Conjugation Of Small Molecules Research Articles

Does glucose-dependent insulinotropic polypeptide receptor blockade as well as agonism have a role to play in management of obesity and diabetes?

Recent approval of the dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist, tirzepatide, for the management of type 2 diabetes mellitus (T2DM) has reinvigorated interest in exploitation of GIP receptor (GIPR) pathways as a means of metabolic disease management. However, debate has long surrounded the use of the GIPR as a therapeutic target and whether agonism or antagonism is of most benefit in management of obesity/diabetes. This controversy appears to be partly resolved by the success of tirzepatide. However, emerging studies indicate that prolonged GIPR agonism may desensitise the GIPR to essentially induce receptor antagonism, with this phenomenon suggested to be more pronounced in the human than rodent setting. Thus, deliberation continues to rage in relation to benefits of GIPR agonism vs antagonism. That said, as with GIPR agonism, it is clear that the metabolic advantages of sustained GIPR antagonism in obesity and obesity-driven forms of diabetes can be enhanced by concurrent GLP-1 receptor (GLP-1R) activation. This narrative review discusses various approaches of pharmacological GIPR antagonism including small molecule, peptide, monoclonal antibody and peptide-antibody conjugates, indicating stage of development and significance to the field. Taken together, there is little doubt that interesting times lie ahead for GIPR agonism and antagonism, either alone or when combined with GLP-1R agonists, as a therapeutic intervention for the management of obesity and associated metabolic disease.

"Off-Label Use" of the Siderophore Enterobactin Enables Targeted Imaging of Cancer with Radioactive Ti(IV).

The development of inert, biocompatible chelation methods is required to harness the emerging positron emitting radionuclide 45Ti for radiopharmaceutical applications. Herein, we evaluate the Ti(IV)-coordination chemistry of four catechol-based, hexacoordinate chelators using synthetic, structural, computational, and radiochemical approaches. The siderophore enterobactin (Ent) and its synthetic mimic TREN-CAM readily form mononuclear Ti(IV) species in aqueous solution at neutral pH. Radiolabeling studies reveal that Ent and TREN-CAM form mononuclear complexes with the short-lived, positron-emitting radionuclide 45Ti(IV), and do not transchelate to plasma proteins in vitro and exhibit rapid renal clearance in naïve mice. These features guide efforts to target the 45Ti isotope to prostate cancer tissue through the design, synthesis, and evaluation of Ent-DUPA, a small molecule conjugate composed of a prostate specific membrane antigen (PSMA) targeting peptide and a monofunctionalized Ent scaffold. The [45Ti][Ti(Ent-DUPA)]2- complex forms readily at room temperature. In a tumor xenograft model in mice, selective tumor tissue accumulation (8±5 %, n=5), and low off-target uptake in other organs is observed. Overall, this work demonstrates targeted imaging with 45Ti(IV), provides a foundation for advancing the application of 45Ti in nuclear medicine, and reveals that Ent can be repurposed as a 45Ti-complexing cargo for targeted nuclear imaging applications.

“Off‐Label Use” of the Siderophore Enterobactin Enables Targeted Imaging of Cancer with Radioactive Ti(IV)

AbstractThe development of inert, biocompatible chelation methods is required to harness the emerging positron emitting radionuclide 45Ti for radiopharmaceutical applications. Herein, we evaluate the Ti(IV)‐coordination chemistry of four catechol‐based, hexacoordinate chelators using synthetic, structural, computational, and radiochemical approaches. The siderophore enterobactin (Ent) and its synthetic mimic TREN‐CAM readily form mononuclear Ti(IV) species in aqueous solution at neutral pH. Radiolabeling studies reveal that Ent and TREN‐CAM form mononuclear complexes with the short‐lived, positron‐emitting radionuclide 45Ti(IV), and do not transchelate to plasma proteins in vitro and exhibit rapid renal clearance in naïve mice. These features guide efforts to target the 45Ti isotope to prostate cancer tissue through the design, synthesis, and evaluation of Ent‐DUPA, a small molecule conjugate composed of a prostate specific membrane antigen (PSMA) targeting peptide and a monofunctionalized Ent scaffold. The [45Ti][Ti(Ent‐DUPA)]2− complex forms readily at room temperature. In a tumor xenograft model in mice, selective tumor tissue accumulation (8±5 %, n=5), and low off‐target uptake in other organs is observed. Overall, this work demonstrates targeted imaging with 45Ti(IV), provides a foundation for advancing the application of 45Ti in nuclear medicine, and reveals that Ent can be repurposed as a 45Ti‐complexing cargo for targeted nuclear imaging applications.

Abstract 3609: Chimeric cytokine receptors increase memory phenotypes and improve expansion and efficacy of CAR-T cell therapies

Abstract Cytokine activation of T cells can promote T cell proliferation, memory and improve T cell function in driving tumor cell killing. However, systemic administration of cytokines at therapeutic doses is also associated with unacceptable toxicities. To overcome this limitation, we have engineered a system of private cytokine signaling to affect T cell homeostasis, identity, and function. Specifically, we have engineered chimeric cytokine receptors (CCRs) composed of single-chain variable fragments (scFvs) that bind specifically to small molecules such as fluorescein or DOTA. These scFvs are fused to transmembrane and intracellular domains of all members of the common gamma-chain (γ-chain) family of cytokine receptors. CCRs that permit private cytokine signaling were introduced into known CAR-T cell therapies and their impact on CAR-T cell phenotype, function, and anti-tumor activity was tested. First, CCR-containing T cells were tested for activation-induced changes in phenotype and expansion following stimulation with small molecule conjugates in vitro. The most promising results were obtained using either the IL-7Rα- or IL-2Rβ/γ-containing CCR’s. CCR-containing constructs were then transduced into one of two different anti-BCMA CAR-T constructs, ide-cel or cilta-cel, using a lentiviral transduction system. CAR-T cells containing each CCR were then compared with CAR-T cells lacking CCRs for phenotype and function in vitro. Results indicated that engineering the IL-7Rα CCR with both of these CAR-T’s led to increased expansion, cytokine secretion and improved tumor cell killing. Next, CAR-T cells expressing the IL-7Rα CCR were compared to CAR-T cells lacking CCR for efficacy and durability in a mouse model of human multiple myeloma. IL-7Rα CCR-expressing CAR-T cells demonstrated improved long-term tumor control compared to ide-cel and cilta-cel alone. In addition, IL-7Rα CCR-expression increased T cell numbers in blood, spleen and bone marrow of tumor-bearing mice. These increases were particularly evident in the CD8+population of CAR-T cells in vivo. These results demostrate that private cytokine signaling using small-molecule activatable CCRs can improve efficacy and durability of CAR-T cells. We are testing these CCRs in mouse models of human solid tumors to assess their ability to overcome the immunosuppressive microenvironment. Citation Format: John R. Newcomb, Jun Ren, Sakeena Syed, Zachary Caruolo, Lindsay Webb, Craig McKay, Stephanie Kwei, Carl Novina, Fred Mermelstein. Chimeric cytokine receptors increase memory phenotypes and improve expansion and efficacy of CAR-T cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3609.

Peptide‐Directed Attachment of Hydroxylamines to Specific Lysines of IgG Antibodies for Bioconjugations with Acylboronates

AbstractThe role of monoclonal antibodies as vehicles to deliver payloads has evolved as a powerful tool in cancer therapy in recent years. The clinical development of therapeutic antibody conjugates with precise payloads holds great promise for targeted therapeutic interventions. The use of affinity‐peptide mediated functionalization of native off‐the‐shelf antibodies offers an effective approach to selectively modify IgG antibodies with a drug–antibody ratio (DAR) of 2. Here, we report the traceless, peptide‐directed attachment of two hydroxylamines to native IgGs followed by chemoselective potassium acyltrifluoroborate (KAT) ligation with quinolinium acyltrifluoroborates (QATs), which provide enhanced ligation rates with hydroxylamines under physiological conditions. By applying KAT ligation to the modified antibodies, conjugation of small molecules, proteins, and oligonucleotides to off‐the‐shelf IgGs proceeds efficiently, in good yields, and with simultaneous cleavage of the affinity peptide‐directing moiety.

Peptide-Directed Attachment of Hydroxylamines to Specific Lysines of IgG Antibodies for Bioconjugations with Acylboronates.

The role of monoclonal antibodies as vehicles to deliver payloads has evolved as a powerful tool in cancer therapy in recent years. The clinical development of therapeutic antibody conjugates with precise payloads holds great promise for targeted therapeutic interventions. The use of affinity-peptide mediated functionalization of native off-the-shelf antibodies offers an effective approach to selectively modify IgG antibodies with a drug-antibody ratio (DAR) of 2. Here, we report the traceless, peptide-directed attachment of two hydroxylamines to native IgGs followed by chemoselective potassium acyltrifluoroborate (KAT) ligation with quinolinium acyltrifluoroborates (QATs), which provide enhanced ligation rates with hydroxylamines under physiological conditions. By applying KAT ligation to the modified antibodies, conjugation of small molecules, proteins, and oligonucleotides to off-the-shelf IgGs proceeds efficiently, in good yields, and with simultaneous cleavage of the affinity peptide-directing moiety.

Peptide-Drug Conjugates: An Emerging Direction for the Next Generation of Peptide Therapeutics.

Building on recent advances in peptide science, medicinal chemists have developed a hybrid class of bioconjugates, called peptide-drug conjugates, that demonstrate improved efficacy compared to peptides and small molecules independently. In this Perspective, we discuss how the conjugation of synergistic peptides and small molecules can be used to overcome complex disease states and resistance mechanisms that have eluded contemporary therapies because of their multi-component activity. We highlight how peptide-drug conjugates display a multi-factor therapeutic mechanism similar to that of antibody-drug conjugates but also demonstrate improved therapeutic properties such as less-severe off-target effects and conjugation strategies with greater site-specificity. The many considerations that go into peptide-drug conjugate design and optimization, such as peptide/small-molecule pairing and chemo-selective chemistries, are discussed. We also examine several peptide-drug conjugate series that demonstrate notable activity toward complex disease states such as neurodegenerative disorders and inflammation, as well as viral and bacterial targets with established resistance mechanisms.

Small molecule conjugation reduces macrophage uptake and increases in vivo blood circulation of polystyrene nanoparticles

Nanomedicine often failed clinically to show therapeutic efficacy due to reduced particle circulation and enhanced capture by the reticuloendothelial system, including the liver. Developing novel immunomodulatory surface coating can prevent macrophage capture and increase the particle circulation of the nanomedicine, resulting in higher therapeutic efficiency. Herein, we demonstrate the development of immunomodulatory small molecule (RZA15) with triazole functionality using copper-catalyzed click chemistry to conjugate onto spherical polystyrene nanoparticles (NPs) using amide coupling reactions, achieving higher blood circulation and lesser macrophage uptake of the nanoconjugates (NCs). In this work, we evaluated the effectiveness of RZA15 coating for the enhanced circulation of polystyrene NPs of 100 nm size, which is commonly utilized for various drug delivery applications, and compared with poly(ethylene)glycol coatings. Several polystyrene NC formulations were analyzed in vitro in normal and macrophage cells for cell viability and cellular uptake studies. In vitro studies demonstrated lesser macrophage uptake of the NCs following RZA15 coating. Finally, in vivo, blood-circulation, pharmacokinetics, and biodistribution studies were performed in the C57BL/6J mouse model that endorsed the substantial role of RZA15 in reducing liver and spleen capture and results in extended circulation in blood. Coating immunomodulatory small molecules to nanoparticles can severely enhance the potential therapeutic effects of nanomedicine at lower doses in near future.

Mono- and multimeric PSMA-targeting small molecule-thorium-227 conjugates for optimized efficacy and biodistribution in preclinical models

PurposePSMA (prostate-specific membrane antigen) is highly expressed on prostate cancer (PrCa) cells and extensively used as a homing target for PrCa treatment. Most prominently, PSMA-targeting conjugate PSMA-617, carrying a DOTA chelator and labeled with therapeutic radionuclides like beta-emitting lutetium-177 or alpha-emitting actinium-225, has shown clinical activity in PrCa patients. We sought to develop PSMA-targeting small molecule (SMOL) conjugates that show high uptake in PSMA-expressing tumors and fast clearance, and can easily be labeled with the alpha emitter thorium-227 (half-life 18.7 days).MethodsA novel linker motif with improved competition against 3H-PSMA-617 on PSMA-expressing LNCaP cells was identified. A 2,3-hydroxypyridinone chelator modified with carboxyl groups (carboxy-HOPO) with increased hydrophilicity and robust labeling with thorium-227 was developed and allowed the synthesis of mono-, di-, tri-, and tetrameric conjugates. The resulting monomeric and multimeric PSMA SMOL-TTCs (targeted thorium conjugate) were evaluated for cellular binding, internalization, and antiproliferative activity. The in vivo antitumor efficacy of the PSMA SMOL-TTCs was determined in ST1273 and KUCaP-1 PrCa models in mice, and their biodistribution was assessed in cynomolgus monkeys, minipigs, and mice.ResultsThe monomeric and multimeric PSMA SMOL conjugates were readily labeled with thorium-227 at room temperature and possessed high stability and good binding, internalization, and antiproliferative activity in vitro. In vivo, the monomeric, dimeric, and trimeric PSMA SMOL-TTCs showed fast clearance, potent antitumor efficacy, and high uptake and retention in prostate tumors in mice. No major uptake or retention in other organs was observed beyond kidneys. Low uptake of free thorium-227 into bone confirmed high complex stability in vivo. Salivary gland uptake remained inconclusive as mini pigs were devalidated as a relevant model and imaging controls failed in cynomolgus monkeys.ConclusionMonomeric and multimeric PSMA SMOL-TTCs show high tumor uptake and fast clearance in preclinical models and warrant further therapeutic exploration.

Synthesis and in vitro cytotoxic activity of dye-linker-macrocycle conjugates with variable linker length and components

The study investigated the structure–activity relationship of newly synthesized dye-linker-macrocycle (DLM) conjugates and the effect of each component on various biological properties, including cytotoxicity, cellular uptake, intracellular localization, interaction with DNA and photodynamic effects. The conjugates were synthesized by combining 1,8-naphthalimide and thioxanthone dyes with 1,4,7,10-tetraazacyclododecane (cyclen) and 1-aza-12-crown-4 (1A12C4) using alkyl linkers of different lengths. The results revealed significant differences in biological activity among the various series of conjugates. Particularly, 1A12C4 conjugates exhibited notably higher cytotoxicity compared to cyclen conjugates. Conjugation with 1A12C4 proved to be an effective strategy for increasing cellular uptake and cytotoxicity of small-molecule conjugates. In addition, the results highlighted the critical role of linker length in modulating the biological activity of DLM conjugates. It became clear that the choice of each component (dye, macrocycle and linker) could significantly alter the biological activity of the conjugates.

Peptide Hydrogels and Nanostructures Controlling Biological Machinery.

Peptides are versatile building blocks for the fabrication of various nanostructures that result in the formation of hydrogels and nanoparticles. Precise chemical functionalization promotes discrete structure formation, causing controlled bioactivity and physical properties for functional materials development. The conjugation of small molecules on amino acid side chains determines their intermolecular interactions in addition to their intrinsic peptide characteristics. Molecular information affects the peptide structure, formation, and activity. In this Perspective, peptide building blocks, nanostructure formation mechanisms, and the properties of these peptide materials are discussed with the results of recent publications. Bioinstructive and stimuli-responsive peptide materials have immense impacts on the nanomedicine field including drug delivery, cellular engineering, regenerative medicine, and biomedicine.

Foldamer Nanotubes Mediated Label-Free Detection of Protein-Small Molecule Interactions.

Development of miniaturized lab-on-chip devices for the detection of rapid and specific small molecule-protein binding interactions at very low concentrations holds significant importance in drug discovery and biomedical applications. Here, the label-free detection of small molecule-protein interactions is reported on the surface functionalizable nanotubes of α,γ-hybrid peptide helical foldamers using nanoscale capacitance and impedance spectroscopy. The 12-helix conformation of the α,γ-hybrid peptide observed in the single crystals, self-assembled into nanotubes in an aqueous environment with exposed cysteine thiols for small molecule conjugation. The binding of streptavidin to the covalently linked biotin on the surface of nanotubes was detected at the picomolar concentrations. No change in the capacitance and impedance were observed in the absence of either immobilized biotin or protein streptavidin. The functionalizable hybrid peptide nanotubes reported here pave the way for the label-free detection of various small molecule protein interactions at very low concentrations.

Doubly Strapped Zwitterionic NIR‐I and NIR‐II Heptamethine Cyanine Dyes for Bioconjugation and Fluorescence Imaging

AbstractHeptamethine cyanine dyes enable deep tissue fluorescence imaging in the near infrared (NIR) window. Small molecule conjugates of the benchmark dye ZW800‐1 have been tested in humans. However, long‐term imaging protocols using ZW800‐1 conjugates are limited by their instability, primarily because the chemically labile C4′‐O‐aryl linker is susceptible to cleavage by biological nucleophiles. Here, we report a modular synthetic method that produces novel doubly strapped zwitterionic heptamethine cyanine dyes, including a structural analogue of ZW800‐1, with greatly enhanced dye stability. NIR‐I and NIR‐II versions of these doubly strapped dyes can be conjugated to proteins, including monoclonal antibodies, without causing undesired fluorophore degradation or dye stacking on the protein surface. The fluorescent antibody conjugates show excellent tumor‐targeting specificity in a xenograft mouse tumor model. The enhanced stability provided by doubly strapped molecular design will enable new classes of in vivo NIR fluorescence imaging experiments with possible translation to humans.

Doubly Strapped Zwitterionic NIR-I and NIR-II Heptamethine Cyanine Dyes for Bioconjugation and Fluorescence Imaging.

Heptamethine cyanine dyes enable deep tissue fluorescence imaging in the near infrared (NIR) window. Small molecule conjugates of the benchmark dye ZW800-1 have been tested in humans. However, long-term imaging protocols using ZW800-1 conjugates are limited by their instability, primarily because the chemically labile C4'-O-aryl linker is susceptible to cleavage by biological nucleophiles. Here, we report a modular synthetic method that produces novel doubly strapped zwitterionic heptamethine cyanine dyes, including a structural analogue of ZW800-1, with greatly enhanced dye stability. NIR-I and NIR-II versions of these doubly strapped dyes can be conjugated to proteins, including monoclonal antibodies, without causing undesired fluorophore degradation or dye stacking on the protein surface. The fluorescent antibody conjugates show excellent tumor-targeting specificity in a xenograft mouse tumor model. The enhanced stability provided by doubly strapped molecular design will enable new classes of in vivo NIR fluorescence imaging experiments with possible translation to humans.

Kinetic analysis of highly effective triplex formation between a small molecule-peptide nucleic acid conjugate probe and the influenza A virus RNA promoter region at neutral pH.

In order to overcome the pH limitations of triplex-forming peptide nucleic acid (PNA) in binding to double-stranded RNA (dsRNA), we have recently proposed a new design of triplex-forming PNA-based fluorogenic probes that work at neutral pH for sensing the panhandle structure of the influenza A virus (IAV) RNA promoter region. Our strategy is based on the conjugation of a small molecule (DPQ) capable of selectively binding to the internal loop structure with the triplex-forming forced intercalation of thiazole orange (tFIT) probe with natural PNA nucleobases. In this work, the triplex formation of tFIT-DPQ conjugate probes with IAV target RNA at neutral pH was examined by means of a stopped-flow technique UV melting and fluorescence titration experiments. The obtained results revealed that (i) the conjugation strategy is responsible for the observed strong binding affinity due to a very fast association rate constant and a slow dissociation rate constant; (ii) the binding follows a pattern of the DPQ unit binding first to the internal loop region, followed by the tFIT unit binding to the complementary dsRNA region. Our results emphasize the importance of both the tFIT and the DPQ components of the conjugate probe design and revealed an association mechanism for the tFIT-DPQ probe-dsRNA triplex formation towards the IAV RNA at neutral pH.

Chemical biology fluorescent tools for in vitro investigation of the multidrug resistant P-glycoprotein (P-gp) expression in tumor cells.

Selective P-glycoprotein (P-gp)-targeted fluorescent conjugates are desirable tools to investigate the role of P-gp, a protein strongly implicated in mediating multidrug resistance and a major cause of chemotherapy failure. Herein, we report the development of 25 novel fluorescent small-molecule conjugates with varying physicochemical and optical properties, and their biological evaluation in a cell model as P-gp targeted constructs. This investigation revealed relationships between molecular structure and cell behavior and uncovered the capacity of conjugates with varying fluorophores to selectively target P-gp. Sulfocyanine 3 labeled conjugates (5, 10, 24, 29, 34) showed a particular intracellular staining pattern. Other conjugates bearing a boron dipyrromethene (BODIPY) core (3, 8, 13, 22, 27 (BODIPY FL), 12 (BODIPY 564/570) and 4, 9 (BODIPY 650/665)) or a 7-nitrobenz-2-oxa-1,3-diazole (NBD) core (11, 30) showed potential for global P-gp direct detection and quantification. These fluorescent conjugates holds key advantages over existing methods for drug resistance evaluation with regards to P-gp expression and could be used as innovative tools in preclinical assays and clinical diagnosis.

Doxorubicin-isoniazid conjugate regulates immune response and tumor microenvironment to enhance cancer therapy

Immune checkpoint inhibitors (ICIs) represent a new class of immunotherapy drugs, and are used to relieve immune suppression or enhance the immune response through the blockade of checkpoint ligands or receptors. ICIs have achieved great success in clinical cancer treatment. Monoamine oxidase A (MAOA) is a potent immune checkpoint of immunotherapy. Recently, it has been reported that MAOA inhibitors could enhance CD8+ T cell activity by upregulating 5-HT autocrine pathway in T cells. In this study, we synthesized doxorubicin (DOX) and isoniazid (INH, a MAOA inhibitor) conjugates through a pH sensitive hydrazone bond. Results of the in vivo studies showed that DOX-INH could effectively enhance the activity of CD8+ T cells and perform a synergistic anti-tumor effect with PD-L1 small molecular inhibitor (BMS202). In addition, in an orthotopic 4T1 breast cancer model, it was demonstrated that DOX-INH could inhibit the epithelial-mesenchymal transition process by blocking Shh, IL-6, and TGF-β signaling pathways, thereby inhibiting the growth and metastasis of breast cancer. Thus, a simple and effective small molecule conjugate produced by the combination of a chemotherapy drug and a MAOA inhibitor shows broad prospect in cancer therapy.

Photocaged DNA-Binding Photosensitizer Enables Photocontrol of Nuclear Entry for Dual-Targeted Photodynamic Therapy.

Photodynamic therapy (PDT) is a clinically approved cancer treatment that requires a photosensitizer (PS), light, and molecular oxygen─a combination which produces reactive oxygen species (ROS) that can induce cancer cell death. To enhance the efficacy of PDT, dual-targeted strategies have been explored where two photosensitizers are administered and localize to different subcellular organelles. To date, a single small-molecule conjugate for dual-targeted PDT with light-controlled nuclear localization has not been achieved. We designed a probe composed of a DNA-binding PS (Br-DAPI) and a photosensitizing photocage (WinterGreen). Illumination with 480 nm light removes WinterGreen from the conjugate and produces singlet oxygen mainly in the cytosol, while Br-DAPI localizes to nuclei, binds DNA, and produces ROS using one- or two-photon illumination. We observe synergistic photocytotoxicity in MCF7 breast cancer cells, and a reduction in size of three-dimensional (3D) tumor spheroids, demonstrating that nuclear/cytosolic photosensitization using a single agent can enhance PDT efficacy.

Medium-Chain Lipid Conjugation Facilitates Cell-Permeability and Bioactivity.

The majority of bioactive molecules act on membrane proteins or intracellular targets and therefore needs to partition into or cross biological membranes. Natural products often exhibit lipid modifications to facilitate critical molecule-membrane interactions, and in many cases their bioactivity is markedly reduced upon removal of a lipid group. However, despite its importance in nature, lipid-conjugation of small molecules is not commonly used in chemical biology and medicinal chemistry, and the effect of such conjugation has not been systematically studied. To understand the composition of lipids found in natural products, we carried out a chemoinformatic characterization of the "natural product lipidome". According to this analysis, lipidated natural products predominantly contain saturated medium-chain lipids (MCLs), which are significantly shorter than the long-chain lipids (LCLs) found in membranes and lipidated proteins. To study the usefulness of such modifications in probe design, we systematically explored the effect of lipid conjugation on five different small molecule chemotypes and find that permeability, cellular retention, subcellular localization, and bioactivity can be significantly modulated depending on the type of lipid tail used. We demonstrate that MCL conjugation can render molecules cell-permeable and modulate their bioactivity. With all explored chemotypes, MCL-conjugates consistently exhibited superior uptake or bioactivity compared to LCL-conjugates and either comparable or superior uptake or bioactivity to short-chain lipid (SCL)-conjugates. Together, our findings suggest that conjugation of small molecules with MCLs could be a powerful strategy for the design of probes and drugs.

A pH-Driven Small-Molecule Nanotransformer Hijacks Lysosomes and Overcomes Autophagy-Induced Resistance in Cancer.

Smart conversion of supramolecular structures in vivo is an attractive strategy in cancer nanomedicine, which is usually achieved via specific peptide sequences. Here we developed a lysosomal targeting small-molecule conjugate, PBC, which self-assembles into nanoparticles at physiological pH and smartly converts to nanofibrils in lysosomes of tumor cells. Such a transformation mechanically leads to lysosomal dysfunction, autophagy inhibition, and unusual cytoplasmic vacuolation, thus granting PBC a unique anticancer activity as a monotherapy. Importantly, the photo-activated PBC elicits significant phototoxicity to lysosomes and shows enormous advantages in overcoming autophagy-caused treatment resistance frequently occurring in conventional phototherapy. This improved phototherapy achieves a complete cure of oral cancer xenografts upon limited administration. Our work provides a new paradigm for the construction of nonpeptide nanotransformers with biomedical activities.