and staff at the University Image Center (#RRID SCR_020997), University of Minnesota.

Footnotes

The authors declare no competing financial interest.

Contributor Information

Tsung-Yun Wong, Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States.

Sachin Sharma, Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States.

Kritee Mehdiratta, Immunometabolism Laboratory, National Institute of Immunology, New Delhi 110067, India.

Rashmi S. Bhosale, Immunometabolism Laboratory, National Institute of Immunology, New Delhi 110067, India

Kaavya Nimmakayala, Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States.

Randall K. Wilharm, Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States

Arnab Chakraborty, Department of Biology, Indian Institute of Science Education and Research, Pune 411008, India.

Moyosore Orimoloye, Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States.

Qiang Liu, Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States.

Siddhesh S. Kamat, Department of Biology, Indian Institute of Science Education and Research, Pune 411008, India

Valérie C. Pierre, Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States; Present Address: Department of Chemistry University of Utah, UT 84112, United States

Rajesh S. Gokhale, Department of Biology, Indian Institute of Science Education and Research, Pune 411008, India

Courtney C. Aldrich, Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States

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Supplementary Materials

Supporting Infomation
NIHMS2118941-supplement-Supporting_Infomation.pdf (5.3MB, pdf)
Compiled raw data
NIHMS2118941-supplement-Compiled_raw_data.xlsx (4.4MB, xlsx)

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