Activation of parkin by a molecular glue.

Mutations in parkin and PINK1 cause early-onset Parkinson's disease (EOPD). The ubiquitin ligase parkin is recruited to damaged mitochondria and activated by PINK1, a kinase that phosphorylates ubiquitin and the ubiquitin-like domain of parkin. Activated phospho-parkin then ubiquitinates mitochondrial proteins to target the damaged organelle for degradation. Here, we present the mechanism of activation of a new class of small molecule allosteric modulators that enhance parkin activity. The compounds act as molecular glues to enhance the ability of phospho-ubiquitin (pUb) to activate parkin. Ubiquitination assays and isothermal titration calorimetry with the most active compound (BIO-2007817) identify the mechanism of action. We present the crystal structure of a closely related compound (BIO-1975900) bound to a complex of parkin and two pUb molecules. The compound binds next to pUb on RING0 and contacts both proteins. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments confirm that activation occurs through release of the catalytic Rcat domain. In organello and mitophagy assays demonstrate that BIO-2007817 partially rescues the activity of parkin EOPD mutants, R42P and V56E, offering a basis for the design of activators as therapeutics for Parkinson's disease.

BAY-9835: Discovery of the First Orally Bioavailable ADAMTS7 Inhibitor.

The matrix metalloprotease ADAMTS7 has been identified by multiple genome-wide association studies as being involved in the development of coronary artery disease. Subsequent research revealed the proteolytic function of the enzyme to be relevant for atherogenesis and restenosis after vessel injury. Based on a publicly known dual ADAMTS4/ADAMTS5 inhibitor, we have in silico designed an ADAMTS7 inhibitor of the catalytic domain, which served as a starting point for an optimization campaign. Initially our inhibitors suffered from low selectivity vs MMP12. An X-ray cocrystal structure inspired us to exploit amino acid differences in the binding site of MMP12 and ADAMTS7 to improve selectivity. Further optimization composed of employing 5-membered heteroaromatic groups as hydantoin substituents to become more potent on ADAMTS7. Finally, fine-tuning of DMPK properties yielded BAY-9835, the first orally bioavailable ADAMTS7 inhibitor. Further optimization to improve selectivity vs ADAMTS12 seems possible, and a respective starting point could be identified.

Discovery of small-molecule positive allosteric modulators of Parkin E3 ligase.

Pharmacological activation of the E3 ligase Parkin represents a rational therapeutic intervention for the treatment of Parkinson's disease. Here we identify several compounds that enhance the activity of wildtype Parkin in the presence of phospho-ubiquitin and act as positive allosteric modulators (PAMs). While these compounds activate Parkin in a series of biochemical assays, they do not act by thermally destabilizing Parkin and fail to enhance the Parkin translocation rate to mitochondria or to enact mitophagy in cell-based assays. We conclude that in the context of the cellular milieu the therapeutic window to pharmacologically activate Parkin is very narrow.

Discovering the anti-cancer potential of non-oncology drugs by systematic viability profiling.

Anti-cancer uses of non-oncology drugs have occasionally been found, but such discoveries have been serendipitous. We sought to create a public resource containing the growth inhibitory activity of 4,518 drugs tested across 578 human cancer cell lines. We used PRISM, a molecular barcoding method, to screen drugs against cell lines in pools. An unexpectedly large number of non-oncology drugs selectively inhibited subsets of cancer cell lines in a manner predictable from the cell lines' molecular features. Our findings include compounds that killed by inducing PDE3A-SLFN12 complex formation; vanadium-containing compounds whose killing depended on the sulfate transporter SLC26A2; the alcohol dependence drug disulfiram, which killed cells with low expression of metallothioneins; and the anti-inflammatory drug tepoxalin, which killed via the multi-drug resistance protein ABCB1. The PRISM drug repurposing resource (https://depmap.org/repurposing) is a starting point to develop new oncology therapeutics, and more rarely, for potential direct clinical translation.

Stabilization of the Max Homodimer with a Small Molecule Attenuates Myc-Driven Transcription.

The transcription factor Max is a basic-helix-loop-helix leucine zipper (bHLHLZ) protein that forms homodimers or interacts with other bHLHLZ proteins, including Myc and Mxd proteins. Among this dynamic network of interactions, the Myc/Max heterodimer has crucial roles in regulating normal cellular processes, but its transcriptional activity is deregulated in a majority of human cancers. Despite this significance, the arsenal of high-quality chemical probes to interrogate these proteins remains limited. We used small molecule microarrays to identify compounds that bind Max in a mechanistically unbiased manner. We discovered the asymmetric polycyclic lactam, KI-MS2-008, which stabilizes the Max homodimer while reducing Myc protein and Myc-regulated transcript levels. KI-MS2-008 also decreases viable cancer cell growth in a Myc-dependent manner and suppresses tumor growth in vivo. This approach demonstrates the feasibility of modulating Max with small molecules and supports altering Max dimerization as an alternative approach to targeting Myc.

Total Synthesis and Structural Reassignment of Lyngbyaloside C Highlighted by Intermolecular Ketene Esterification.

Lyngbyaloside C, a classic macrolide, isolated from Lyngbya bouilloni, has shown moderate anticancer activity against several cancer cell lines. Here, we report the first total synthesis and stereochemical configuration reassignment of lyngbyaloside C. The synthesis highlights a one-pot intermolecular ketene esterification reaction to form the crucial tertiary ester and tetrahydropyran. In addition, a novel and concise synthetic pathway towards the 1,3-syn secondary, tertiary diol fragment is described using a regio- and stereospecific electrophilic ether transfer reaction.

Concise enantioselective synthesis of diospongins A and B.

Ether transfer methodology is capable of stereoselectively generating 1,3-diol mono- and diethers in good yield. Surprisingly, allylic and benzylic substrates provide none of the desired products when exposed to previously optimized conditions of iodine monochloride. Herein, second-generation activation conditions for ether transfer have been developed that circumvents undesired side reactions for these substrates. The application of this chemistry to the enantioselective synthesis of diospongins A and B has now been accomplished.

Stereoselective synthesis of the C9-C19 fragment of lyngbyaloside B and C via ether transfer.

A stereoselective synthesis of the C9-C19 fragment of lyngbyaloside B and C highlighted, by an extension of our ether transfer methodology, enables the formation of tertiary ethers. 2-Naphthylmethyl ethers have been shown to proceed efficiently through ether transfer with high stereoselectivity and are easily deprotected by DDQ oxidation. Variation of the workup conditions results in the stereoselective formation of syn-1,3-diol mono- or diethers.

Synthesis and SAR of inhibitors of protein kinase CK2: novel tricyclic quinoline analogs.

Protein kinase CK2 is a potential drug target for many diseases including cancer and inflammation disorders. The crystal structure of clinical candidate CX-4945 1 with CK2 revealed an indirect interaction with the protein through hydrogen bonding between the NH of the 3-chlorophenyl amine and a water molecule. Herein, we investigate the relevance of this hydrogen bond by preparing several novel tricyclic derivatives lacking a NH moiety at the same position. This SAR study allowed the discovery of highly potent CK2 inhibitors.

7-(4H-1,2,4-Triazol-3-yl)benzo[c][2,6]naphthyridines: a novel class of Pim kinase inhibitors with potent cell antiproliferative activity.

A novel class of pan-Pim kinase inhibitors was designed by modifying the CK2 inhibitor CX-4945. Introduction of a triazole or secondary amide functionality on the C-7 position and 2'-halogenoanilines on C-5 resulted in potent inhibitors of the Pim-1 and Pim-2 isoforms, with many analogs active at single digit nanomolar concentrations. The molecules inhibited the phosphorylation at Serine 112 of the apoptosis effector BAD, and had potent antiproliferative effects on the AML cell line MV-4-11 (IC(50) <30 nM). This work delivers an excellent lead-optimization platform for Pim targeting anticancer therapies.

Pre-clinical characterization of CX-4945, a potent and selective small molecule inhibitor of CK2 for the treatment of cancer.

In this article we describe the preclinical characterization of 5-(3-chlorophenylamino) benzo[c][2,6]naphthyridine-8-carboxylic acid (CX-4945), the first orally available small molecule inhibitor of protein CK2 in clinical trials for cancer. CX-4945 was optimized as an ATP-competitive inhibitor of the CK2 holoenzyme (Ki = 0.38 nM). Iterative synthesis and screening of analogs, guided by molecular modeling, led to the discovery of orally available CX-4945. CK2 promotes signaling in the Akt pathway and CX-4945 suppresses the phosphorylation of Akt as well as other key downstream mediators of the pathway such as p21. CX-4945 induced apoptosis and caused cell cycle arrest in cancer cells in vitro. CX-4945 exhibited a dose-dependent antitumor activity in a xenograft model of PC3 prostate cancer model and was well tolerated. In vivo time-dependent reduction in the phosphorylation of the biomarker p21 at T145 was observed by immunohistochemistry. Inhibition of the newly validated CK2 target by CX-4945 represents a fresh therapeutic strategy for cancer.

Novel potent pyrimido[4,5-c]quinoline inhibitors of protein kinase CK2: SAR and preliminary assessment of their analgesic and anti-viral properties.

We describe the discovery of novel potent substituted pyrimido[4,5-c]quinoline ATP-competitive inhibitors of protein kinase CK2. A binding model of the inhibitors with the protein was elaborated on the basis of SAR and revealed various modes of interaction with the hinge region. Representative analog 14k (CK2 IC(50)=9 nM) showed anti-viral activity at nanomolar concentrations against HIV-1. Orally available compound 7e (CK2 IC(50)=3 nM) reduced pain in the phase II of a murine formalin model. These preliminary data confirm that properly optimized CK2 inhibitors may be used for anti-viral and pain therapy.

Discovery and SAR of 5-(3-chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic acid (CX-4945), the first clinical stage inhibitor of protein kinase CK2 for the treatment of cancer.

Herein we chronicle the discovery of CX-4945 (25n), a first-in-class, orally bioavailable ATP-competitive inhibitor of protein kinase CK2 in clinical trials for cancer. CK2 has long been considered a prime cancer drug target because of the roles of deregulated and overexpressed CK2 in cancer-promoting prosurvival and antiapoptotic pathways. These biological properties as well as the suitability of CK2's small ATP binding site for the design of selective inhibitors, led us to fashion novel therapeutic agents for cancer. The optimization leading to 25n (K(i) = 0.38 nM) was guided by molecular modeling, suggesting a strong binding of 25n resulting from a combination of hydrophobic interactions, an ionic bridge with Lys68, and hydrogen bonding with the hinge region. 25n was found to be highly selective, orally bioavailable across species (20-51%) and efficacious in xenograft models. The discovery of 25n will allow the therapeutic targeting of CK2 in humans for the first time.