Targeted protein degradation via intramolecular bivalent glues.

Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)-bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target1-4. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change 'glues' BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4-IBG1-DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.

Functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders.

Targeted protein degradation is a novel pharmacology established by drugs that recruit target proteins to E3 ubiquitin ligases. Based on the structure of the degrader and the target, different E3 interfaces are critically involved, thus forming defined 'functional hotspots'. Understanding disruptive mutations in functional hotspots informs on the architecture of the assembly, and highlights residues susceptible to acquire resistance phenotypes. Here we employ haploid genetics to show that hotspot mutations cluster in substrate receptors of hijacked ligases, where mutation type and frequency correlate with gene essentiality. Intersection with deep mutational scanning revealed hotspots that are conserved or specific for chemically distinct degraders and targets. Biophysical and structural validation suggests that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and widely accessible methodology to characterize small-molecule degraders and associated resistance mechanisms.

Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity.

Bivalent proteolysis-targeting chimeras (PROTACs) drive protein degradation by simultaneously binding a target protein and an E3 ligase and forming a productive ternary complex. We hypothesized that increasing binding valency within a PROTAC could enhance degradation. Here, we designed trivalent PROTACs consisting of a bivalent bromo and extra terminal (BET) inhibitor and an E3 ligand tethered via a branched linker. We identified von Hippel-Lindau (VHL)-based SIM1 as a low picomolar BET degrader with preference for bromodomain containing 2 (BRD2). Compared to bivalent PROTACs, SIM1 showed more sustained and higher degradation efficacy, which led to more potent anticancer activity. Mechanistically, SIM1 simultaneously engages with high avidity both BET bromodomains in a cis intramolecular fashion and forms a 1:1:1 ternary complex with VHL, exhibiting positive cooperativity and high cellular stability with prolonged residence time. Collectively, our data along with favorable in vivo pharmacokinetics demonstrate that augmenting the binding valency of proximity-induced modalities can be an enabling strategy for advancing functional outcomes.

Surface Passivation Method for the Super-repellence of Aqueous Macromolecular Condensates.

Solutions of macromolecules can undergo liquid-liquid phase separation to form droplets with ultralow surface tension. Droplets with such low surface tension wet and spread over common surfaces such as test tubes and microscope slides, complicating in vitro experiments. The development of a universal super-repellent surface for macromolecular droplets has remained elusive because their ultralow surface tension requires low surface energies. Furthermore, the nonwetting of droplets containing proteins poses additional challenges because the surface must remain inert to a wide range of chemistries presented by the various amino acid side chains at the droplet surface. Here, we present a method to coat microscope slides with a thin transparent hydrogel that exhibits complete dewetting (contact angles θ ≈ 180°) and minimal pinning of phase-separated droplets in aqueous solution. The hydrogel is based on a swollen matrix of chemically cross-linked polyethylene glycol diacrylate of molecular weight 12 kDa (PEGDA), and can be prepared with basic chemistry laboratory equipment. The PEGDA hydrogel is a powerful tool for in vitro studies of weak interactions, dynamics, and the internal organization of phase-separated droplets in aqueous solutions.

Stereoselective synthesis of allele-specific BET inhibitors.

Developing stereoselective synthetic routes that are efficient and cost-effective allows easy access to biologically active molecules. Our previous syntheses of allele-selective bumped inhibitors of the Bromo and Extra-Terminal (BET) domain proteins, Brd2, Brd3, Brd4 and BrdT, required a wasteful, late-stage alkylation step and expensive chiral separation. To circumvent these limitations, we developed a route based on stereocontrolled alkylation of an N-Pf protected aspartic acid derivative that was used in a divergent, racemisation-free protocol to yield structurally diverse and enantiopure triazolodiazepines. With this approach, we synthesized bumped thienodiazepine-based BET inhibitor, ET-JQ1-OMe, in five steps and 99% ee without the need for chiral chromatography. Exquisite selectivity of ET-JQ1-OMe for Leu-Ala and Leu-Val mutants over wild-type bromodomain was established by isothermal titration calorimetry and X-ray crystallography. Our new approach provides unambiguous chemical evidence for the absolute stereochemistry of the active, allele-specific BET inhibitors and a viable route that will open wider access to this compound class.

Structure-Based Design of a Macrocyclic PROTAC.

Constraining a molecule in its bioactive conformation via macrocyclization represents an attractive strategy to rationally design functional chemical probes. While this approach has been applied to enzyme inhibitors or receptor antagonists, to date it remains unprecedented for bifunctional molecules that bring proteins together, such as PROTAC degraders. Herein, we report the design and synthesis of a macrocyclic PROTAC by adding a cyclizing linker to the BET degrader MZ1. A co-crystal structure of macroPROTAC-1 bound in a ternary complex with VHL and the second bromodomain of Brd4 validated the rational design. Biophysical studies revealed enhanced discrimination between the second and the first bromodomains of BET proteins. Despite a 12-fold loss of binary binding affinity for Brd4, macroPROTAC-1 exhibited cellular activity comparable to MZ1. Our findings support macrocyclization as an advantageous strategy to enhance PROTAC degradation potency and selectivity between homologous targets.

Structural basis of PROTAC cooperative recognition for selective protein degradation.

Inducing macromolecular interactions with small molecules to activate cellular signaling is a challenging goal. PROTACs (proteolysis-targeting chimeras) are bifunctional molecules that recruit a target protein in proximity to an E3 ubiquitin ligase to trigger protein degradation. Structural elucidation of the key ternary ligase-PROTAC-target species and its impact on target degradation selectivity remain elusive. We solved the crystal structure of Brd4 degrader MZ1 in complex with human VHL and the Brd4 bromodomain (Brd4BD2). The ligand folds into itself to allow formation of specific intermolecular interactions in the ternary complex. Isothermal titration calorimetry studies, supported by surface mutagenesis and proximity assays, are consistent with pronounced cooperative formation of ternary complexes with Brd4BD2. Structure-based-designed compound AT1 exhibits highly selective depletion of Brd4 in cells. Our results elucidate how PROTAC-induced de novo contacts dictate preferential recruitment of a target protein into a stable and cooperative complex with an E3 ligase for selective degradation.

Cereblon versus VHL: Hijacking E3 ligases against each other using PROTACs.

The von Hippel-Lindau (VHL) and cereblon (CRBN) proteins are substrate recognition subunits of two ubiquitously expressed and biologically important Cullin RING E3 ubiquitin ligase complexes. VHL and CRBN are also the two most popular E3 ligases being recruited by bifunctional Proteolysis-targeting chimeras (PROTACs) to induce ubiquitination and subsequent proteasomal degradation of a target protein. Using homo-PROTACs, VHL and CRBN have been independently dimerized to induce their own degradation. Here we report the design, synthesis and cellular activity of VHL-CRBN hetero-dimerizing PROTACs featuring diverse conjugation patterns. We found that the most active compound 14a induced potent, rapid and profound preferential degradation of CRBN over VHL in cancer cell lines. At lower concentrations, weaker degradation of VHL was instead observed. This work demonstrates proof of concept of designing PROTACs to hijack different E3 ligases against each other, and highlights a powerful and generalizable proximity-induced strategy to achieve E3 ligase knockdown.

3-Fluoro-4-hydroxyprolines: Synthesis, Conformational Analysis, and Stereoselective Recognition by the VHL E3 Ubiquitin Ligase for Targeted Protein Degradation.

Hydroxylation and fluorination of proline alters the pyrrolidine ring pucker and the trans:cis amide bond ratio in a stereochemistry-dependent fashion, affecting molecular recognition of proline-containing molecules by biological systems. While hydroxyprolines and fluoroprolines are common motifs in medicinal and biological chemistry, the synthesis and molecular properties of prolines containing both modifications, i.e., fluoro-hydroxyprolines, have not been described. Here we present a practical and facile synthesis of all four diastereoisomers of 3-fluoro-4-hydroxyprolines (F-Hyps), starting from readily available 4-oxo-l-proline derivatives. Small-molecule X-ray crystallography, NMR spectroscopy, and quantum mechanical calculations are consistent with fluorination at C3 having negligible effects on the hydrogen bond donor capacity of the C4 hydroxyl, but inverting the natural preference of Hyp from C4-exo to C4-endo pucker. In spite of this, F-Hyps still bind to the von Hippel-Lindau (VHL) E3 ligase, which naturally recognizes C4-exo Hyp in a stereoselective fashion. Co-crystal structures and electrostatic potential calculations support and rationalize the observed preferential recognition for (3 R,4 S)-F-Hyp over the corresponding (3 S,4 S) epimer by VHL. We show that (3 R,4 S)-F-Hyp provides bioisosteric Hyp substitution in both hypoxia-inducible factor 1 alpha (HIF-1α) substrate peptides and peptidomimetic ligands that form part of PROTAC (proteolysis targeting chimera) conjugates for targeted protein degradation. Despite a weakened affinity, Hyp substitution with (3 S,4 S)-F-Hyp within the PROTAC MZ1 led to Brd4-selective cellular degradation at concentrations >100-fold lower than the binary Kd for VHL. We anticipate that the disclosed chemistry of 3-fluoro-4-hydroxyprolines and their application as VHL ligands for targeted protein degradation will be of wide interest to medicinal organic chemists, chemical biologists, and drug discoverers alike.

Synthesis and Superpotent Anticancer Activity of Tubulysins Carrying Non-hydrolysable N-Substituents on Tubuvaline.

Synthetic tubulysins 24 a-m, containing non-hydrolysable N-substituents on tubuvaline (Tuv), were obtained in high purity and good overall yields using a multistep synthesis. A key step was the formation of differently N-substituted Ile-Tuv fragments 10 by using an aza-Michael reaction of azido-Ile derivatives 8 with the α,ß-unsaturated oxo-thiazole 5. A structure-activity relationship study using a panel of human tumour cell lines showed strong anti-proliferative activity for all compounds 24 a-m, with IC50 values in the sub-nanomolar range, which were distinctly lower than those of tubulysin A, vinorelbine and paclitaxel. Furthermore, 24 a-m were able to overcome cross-resistance to paclitaxel and vinorelbine in two tumour cell lines with acquired resistance to doxorubicin. Compounds 24 e and 24 g were selected as leads to evaluate their mechanism of action. In vitro assays showed that both 24 e and 24 g interfere with tubulin polymerization in a vinca alkaloid-like manner and prevent paclitaxel-induced assembly of tubulin polymers. Both compounds exerted antimitotic activity and induced apoptosis in cancer cells at very low concentrations. Compound 24 e also exhibited potent antitumor activity at well tolerated doses on in vivo models of diffuse malignant peritoneal mesothelioma, such as MESOII peritoneal mesothelioma xenografts, the growth of which was not significantly affected by vinorelbine. These results indicate that synthetic tubulysins 24 could be used as standalone chemotherapeutic agents in difficult-to-treat cancers.

Design, synthesis, in vitro characterization and preliminary imaging studies on fluorinated bile acid derivatives as PET tracers to study hepatic transporters.

With the aim of identifying a fluorinated bile acid derivative that could be used as [18F]-labeled Positron Emission Tomography (PET) tracer for imaging the in vivo functioning of liver transporter proteins, and particularly of OATP1B1, three fluorinated bile acid triazole derivatives of cholic, deoxycholic and lithocholic acid (CATD, DCATD and LCATD 4a-c, respectively) were synthesized and labeled with tritium. In vitro transport properties were studied with cell-based assays to identify the best substrate for OATP1B1. In addition, the lead compound, LCATD (4c), was tested as a substrate of other liver uptake transporters OATP1B3, NTCP and efflux transporter BSEP to evaluate its specificity of liver transport. The results suggest that 4c is a good substrate of OATP1B1 and NTCP, whereas it is a poor substrate of OATP1B3. The efflux transporter BSEP also appears to be involved in the excretion of 4c from hepatocytes. The automated radiosynthesis of [18F]-4c was accomplished in a multi-GBq scale and a pilot imaging experiment in a wild type rat was performed after i.v. administration to assess the biodistribution and clearance of the tracer. PET imaging revealed that radioactivity was primarily located in the liver (tmax=75s) and cleared exclusively through the bile, thus allowing to image the hepatobiliary excretion of bile acids in the animal model. These findings suggest that [18F]-LCATD 4c is a promising PET probe for the evaluation of hepatic transporters OATP1B1, NTCP and BSEP activity with potential for studying drug-drug interactions and drug-induced toxicity involving these transporters.

PET Tracers To Study Clinically Relevant Hepatic Transporters.

Transporter proteins expressed on the cell membranes of hepatocytes are directly involved in the hepatic clearance, mediating the transport of drugs and metabolites through the hepatocyte, from the bloodstream into the bile. Reduction of hepatic transporter activity (due to chemical inhibition, genetic polymorphism, or low expression) can increase systemic or liver exposure to potentially toxic compounds, causing adverse effects. Many clinically used drugs have been associated with inhibition of hepatic transporters in vitro, suggesting the potential involvement of liver transporters in drug-drug interactions (DDIs). Recently, radiolabeled hepatic transporter substrates have been successfully employed in positron emission tomography (PET) imaging to demonstrate inhibition of clinically relevant hepatic transporters. The present article briefly describes the clinical relevance of hepatic transporters followed by a review of the application of PET imaging for the determination of pharmacokinetic parameters useful to describe the transporter activity and the design, accessibility, and preclinical and clinical applications of available radiotracers. Finally, based on the analysis of the strengths and limitations of the available tracers, some criteria for the development of novel PET probes for hepatic transporters and new potential applications are suggested.

Phase-separated droplets swim to their dissolution.

Biological macromolecules can condense into liquid domains. In cells, these condensates form membraneless organelles that can organize chemical reactions. However, little is known about the physical consequences of chemical activity in and around condensates. Working with model bovine serum albumin (BSA) condensates, we show that droplets swim along chemical gradients. Active BSA droplets loaded with urease swim toward each other. Passive BSA droplets show diverse responses to externally applied gradients of the enzyme's substrate and products. In all these cases, droplets swim toward solvent conditions that favor their dissolution. We call this behavior "dialytaxis", and expect it to be generic, as conditions which favor dissolution typically reduce interfacial tension, whose gradients are well-known to drive droplet motion through the Marangoni effect. These results could potentially suggest alternative physical mechanisms for active transport in living cells, and may enable the design of fluid micro-robots.

Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2.

The Src homology 2 (SH2) domain recognizes phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signaling. Drug discovery efforts targeting the SH2 domains have long been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we use structure-based design to target the SH2 domain of the E3 ligase suppressor of cytokine signaling 2 (SOCS2). Starting from the highly ligand-efficient pY amino acid, a fragment growing approach reveals covalent modification of Cys111 in a co-crystal structure, which we leverage to rationally design a cysteine-directed electrophilic covalent inhibitor MN551. We report the prodrug MN714 containing a pivaloyloxymethyl (POM) protecting group and evidence its cell permeability and capping group unmasking using cellular target engagement and in-cell 19F NMR spectroscopy. Covalent engagement at Cys111 competitively blocks recruitment of cellular SOCS2 protein to its native substrate. The qualified inhibitors of SOCS2 could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation.

The 17th EFMC Short Course on Medicinal Chemistry on Small Molecule Protein Degraders.

The 17th EFMC Short Course on Medicinal Chemistry took place April 23-26, 2023 in Oegstgeest, near Leiden in the Netherlands. It covered for the first time the exciting topic of Targeted Protein Degradation (full title: Small Molecule Protein Degraders: A New Opportunity for Drug Design and Development). The course was oversubscribed, with 35 attendees and 6 instructors mainly from Europe but also from the US and South Africa, and representing both industry and academia. This report summarizes the successful event, key lectures given and topics discussed.

"Stay at Home" during the COVID-19 Pandemic: Effects on Physical Activity and Sedentary Behavior in an Italian Academic Community.

This study investigates changes in physical activity (PA) and sedentary behavior in an Italian university community during Phase 1 of SARS-CoV-2 lockdown ("stay at home" government decree, from March 8th to May 4th, 2020) compared to their habits prior to the COVID-19 pandemic. We also examine differences according to gender, university position, BMI categories, and sport participation. A total of 2596 people (median age 24, IQR 11 years; 70.8% women) filled out a survey after eight weeks of statutory confinement at home. The International Physical Activity Questionnaire measured PA and sedentary behavior in a typical week before and during lockdown. Physically inactive people passed from 10.9% to 35.0% before−during the lockdown. The total amount of PA decreased (median 2307 vs. 1367 MET-min/week; p < 0.001), while sedentary behavior increased (median 349 vs. 440 min/day; p < 0.001) between before and during the lockdown. The following categories showed a higher reduction in the total amount of PA: men when compared to women; people with normal weight when compared to pre-obese/obese people; and people who played sports when compared to those who did not play sports. There is a need to propose PA/exercise programs to counteract physical inactivity and sedentarism during a social emergency, with special attention to people who showed higher PA reduction.

Diastereoselective protocols for the synthesis of 2,3-trans- and 2,3-cis-6-methoxy-morpholine-2-carboxylic acid derivatives.

Two diastereoselective and straightforward protocols for the high-yielding synthesis of 2,3-trans- and 2,3-cis-6-methoxy-3-substituted morpholine-2-carboxylic esters were realized in few steps, through the condensation between 5,6-diethoxy-5,6-dimethyl-1,4-dioxan-2-one and an appropriate imine, which is the key reaction to control the C2-C3 relative stereochemistry, followed by a methanolysis/ring-closure tandem reaction sequence. In particular, 2,3-trans-morpholines derive from the R*,S*-product of the acid condensation of N-functionalized alkylimines with the silylketene acetal of the above lactone, whereas 2,3-cis-morpholines derive from the R*,R*-product of basic condensation of an N-tosylimines with the lactone.