Discovery of Monovalent Direct Degraders of BRD4 that Act via the Recruitment of DCAF11.

Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extraterminal protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent antitumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent antitumor activity in vivo.

Discovery of Monovalent Direct Degraders of BRD4 That Act Via the Recruitment of DCAF11.

Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extra-terminal (BET) protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent anti-tumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent anti-tumor activity in vivo.

Antivesiculation and Complete Unbinding of Tail-Tethered Lipids.

We report the effect of tail-tethering on vesiculation and complete unbinding of bilayered membranes. Amphiphilic molecules of a bolalipid, resembling the tail-tethered molecular structure of archaeal lipids, with two identical zwitterionic phosphatidylcholine headgroups self-assemble into a large flat lamellar membrane, in contrast to the multilamellar vesicles (MLVs) observed in its counterpart, monopolar nontethered zwitterionic lipids. The antivesiculation is confirmed by small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cyro-TEM). With the net charge of zero and higher bending rigidity of the membrane (confirmed by neutron spin echo (NSE) spectroscopy), the current membrane theory would predict that membranes should stack with each other (aka "bind") due to dominant van der Waals attraction, while the outcome of the nonstacking ("unbinding") membrane suggests that the theory needs to include entropic contribution for the nonvesicular structures. This report pioneers an understanding of how the tail-tethering of amphiphiles affects the structure, enabling better control over the final nanoscale morphology.

Artificial intelligence-rationalized balanced PPARα/γ dual agonism resets dysregulated macrophage processes in inflammatory bowel disease.

A computational platform, Boolean network explorer (BoNE), has recently been developed to infuse AI-enhanced precision into drug discovery; it enables invariant Boolean Implication Networks of disease maps for prioritizing high-value targets. Here we used BoNE to query an Inflammatory Bowel Disease (IBD)-map and prioritize a therapeutic strategy that involves dual agonism of two nuclear receptors, PPARα/γ. Balanced agonism of PPARα/γ was predicted to modulate macrophage processes, ameliorate colitis, 'reset' the gene expression network from disease to health. Predictions were validated using a balanced and potent PPARα/γ-dual-agonist (PAR5359) in Citrobacter rodentium- and DSS-induced murine colitis models. Using inhibitors and agonists, we show that balanced-dual agonism promotes bacterial clearance efficiently than individual agonists, both in vivo and in vitro. PPARα is required and sufficient to induce the pro-inflammatory cytokines and cellular ROS, which are essential for bacterial clearance and immunity, whereas PPARγ-agonism blunts these responses, delays microbial clearance; balanced dual agonism achieved controlled inflammation while protecting the gut barrier and 'reversal' of the transcriptomic network. Furthermore, dual agonism reversed the defective bacterial clearance observed in PBMCs derived from IBD patients. These findings not only deliver a macrophage modulator for use as barrier-protective therapy in IBD, but also highlight the potential of BoNE to rationalize combination therapy.

Fusion of Bipolar Tetraether Lipid Membranes Without Enhanced Leakage of Small Molecules.

A major challenge in liposomal research is to minimize the leakage of encapsulated cargo from either uncontrolled passive permeability across the liposomal membrane or upon fusion with other membranes. We previously showed that liposomes made from pure Archaea-inspired bipolar tetraether lipids exhibit exceptionally low permeability of encapsulated small molecules due to their capability to form more tightly packed membranes compared to typical monopolar lipids. Here, we demonstrate that liposomes made of synthetic bipolar tetraether lipids can also undergo membrane fusion, which is commonly accompanied by content leakage of liposomes when using typical bilayer-forming lipids. Importantly, we demonstrate calcium-mediated fusion events between liposome made of glycerolmonoalkyl glycerol tetraether lipids with phosphatidic acid headgroups (GMGTPA) occur without liposome content release, which contrasts with liposomes made of bilayer-forming EggPA lipids that displayed ~80% of content release under the same fusogenic conditions. NMR spectroscopy studies of a deuterated analog of GMGTPA lipids reveal the presence of multiple rigid and dynamic conformations, which provide evidence for the possibility of these lipids to form intermediate states typically associated with membrane fusion events. The results support that biomimetic GMGT lipids possess several attractive properties (e.g., low permeability and non-leaky fusion capability) for further development in liposome-based technologies.

Entropic effects enable life at extreme temperatures.

Maintaining membrane integrity is a challenge at extreme temperatures. Biochemical synthesis of membrane-spanning lipids is one adaptation that organisms such as thermophilic archaea have evolved to meet this challenge and preserve vital cellular function at high temperatures. The molecular-level details of how these tethered lipids affect membrane dynamics and function, however, remain unclear. Using synthetic monolayer-forming lipids with transmembrane tethers, here, we reveal that lipid tethering makes membrane permeation an entropically controlled process that helps to limit membrane leakage at elevated temperatures relative to bilayer-forming lipid membranes. All-atom molecular dynamics simulations support a view that permeation through membranes made of tethered lipids reduces the torsional entropy of the lipids and leads to tighter lipid packing, providing a molecular interpretation for the increased transition-state entropy of leakage.

Fluid surface coatings for solid-state nanopores: comparison of phospholipid bilayers and archaea-inspired lipid monolayers.

In the context of sensing and characterizing single proteins with synthetic nanopores, lipid bilayer coatings provide at least four benefits: first, they minimize unwanted protein adhesion to the pore walls by exposing a zwitterionic, fluid surface. Second, they can slow down protein translocation and rotation by the opportunity to tether proteins with a lipid anchor to the fluid bilayer coating. Third, they provide the possibility to impart analyte specificity by including lipid anchors with a specific receptor or ligand in the coating. Fourth, they offer a method for tuning nanopore diameters by choice of the length of the lipid's acyl chains. The work presented here compares four properties of various lipid compositions with regard to their suitability as nanopore coatings for protein sensing experiments: (1) electrical noise during current recordings through solid-state nanopores before and after lipid coating, (2) long-term stability of the recorded current baseline and, by inference, of the coating, (3) viscosity of the coating as quantified by the lateral diffusion coefficient of lipids in the coating, and (4) the success rate of generating a suitable coating for quantitative nanopore-based resistive pulse recordings. We surveyed lipid coatings prepared from bolaamphiphilic, monolayer-forming lipids inspired by extremophile archaea and compared them to typical bilayer-forming phosphatidylcholine lipids containing various fractions of curvature-inducing lipids or cholesterol. We found that coatings from archaea-inspired lipids provide several advantages compared to conventional phospholipids; the stable, low noise baseline qualities and high viscosity make these membranes especially suitable for analysis that estimates physical protein parameters such as the net charge of proteins as they enable translocation events with sufficiently long duration to time-resolve dwell time distributions completely. The work presented here reveals that the ease or difficulty of coating a nanopore with lipid membranes did not depend significantly on the composition of the lipid mixture, but rather on the geometry and surface chemistry of the nanopore in the solid state substrate. In particular, annealing substrates containing the nanopore increased the success rate of generating stable lipid coatings.

The small molecule CA140 inhibits the neuroinflammatory response in wild-type mice and a mouse model of AD.

BACKGROUND: Neuroinflammation is associated with neurodegenerative diseases, including Alzheimer's disease (AD). Thus, modulating the neuroinflammatory response represents a potential therapeutic strategy for treating neurodegenerative diseases. Several recent studies have shown that dopamine (DA) and its receptors are expressed in immune cells and are involved in the neuroinflammatory response. Thus, we recently developed and synthesized a non-self-polymerizing analog of DA (CA140) and examined the effect of CA140 on neuroinflammation. METHODS: To determine the effects of CA140 on the neuroinflammatory response, BV2 microglial cells were pretreated with lipopolysaccharide (LPS, 1 µg/mL), followed by treatment with CA140 (10 µM) and analysis by reverse transcription-polymerase chain reaction (RT-PCR). To examine whether CA140 alters the neuroinflammatory response in vivo, wild-type mice were injected with both LPS (10 mg/kg, intraperitoneally (i.p.)) and CA140 (30 mg/kg, i.p.), and immunohistochemistry was performed. In addition, familial AD (5xFAD) mice were injected with CA140 or vehicle daily for 2 weeks and examined for microglial and astrocyte activation. RESULTS: Pre- or post-treatment with CA140 differentially regulated proinflammatory responses in LPS-stimulated microglia and astrocytes. Interestingly, CA140 regulated D1R levels to alter LPS-induced proinflammatory responses. CA140 significantly downregulated LPS-induced phosphorylation of ERK and STAT3 in BV2 microglia cells. In addition, CA140-injected wild-type mice exhibited significantly decreased LPS-induced microglial and astrocyte activation. Moreover, CA140-injected 5xFAD mice exhibited significantly reduced microglial and astrocyte activation. CONCLUSIONS: CA140 may be beneficial for preventing and treating neuroinflammatory-related diseases, including AD.

Evaluation of tetraether lipid-based liposomal carriers for encapsulation and retention of nucleoside-based drugs.

Although liposomal nanoparticles are one of the most versatile class of drug delivery systems, stable liposomal formulation of small neutral drug molecules still constitutes a challenge due to the low drug retention of current lipid membrane technologies. In this study, we evaluate the encapsulation and retention of seven nucleoside analog-based drugs in liposomes made of archaea-inspired tetraether lipids, which are known to enhance packing and membrane robustness compared to conventional bilayer-forming lipids. Liposomes comprised of the pure tetraether lipid generally showed improved retention of drugs (up to 4-fold) compared with liposomes made from a commercially available diacyl lipid. Interestingly, we did not find a significant correlation between the liposomal leakage rates of the molecules with typical parameters used to assess lipophilicity of drugs (such logD or topological polar surface area), suggesting that specific structural elements of the drug molecules can have a dominant effect on leakage from liposomes over general lipophilic character.

Thiol-Triggered Release of Intraliposomal Content from Liposomes Made of Extremophile-Inspired Tetraether Lipids.

Liposomal drug-delivery systems have been used for delivery of drugs to targeted tissues while reducing unwanted side effects. DOXIL, for instance, is a liposomal formulation of the anticancer agent doxorubicin (DOX) that has been used to address problems associated with nonspecific toxicity of free DOX. However, while this liposomal formulation allows for a more-stable circulation of doxorubicin in the body compared to free drug, the efficacy for cancer therapy is reduced in comparison with systemic injections of free drug. A robust liposomal system that can be triggered to release DOX in cancer cells could mitigate problems associated with reduced drug efficacy. In this work, we present a serum-stable, cholesterol-integrated tetraether lipid comprising of a cleavable disulfide bond, {GcGT(S-S)PC-CH}, that is designed to respond to the reducing environment of the cell to trigger the release intraliposomal content upon cellular uptake by cancer cells. A cell viability assay revealed that DOX- loaded liposomes composed of pure GcGT(S-S)PC-CH lipids were ∼20 times more toxic than DOXIL, with an IC50 value comparable to that of free DOX. The low inherent membrane-leakage properties of GcGT(S-S)PC-CH liposomes in the presence of serum, combined with an intracellular triggered release of encapsulated cargo, represents a promising approach for developing improved drug-delivery formulations for the treatment of cancer and possibly other diseases.

Hybrid Lipids Inspired by Extremophiles and Eukaryotes Afford Serum-Stable Membranes with Low Leakage.

This paper presents a new hybrid lipid that fuses the ideas of molecular tethering of lipid tails used by archaea and the integration of cholesterol groups used by eukaryotes, thereby leveraging two strategies employed by nature to increase lipid packing in membranes. Liposomes comprised of pure hybrid lipids exhibited a 5-30-fold decrease in membrane leakage of small ions and molecules compared to liposomes that used only one strategy (lipid tethering or cholesterol incorporation) to increase membrane integrity. Molecular dynamics simulations reveal that tethering of lipid tails and integration of cholesterol both reduce the disorder in lipid tails and time-dependent variance in area per lipid within a membrane, leading to tighter lipid packing. These hybrid lipid membranes have exceptional stability in serum, yet can support functional ion channels, can serve as a substrate for phospholipase enzymes, and can be used for liposomal delivery of molecules into living cells.

Hydrophobic Nanoparticles Reduce the ß-Sheet Content of SEVI Amyloid Fibrils and Inhibit SEVI-Enhanced HIV Infectivity.

Semen-derived enhancer of virus infection (SEVI) fibrils are naturally abundant amyloid aggregates found in semen that facilitate viral attachment and internalization of human immunodeficiency virus (HIV) in cells, thereby increasing the probability of infection. Mature SEVI fibrils are composed of aggregated peptides exhibiting high ß-sheet secondary structural characteristics. Herein, we show that polymers containing hydrophobic side chains can interact with SEVI and reduce its ß-sheet content by ∼45% compared with the ß-sheet content of SEVI in the presence of polymers with hydrophilic side chains, as estimated by polarization modulation-infrared reflectance absorption spectroscopy measurements. A nanoparticle (NP) formulation of this hydrophobic polymer reduced SEVI-mediated HIV infection in TMZ-bl cells by 60% compared with the control treatment. Although these NPs lacked specific amyloid-targeting groups, thus requiring high concentrations to observe biological activity, the use of hydrophobic interactions to alter the secondary structure of amyloids represents a useful approach to neutralizing the SEVI function. These results could, therefore, have general implications in the design of novel materials that can modify the activity of amyloids associated with a variety of other neurological and systemic diseases.

Characterization of drug encapsulation and retention in archaea-inspired tetraether liposomes.

The passive leakage of small molecules across membranes is a major limitation of liposomal drug formulations. Here, we evaluate the leakage of 3 clinically used chemotherapeutic agents (cytarabine, methotrexate and vincristine) encapsulated in liposomes comprised of a synthetic, archaea-inspired, membrane-spanning tetraether lipid. Liposomes comprised of the pure tetraether lipid exhibited superior retention of both a neutrally and positively charged drug (up to an ∼9-fold decrease in the rate of drug leakage) compared to liposomes formed from a commercial diacyl lipid, while exhibiting a similar retention of a negatively charged drug that did not appreciably leak from either type of liposome. We also demonstrate that liposomes made of the archaea-inspired lipid can be used for the delivery of encapsulated small molecules into living cells.

Effects of Lipid Tethering in Extremophile-Inspired Membranes on H(+)/OH(-) Flux at Room Temperature.

This work explores the proton/hydroxide permeability (PH+/OH-) of membranes that were made of synthetic extremophile-inspired phospholipids with systematically varied structural elements. A fluorescence-based permeability assay was optimized to determine the effects on the PH+/OH- through liposome membranes with variations in the following lipid attributes: transmembrane tethering, tether length, and the presence of isoprenoid methyl groups on one or both lipid tails. All permeability assays were performed in the presence of a low concentration of valinomycin (10 nM) to prevent buildup of a membrane potential without artificially increasing the measured PH+/OH-. Surprisingly, the presence of a transmembrane tether did not impact PH+/OH- at room temperature. Among tethered lipid monolayers, PH+/OH- increased with increasing tether length if the number of carbons in the untethered acyl tail was constant. Untethered lipids with two isoprenoid methyl tails led to lower PH+/OH- values than lipids with only one or no isoprenoid tails. Molecular dynamics simulations revealed a strong positive correlation between the probability of observing water molecules in the hydrophobic core of these lipid membranes and their proton permeability. We propose that water penetration as revealed by molecular dynamics may provide a general strategy for predicting proton permeability through various lipid membranes without the need for experimentation.

Effect of Headgroups on Small-Ion Permeability across Archaea-Inspired Tetraether Lipid Membranes.

This paper examines the effects of four different polar headgroups on small-ion membrane permeability from liposomes comprised of Archaea-inspired glycerolmonoalkyl glycerol tetraether (GMGT) lipids. We found that the membrane-leakage rate across GMGT lipid membranes varied by a factor of ≤1.6 as a function of headgroup structure. However, the leakage rates of small ions across membranes comprised of commercial bilayer-forming 1-palmitoyl-2-oleoyl-sn-glycerol (PO) lipids varied by as much as 32-fold within the same series of headgroups. These results demonstrate that membrane leakage from GMGT lipids is less influenced by headgroup structure, making it possible to tailor the structure of the polar headgroups on GMGT lipids while retaining predictable leakage properties of membranes comprised of these tethered lipids.

From solution to in-cell study of the chemical reactivity of acid sensitive functional groups: a rational approach towards improved cleavable linkers for biospecific endosomal release.

pH-Sensitive linkers designed to undergo selective hydrolysis at acidic pH compared to physiological pH can be used for the selective release of therapeutics at their site of action. In this paper, the hydrolytic cleavage of a wide variety of molecular structures that have been reported for their use in pH-sensitive delivery systems was examined. A wide variety of hydrolytic stability profiles were found among the panel of tested chemical functionalities. Even within a structural family, a slight modification of the substitution pattern has an unsuspected outcome on the hydrolysis stability. This work led us to establish a first classification of these groups based on their reactivities at pH 5.5 and their relative hydrolysis at pH 5.5 vs. pH 7.4. From this classification, four representative chemical functions were selected and studied in-vitro. The results revealed that only the most reactive functions underwent significant lysosomal cleavage, according to flow cytometry measurements. These last results question the acid-based mechanism of action of known drug release systems and advocate for the importance of an in-depth structure-reactivity study, using a tailored methodology, for the rational design and development of bio-responsive linkers.

Fluorescent Analogue of Batimastat Enables Imaging of α-Secretase in Living Cells.

The ADAM family of metalloproteases cleave a diverse range of transmembrane substrates, resulting in the release of their soluble ectodomains. This process of protein shedding, termed α-secretase processing, is involved in many facets of both normal and disease related cellular function. While the processing of substrates has been well documented, the regulation and trafficking of the ADAMs are less well understood. Tools that allow for the study of ADAMs under their native environment will allow for a better understanding of their regulation and activity. Here we describe the design and evaluation of a novel fluorescent analogue of a well-characterized ADAM inhibitor, Batimastat. This probe exhibited similar activity for inhibiting α-secretase processing in cells as did Batimastat. Importantly, this probe specifically labeled ADAMs fluorescently in both fixed and living cells, enabling the possibility to study the trafficking of α-secretase proteins in a dynamic environment.

Cyclohexane Rings Reduce Membrane Permeability to Small Ions in Archaea-Inspired Tetraether Lipids.

Extremophile archaeal organisms overcome problems of membrane permeability by producing lipids with structural elements that putatively improve membrane integrity compared to lipids from other life forms. Herein, we describe a series of lipids that mimic some key structural features of archaeal lipids, such as: 1) single tethering of lipid tails to create fully transmembrane tetraether lipids and 2) the incorporation of small rings into these tethered segments. We found that membranes formed from pure tetraether lipids leaked small ions at a rate that was about two orders of magnitude slower than common bilayer-forming lipids. Incorporation of cyclopentane rings into the tetraether lipids did not affect membrane leakage, whereas a cyclohexane ring reduced leakage by an additional 40 %. These results show that mimicking certain structural features of natural archaeal lipids results in improved membrane integrity, which may help overcome limitations of many current lipid-based technologies.

Spiro Diorthoester (SpiDo), a Human Plasma Stable Acid-Sensitive Cleavable Linker for Lysosomal Release.

pH-sensitive linkers designed to undergo selective hydrolysis at acidic pH compared to physiological pH can be used for selective release of therapeutics selectively at targets and orthoesters have been demonstrated to be good candidates for such linkers. Following an HPLC screening, a Spiro Diorthoester (SpiDo) derivative was identified as a potent acid-labile group for the development of pH-sensitive targeted systems. After incorporation of this linker into activatable FRET-based probe and side-by-side comparison to a well-known alkylhydrazone linker, this SpiDo linker has shown a fast and pH sensitive hydrolysis for mild acidic conditions, a pH sensitive lysosomal hydrolysis, and high stability in human plasma.