Clp-targeting BacPROTACs impair mycobacterial proteostasis and survival.

The ClpC1:ClpP1P2 protease is a core component of the proteostasis system in mycobacteria. To improve the efficacy of antitubercular agents targeting the Clp protease, we characterized the mechanism of the antibiotics cyclomarin A and ecumicin. Quantitative proteomics revealed that the antibiotics cause massive proteome imbalances, including upregulation of two unannotated yet conserved stress response factors, ClpC2 and ClpC3. These proteins likely protect the Clp protease from excessive amounts of misfolded proteins or from cyclomarin A, which we show to mimic damaged proteins. To overcome the Clp security system, we developed a BacPROTAC that induces degradation of ClpC1 together with its ClpC2 caretaker. The dual Clp degrader, built from linked cyclomarin A heads, was highly efficient in killing pathogenic Mycobacterium tuberculosis, with >100-fold increased potency over the parent antibiotic. Together, our data reveal Clp scavenger proteins as important proteostasis safeguards and highlight the potential of BacPROTACs as future antibiotics.

BacPROTACs mediate targeted protein degradation in bacteria.

Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis-targeting chimeras. Despite their great promise for medical chemistry, so far, it has not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, drug susceptibility and degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, allowing selective targeting of endogenous proteins via fusion to an established degron. In addition to guiding antibiotic discovery, the BacPROTAC technology presents a versatile research tool enabling the inducible degradation of bacterial proteins.

Synthesis of a 13C-methylene-labeled isoleucine precursor as a useful tool for studying protein side-chain interactions and dynamics.

In this study, we present the synthesis and incorporation of a metabolic isoleucine precursor compound for selective methylene labeling. The utility of this novel α-ketoacid isotopologue is shown by incorporation into the protein Brd4-BD1, which regulates gene expression by binding to acetylated histones. High quality single quantum 13C-1 H-HSQC were obtained, as well as triple quantum HTQC spectra, which are superior in terms of significantly increased 13C-T2 times. Additionally, large chemical shift perturbations upon ligand binding were observed. Our study thus proves the great sensitivity of this precursor as a reporter for side-chain dynamic studies and for investigations of CH-π interactions in protein-ligand complexes.

13 Cß-Valine and 13 Cγ-Leucine Methine Labeling To Probe Protein Ligand Interaction.

Precise information regarding the interaction between proteins and ligands at molecular resolution is crucial for effectively guiding the optimization process from initial hits to lead compounds in early stages of drug development. In this study, we introduce a novel aliphatic side chain isotope-labeling scheme to directly probe interactions between ligands and aliphatic sidechains using NMR techniques. To demonstrate the applicability of this method, we selected a set of Brd4-BD1 binders and analyzed 1 H chemical shift perturbation resulting from CH-π interaction of Hß -Val and Hγ -Leu as CH donors with corresponding ligand aromatic moieties as π acceptors.

Electrochemical Umpolung C-H Functionalization of Oxindoles.

Herein, we present a general electrochemical method to access unsymmetrical 3,3-disubstituted oxindoles by direct C-H functionalization where the oxindole fragment behaves as an electrophile. This Umpolung approach does not rely on stoichiometric oxidants and proceeds under mild, environmentally benign conditions. Importantly, it enables the functionalization of these scaffolds through C-O, and by extension to C-C or even C-N bond formation.

BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC design.

Targeting subunits of BAF/PBAF chromatin remodeling complexes has been proposed as an approach to exploit cancer vulnerabilities. Here, we develop proteolysis targeting chimera (PROTAC) degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided rational and efficient optimization toward ACBI1, a potent and cooperative degrader of SMARCA2, SMARCA4 and PBRM1. ACBI1 induced anti-proliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics- and structure-based PROTAC design approach to degrade high profile drug targets, and pave the way toward new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.

Publisher Correction: BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC design.

In the version of this article originally published, several lines of text in the last paragraph of the right column on page 1 of the PDF were transposed into the bottom paragraph of the left column. The affected text of the left column should read "The ATP-dependent activities of the BAF (SWI/SNF) chromatin remodeling complexes affect the positioning of nucleosomes on DNA and thereby many cellular processes related to chromatin structure, including transcription, DNA repair and decatenation of chromosomes during mitosis12,13." The affected text of the right column should read "SMARCA2/4BD inhibitors are thus precluded from use for the treatment of SMARCA4 mutant cancers but could provide attractive ligands for PROTAC conjugation. Small molecules binding to other bromodomains have been successfully converted into PROTACs by conjugating them with structures capable of binding to the E3 ligases von Hippel-Lindau (VHL) or cereblon5,6,10,11,25,26,27." The errors have been corrected in the PDF version of the paper.

Additive effects in the palladium-catalyzed carboiodination of chiral N-allyl carboxamides.

The use of Pd catalysis as a means to synthesize organic halides has recently received increased attention. Among the reported methods is the Pd-catalyzed carboiodination, which uses extremely bulky ligands to facilitate carbon-halogen reductive elimination from Pd(II) as the key catalytic step. When approaching substrates exhibiting low stereoselectivity, catalyst troubleshooting becomes difficult as there are few ligands known to promote the key reductive elimination. Herein, we present our finding that tertiary amines act as weakly coordinating ligands which significantly enhance diastereoselectivity in the Pd/QPhos-catalyzed carboiodination of chiral N-allyl carboxamides. This methodology allows efficient access to enantioenriched and densely functionalized dihydroisoquinolinones, and has been applied toward the asymmetric formal synthesis of (+)-corynoline.

Light-mediated total synthesis of 17-deoxyprovidencin.

An asymmetric synthesis of the diterpenoid 17-deoxyprovidencin is described. Key steps include an aldol addition, a base-catalyzed Wipf-type furan formation, a Z-selective ring-closing metathesis for macrocyclization, a photochemical E/Z isomerization to a highly strained and conformationally restricted ring system, and the stereoselective formation of two epoxides on the ring.

Intermolecular domino reaction of two aryl iodides involving two C-H functionalizations.

A palladium-catalyzed intermolecular cross-coupling of two aryl iodides is reported, giving polycyclic ring systems with a high level of convergence and efficiency.

Synthesis of Complex Tetracyclic Fused Scaffolds Enabled by (3 + 2) Cycloaddition.

We describe the single-step formation of complex tetracyclic fused scaffolds enabled by (3 + 2) cycloaddition of azomethine ylides. Various indoles, N-protecting groups, and amino acids are well tolerated. The products are obtained in a catalyst-free manner with moderate to excellent yield and high diastereoselectivity. Representing a new scaffold that is not yet found in nature, the construction of pyrrolidine-fused cyclohepta-, azepino-, or oxepinoindoles could be found valuable in the synthesis of new pseudo-natural products.

Inhibition of p53-MDM2 binding reduces senescent cell abundance and improves the adaptive responses of skeletal muscle from aged mice.

Skeletal muscle adaptation to external stimuli, such as regeneration following injury and hypertrophy in response to resistance exercise, are blunted with advanced age. The accumulation of senescent cells, along with defects in myogenic progenitor cell (MPC) proliferation, have been strongly linked as contributing factors to age-associated impairment in muscle adaptation. p53 plays an integral role in all these processes, as upregulation of p53 causes apoptosis in senescent cells and prevents mitotic catastrophe in MPCs from old mice. The goal of this study was to determine if a novel pharmaceutical agent (BI01), which functions by upregulating p53 through inhibition of binding to MDM2, the primary p53 regulatory protein, improves muscle regeneration and hypertrophy in old mice. BI01 effectively reduced the number of senescent cells in vitro but had no effect on MPC survival or proliferation at a comparable dose. Following repeated oral gavage with 2 mg/kg of BI01 (OS) or vehicle (OV), old mice (24 months) underwent unilateral BaCl2 injury in the tibialis anterior (TA) muscle, with PBS injections serving as controls. After 7 days, satellite cell number was higher in the TA of OS compared to OV mice, as was the expression of genes involved in ATP production. By 35 days, old mice treated with BI01 displayed reduced senescent cell burden, enhanced regeneration (higher muscle mass and fiber cross-sectional area) and restoration of muscle function relative to OV mice. To examine the impact of 2 mg/kg BI01 on muscle hypertrophy, the plantaris muscle was subjected to 28 days of mechanical overload (MOV) in OS and OV mice. In response to MOV, OS mice had larger plantaris muscles and muscle fibers than OV mice, particularly type 2b + x fibers, associated with reduced senescent cells. Together our data show that BI01 is an effective senolytic agent that may also augment muscle metabolism to enhance muscle regeneration and hypertrophy in old mice.

Discovery and Characterization of Brigimadlin, a Novel and Highly Potent MDM2-p53 Antagonist Suitable for Intermittent Dose Schedules.

p53 is known as the guardian of the genome and is one of the most important tumor-suppressors. It is inactivated in most tumors, either via tumor protein p53 (TP53) gene mutation or copy number amplification of key negative regulators, e.g., mouse double minute 2 (MDM2). Compounds that bind to the MDM2 protein and disrupt its interaction with p53 restore p53 tumor suppressor activity, thereby promoting cell cycle arrest and apoptosis. Previous clinical experience with MDM2-p53 protein-protein interaction antagonists (MDM2-p53 antagonists) have demonstrated that thrombocytopenia and neutropenia represent on-target dose-limiting toxicities that might restrict their therapeutic utility. Dosing less frequently, while maintaining efficacious exposure, represents an approach to mitigate toxicity and improve the therapeutic window of MDM2-p53 antagonists. However, to achieve this, a molecule possessing excellent potency and ideal pharmacokinetic properties is required. Here, we present the discovery and characterization of brigimadlin (BI 907828), a novel, investigational spiro-oxindole MDM2-p53 antagonist. Brigimadlin exhibited high bioavailability and exposure, as well as dose-linear pharmacokinetics in preclinical models. Brigimadlin treatment restored p53 activity and led to apoptosis induction in preclinical models of TP53 wild-type, MDM2-amplified cancer. Oral administration of brigimadlin in an intermittent dosing schedule induced potent tumor growth inhibition in several TP53 wild-type, MDM2-amplified xenograft models. Exploratory clinical pharmacokinetic studies (NCT03449381) showed high systemic exposure and a long plasma elimination half-life in cancer patients who received oral brigimadlin. These findings support the continued clinical evaluation of brigimadlin in patients with MDM2-amplified cancers, such as dedifferentiated liposarcoma.

Homo-BacPROTAC-induced degradation of ClpC1 as a strategy against drug-resistant mycobacteria.

Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be designed to slow down the emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We are extending this concept by developing proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti-Mycobacterium tuberculosis (Mtb) BacPROTACs are derived from cyclomarins which, when dimerized, generate compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduce levels of endogenous ClpC1 in Mycobacterium smegmatis and display minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug-resistant Mtb isolates. The compounds also kill Mtb residing in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance.

Confounding Factors in Targeted Degradation of Short-Lived Proteins.

Targeted protein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but to what extent it influences target protein degradation has not been systematically explored. Using simple mathematical modeling of protein degradation, we find that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a degrader agent which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein-degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required to conclude that a novel agent works as a bona fide targeted protein degrader.

Targeting cancer with small-molecule pan-KRAS degraders.

Mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) protein are highly prevalent in cancer. However, small-molecule concepts that address oncogenic KRAS alleles remain elusive beyond replacing glycine at position 12 with cysteine (G12C), which is clinically drugged through covalent inhibitors. Guided by biophysical and structural studies of ternary complexes, we designed a heterobifunctional small molecule that potently degrades 13 out of 17 of the most prevalent oncogenic KRAS alleles. Compared with inhibition, KRAS degradation results in more profound and sustained pathway modulation across a broad range of KRAS mutant cell lines, killing cancer cells while sparing models without genetic KRAS aberrations. Pharmacological degradation of oncogenic KRAS was tolerated and led to tumor regression in vivo. Together, these findings unveil a new path toward addressing KRAS-driven cancers with small-molecule degraders.

European Medicinal Chemistry Leaders in Industry (EMCL) - On the Status and Future of Medicinal Chemistry Research in Europe.

The status of industrial Medicinal Chemistry was discussed with European Medicinal Chemistry Leaders from large to mid-sized pharma and CRO companies as well as biotechs. The chemical modality space has expanded recently from small molecules to address new challenging targets. Besides the classical SAR/SPR optimization of drug molecules also their 'greenness' has increasing importance. The entire pharma discovery ecosystem has developed significantly. Beyond pharma and academia new key players such as Biotech and integrated CROs as well as Digital companies have appeared and are now to a large extend fueled by VC money. Digitalization is happening everywhere but surprisingly did not change speed and success rates of projects so far. Future Medicinal Chemists will still have to be excellent synthetic chemists but in addition they must be knowledgeable in new computational areas such as data sciences. Their ability to collaborate and to work in teams is key.

Synthesis of MDM2-p53 Inhibitor BI-0282 via a Dipolar Cycloaddition and Late-Stage Davis-Beirut Reaction.

Herein, we report the structure and synthesis of the potent MDM2-p53 inhibitor BI-0282. The complex spirooxindole scaffold bearing four stereocenters embedded in a rigid polycyclic ring-system was effectively prepared on a multi-gram scale in only five synthesis steps employing a three-component 1,3-dipolar cycloaddition and a late-stage Davis-Beirut reaction as key steps.

A selective and orally bioavailable VHL-recruiting PROTAC achieves SMARCA2 degradation in vivo.

Targeted protein degradation offers an alternative modality to classical inhibition and holds the promise of addressing previously undruggable targets to provide novel therapeutic options for patients. Heterobifunctional molecules co-recruit a target protein and an E3 ligase, resulting in ubiquitylation and proteosome-dependent degradation of the target. In the clinic, the oral route of administration is the option of choice but has only been achieved so far by CRBN- recruiting bifunctional degrader molecules. We aimed to achieve orally bioavailable molecules that selectively degrade the BAF Chromatin Remodelling complex ATPase SMARCA2 over its closely related paralogue SMARCA4, to allow in vivo evaluation of the synthetic lethality concept of SMARCA2 dependency in SMARCA4-deficient cancers. Here we outline structure- and property-guided approaches that led to orally bioavailable VHL-recruiting degraders. Our tool compound, ACBI2, shows selective degradation of SMARCA2 over SMARCA4 in ex vivo human whole blood assays and in vivo efficacy in SMARCA4-deficient cancer models. This study demonstrates the feasibility for broadening the E3 ligase and physicochemical space that can be utilised for achieving oral efficacy with bifunctional molecules.

Fragment Optimization of Reversible Binding to the Switch II Pocket on KRAS Leads to a Potent, In Vivo Active KRASG12C Inhibitor.

Activating mutations in KRAS are the most frequent oncogenic alterations in cancer. The oncogenic hotspot position 12, located at the lip of the switch II pocket, offers a covalent attachment point for KRASG12C inhibitors. To date, KRASG12C inhibitors have been discovered by first covalently binding to the cysteine at position 12 and then optimizing pocket binding. We report on the discovery of the in vivo active KRASG12C inhibitor BI-0474 using a different approach, in which small molecules that bind reversibly to the switch II pocket were identified and then optimized for non-covalent binding using structure-based design. Finally, the Michael acceptor containing warhead was attached. Our approach offers not only an alternative approach to discovering KRASG12C inhibitors but also provides a starting point for the discovery of inhibitors against other oncogenic KRAS mutants.