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.

Electrochemical Rearrangement of 3-Hydroxyoxindoles into Benzoxazinones.

We report an unexpected rearrangement of 3-hydroxyoxindoles into benzoxazinones using electrochemistry. Our reaction employs mild and environmentally friendly conditions, and the benzoxazinone products are obtained in moderate to excellent yields. Mechanistic experiments suggest that a peroxide intermediate is likely involved.

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.

Getting a Grip on the Undrugged: Targeting ß-Catenin with Fragment-Based Methods.

Aberrant WNT pathway activation, leading to nuclear accumulation of ß-catenin, is a key oncogenic driver event. Mutations in the tumor suppressor gene APC lead to impaired proteasomal degradation of ß-catenin and subsequent nuclear translocation. Restoring cellular degradation of ß-catenin represents a potential therapeutic strategy. Here, we report the fragment-based discovery of a small molecule binder to ß-catenin, including the structural elucidation of the binding mode by X-ray crystallography. The difficulty in drugging ß-catenin was confirmed as the primary screening campaigns identified only few and very weak hits. Iterative virtual and NMR screening techniques were required to discover a compound with sufficient potency to be able to obtain an X-ray co-crystal structure. The binding site is located between armadillo repeats two and three, adjacent to the BCL9 and TCF4 binding sites. Genetic studies show that it is unlikely to be useful for the development of protein-protein interaction inhibitors but structural information and established assays provide a solid basis for a prospective optimization towards ß-catenin proteolysis targeting chimeras (PROTACs) as alternative modality.

One Atom Makes All the Difference: Getting a Foot in the Door between SOS1 and KRAS.

KRAS, the most common oncogenic driver in human cancers, is controlled and signals primarily through protein-protein interactions (PPIs). The interaction between KRAS and SOS1, crucial for the activation of KRAS, is a typical, challenging PPI with a large contact surface area and high affinity. Here, we report that the addition of only one atom placed between Y884SOS1 and A73KRAS is sufficient to convert SOS1 activators into SOS1 inhibitors. We also disclose the discovery of BI-3406. Combination with the upstream EGFR inhibitor afatinib shows in vivo efficacy against KRASG13D mutant colorectal tumor cells, demonstrating the utility of BI-3406 to probe SOS1 biology. These findings challenge the dogma that large molecules are required to disrupt challenging PPIs. Instead, a "foot in the door" approach, whereby single atoms or small functional groups placed between key PPI interactions, can lead to potent inhibitors even for challenging PPIs such as SOS1-KRAS.

BI-3406, a Potent and Selective SOS1-KRAS Interaction Inhibitor, Is Effective in KRAS-Driven Cancers through Combined MEK Inhibition.

KRAS is the most frequently mutated driver of pancreatic, colorectal, and non-small cell lung cancers. Direct KRAS blockade has proved challenging, and inhibition of a key downstream effector pathway, the RAF-MEK-ERK cascade, has shown limited success because of activation of feedback networks that keep the pathway in check. We hypothesized that inhibiting SOS1, a KRAS activator and important feedback node, represents an effective approach to treat KRAS-driven cancers. We report the discovery of a highly potent, selective, and orally bioavailable small-molecule SOS1 inhibitor, BI-3406, that binds to the catalytic domain of SOS1, thereby preventing the interaction with KRAS. BI-3406 reduces formation of GTP-loaded RAS and limits cellular proliferation of a broad range of KRAS-driven cancers. Importantly, BI-3406 attenuates feedback reactivation induced by MEK inhibitors and thereby enhances sensitivity of KRAS-dependent cancers to MEK inhibition. Combined SOS1 and MEK inhibition represents a novel and effective therapeutic concept to address KRAS-driven tumors. SIGNIFICANCE: To date, there are no effective targeted pan-KRAS therapies. In-depth characterization of BI-3406 activity and identification of MEK inhibitors as effective combination partners provide an attractive therapeutic concept for the majority of KRAS-mutant cancers, including those fueled by the most prevalent mutant KRAS oncoproteins, G12D, G12V, G12C, and G13D.See related commentary by Zhao et al., p. 17.This article is highlighted in the In This Issue feature, p. 1.

Iron-catalyzed aryl-aryl cross-coupling reaction tolerating amides and unprotected quinolinones.

The iron(III)-catalyzed cross-coupling reaction between functionalized arylcopper reagents and aromatic iodides bearing an amide function or an unprotected quinolinone leads smoothly to polyfunctionalized biphenyls in excellent yields due to an intramolecular chelating effect of the amide group.

Synthesis of functionalized diarylmethanes via a copper-catalyzed cross-coupling of arylmagnesium reagents with benzylic phosphates.

A combination of copper chloride, triethyl phosphite, and tetrabutylammonium iodide is a very efficient catalytic system for the synthesis of polyfunctionalized diarylmethanes, using the cross-coupling reaction of arylmagnesium halides with benzylic phosphates. The antibiotic Trimethoprim has been prepared using this Cu(I)-catalyzed cross-coupling in 52% overall yield (four steps).

Preparation of aryl-alkylamines via electrophilic amination of functionalized arylazo tosylates with alkylzinc reagents.

[reaction: see text] A new electrophilic amination reaction of functionalized arylazo tosylates with alkylzinc halides or dialkylzinc reagents in THF leads to the corresponding hydrazines. A facile cleavage of the N-N bond is achieved using Raney nickel in refluxing ethanol, leading to substituted secondary aryl-alkylamines in 45-79% yield.

Discovery of Novel Spiro[3H-indole-3,2'-pyrrolidin]-2(1H)-one Compounds as Chemically Stable and Orally Active Inhibitors of the MDM2-p53 Interaction.

Scaffold modification based on Wang's pioneering MDM2-p53 inhibitors led to novel, chemically stable spiro-oxindole compounds bearing a spiro[3H-indole-3,2'-pyrrolidin]-2(1H)-one scaffold that are not prone to epimerization as observed for the initial spiro[3H-indole-3,3'-pyrrolidin]-2(1H)-one scaffold. Further structure-based optimization inspired by natural product architectures led to a complex fused ring system ideally suited to bind to the MDM2 protein and to interrupt its protein-protein interaction (PPI) with TP53. The compounds are highly selective and show in vivo efficacy in a SJSA-1 xenograft model even when given as a single dose as demonstrated for 4-[(3S,3'S,3'aS,5'R,6'aS)-6-chloro-3'-(3-chloro-2-fluorophenyl)-1'-(cyclopropylmethyl)-2-oxo-1,2,3',3'a,4',5',6',6'a-octahydro-1'H-spiro[indole-3,2'-pyrrolo[3,2-b]pyrrole]-5'-yl]benzoic acid (BI-0252).

Efficient cross-coupling of functionalized arylzinc halides catalyzed by a nickel chloride-diethyl phosphite system.

[reaction: see text] The combination of diethyl phosphite and DMAP as ligands for nickel in an 8:1 THF-N-ethylpyrrolidinone (NEP) mixture allows a very efficient cross-coupling reaction to be performed between various functionalized arylzinc halides and aryl bromides, triflates and activated chlorides. The reaction proceeds at 25 degrees C within 1-48 h and requires only 0.05 mol % of the nickel catalyst.

Direct preparation of polyfunctional amino-substituted arylmagnesium reagents via an iodine-magnesium exchange reaction.

The successive addition of PhMgCl and i-PrMgCl to functionalised iodoanilines allows their conversion to the corresponding amino-functionalised Grignard reagents, which react smoothly with a range of electrophiles in high yield.

Asymmetric construction of rings A-D of daphnicyclidin-type alkaloids.

The aza-Cope-Mannich reaction and ring-closing metathesis are key steps in the assembly of intermediates containing rings A-D of Daphniphyllum alkaloids of the daphnicyclidin type such as daphnipaxinin and oldhamine A.