Drugging an undruggable pocket on KRAS.

The 3 human RAS genes, KRAS, NRAS, and HRAS, encode 4 different RAS proteins which belong to the protein family of small GTPases that function as binary molecular switches involved in cell signaling. Activating mutations in RAS are among the most common oncogenic drivers in human cancers, with KRAS being the most frequently mutated oncogene. Although KRAS is an excellent drug discovery target for many cancers, and despite decades of research, no therapeutic agent directly targeting RAS has been clinically approved. Using structure-based drug design, we have discovered BI-2852 (1), a KRAS inhibitor that binds with nanomolar affinity to a pocket, thus far perceived to be "undruggable," between switch I and II on RAS; 1 is mechanistically distinct from covalent KRASG12C inhibitors because it binds to a different pocket present in both the active and inactive forms of KRAS. In doing so, it blocks all GEF, GAP, and effector interactions with KRAS, leading to inhibition of downstream signaling and an antiproliferative effect in the low micromolar range in KRAS mutant cells. These findings clearly demonstrate that this so-called switch I/II pocket is indeed druggable and provide the scientific community with a chemical probe that simultaneously targets the active and inactive forms of KRAS.

KRAS Binders Hidden in Nature.

Natural products have proven to be a rich source of molecular architectures for drugs. Here, an integrated approach to natural product screening is proposed, which uncovered eight new natural product scaffolds for KRAS-the most frequently mutated oncogenic driver in human cancers, which has remained thus far undrugged. The approach combines aspects of virtual screening, fragment-based screening, structure-activity relationships (SAR) by NMR, and structure-based drug discovery to overcome the limitations in traditional natural product approaches. By using our approach, a new "snugness of fit" scoring function and the first crystal-soaking system of the active form of KRASG12D , the protein-ligand X-ray structures of a tricyclic indolopyrrole fungal alkaloid and an indoloisoquinolinone have been successfully elucidated. The natural product KRAS hits discovered provide fruitful ground for the optimization of highly potent natural-product-based inhibitors of the active form of oncogenic RAS. This integrated approach for screening natural products also holds promise for other "undruggable" targets.

Sensitivity and engineered resistance of myeloid leukemia cells to BRD9 inhibition.

Here we show that acute myeloid leukemia (AML) cells require the BRD9 subunit of the SWI-SNF chromatin-remodeling complex to sustain MYC transcription, rapid cell proliferation and a block in differentiation. Based on these observations, we derived small-molecule inhibitors of the BRD9 bromodomain that selectively suppress the proliferation of mouse and human AML cell lines. To establish these effects as on-target, we engineered a bromodomain-swap allele of BRD9 that retains functionality despite a radically altered bromodomain pocket. Expression of this allele in AML cells confers resistance to the antiproliferative effects of our compound series, thus establishing BRD9 as the relevant cellular target. Furthermore, we used an analogous domain-swap strategy to generate an inhibitor-resistant allele of EZH2. To our knowledge, our study provides the first evidence for a role of BRD9 in cancer and reveals a simple genetic strategy for constructing resistance alleles to demonstrate on-target activity of chemical probes in cells.

Chasing Red Herrings: Palladium Metal Salt Impurities Feigning KRAS Activity in Biochemical Assays.

Identifying promising chemical starting points for small molecule inhibitors of active, GTP-loaded KRAS "on" remains of great importance to clinical oncology and represents a significant challenge in medicinal chemistry. Here, we describe broadly applicable learnings from a KRAS hit finding campaign: While we initially identified KRAS inhibitors in a biochemical high-throughput screen, we later discovered that compound potencies were all but assay artifacts linked to metal salts interfering with KRAS AlphaScreen assay technology. The source of the apparent biochemical KRAS inhibition was ultimately traced to unavoidable palladium impurities from chemical synthesis. This discovery led to the development of a Metal Ion Interference Set (MIIS) for up-front assay development and testing. Profiling of the MIIS across 74 assays revealed a reduced interference liability of label-free biophysical assays and, as a result, provided general estimates for luminescence- and fluorescence-based assay susceptibility to metal salt interference.

Discovery of a Chemical Probe to Study Implications of BPTF Bromodomain Inhibition in Cellular and in†vivo Experiments.

The bromodomain and PHD-finger containing transcription factor (BPTF) is part of the nucleosome remodeling factor (NURF) complex and has been implicated in multiple cancer types. Here, we report the discovery of a potent and selective chemical probe targeting the bromodomain of BPTF with an attractive pharmacokinetic profile enabling cellular and in vivo experiments in mice. Microarray-based transcriptomics in presence of the probe in two lung cancer cell lines revealed only minor effects on the transcriptome. Profiling against a panel of cancer cell lines revealed that the antiproliferative effect does not correlate with BPTF dependency score in depletion screens. Both observations and the multi-domain architecture of BPTF suggest that depleting the protein by proteolysis targeting chimeras (PROTACs) could be a promising strategy to target cancer cell proliferation. We envision that the presented chemical probe and the related negative control will enable the research community to further explore scientific hypotheses with respect to BPTF bromodomain inhibition.

(±)-trans,cis-4-Hydroxy-5,6-di-O-isopropylidenecyclohex-2-ene-1-one: synthesis and facile dimerization to decahydrodibenzofurans.

An efficient synthesis of (±)-trans,cis-4-hydroxy-5,6-di-O-isopropylidenecyclohex-2-ene-1-one (3) has been developed from acetonide-protected meso-1,2-dihydrocatechol derivative 1 via photooxygenation, then Kornblum-DeLaMare rearrangement. The product is unstable unless its 4-hydroxy group is protected, as it undergoes facile dimerization in solution to a 1:1 mixture of diastereoisomeric decahydrodibenzofurans 8 and 9. A new synthesis of the dihydrocatechol 1 from 1,3-cyclohexadiene has also been developed.

A strategy for isotope containment during radiosynthesis--devolatilisation of bromobenzene by fluorous-tagging-Ir-catalysed borylation en route to the 4-phenylpiperidine pharmacophore.

Syntheses of two 4-phenylpiperidines from bromobenzene have been developed involving anchoring to a fluorous-tag, Ir-catalysed borylation, Pd- and Co-catalysed elaboration then traceless cleavage. Although performed using 'cold' (i.e. unlabelled) bromobenzene as the starting material, these routes have been designed to minimise material loss via volatile intermediates and expedite purification during radiosynthesis from 'hot' (i.e. [(14)C] labelled) bromobenzene.

Cis and trans selective 1,4-addition of a lithium dithioester enolate to 4-O-TBS-2-cyclohexenone.

The 1,4-addition of the lithium enolate of methyldithioacetate (LMDTA) to (+/-)-4-O-TBS-2-cyclohexenone (3) can be varied from being highly 3,4-trans selective to being highly 3,4-cis selective simply by varying the reaction temperature. This stereodivergency allows expedient syntheses of the corresponding trans and cis methyl esters 6t and 6c and derived bicyclic ketolactones 7t and 7c.

Structure-Based Design of an in Vivo Active Selective BRD9 Inhibitor.

Components of the chromatin remodelling switch/sucrose nonfermentable (SWI/SNF) complex are recurrently mutated in tumors, suggesting that altering the activity of the complex plays a role in oncogenesis. However, the role that the individual subunits play in this process is not clear. We set out to develop an inhibitor compound targeting the bromodomain of BRD9 in order to evaluate its function within the SWI/SNF complex. Here, we present the discovery and development of a potent and selective BRD9 bromodomain inhibitor series based on a new pyridinone-like scaffold. Crystallographic information on the inhibitors bound to BRD9 guided their development with respect to potency for BRD9 and selectivity against BRD4. These compounds modulate BRD9 bromodomain cellular function and display antitumor activity in an AML xenograft model. Two chemical probes, BI-7273 (1) and BI-9564 (2), were identified that should prove to be useful in further exploring BRD9 bromodomain biology in both in vitro and in vivo settings.

Safe and reliable synthesis of diazoketones and quinoxalines in a continuous flow reactor.

A flow method for the synthesis of aliphatic and aromatic diazoketones from acyl chloride precursors has been developed and used to prepare quinoxalines in a multistep sequence without isolation of the potentially explosive diazoketone. The protocol showcases an efficient in-line purification using supported scavengers with time-saving and safety benefits and in particular a reduction in the operator's exposure to carcinogenic phenylenediamines.