Rohan Eric John Beckwith

Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide

In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4–RBX1–DDB1–CRBN (known as CRL4CRBN) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4CRBN. Here we present crystal structures of the DDB1–CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4CRBN and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN. Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.

Plate-Based Diversity Selection Based on Empirical HTS Data to Enhance the Number of Hits and Their Chemical Diversity

Typically, screening collections of pharmaceutical companies contain more than a million compounds today. However, for certain high-throughput screening (HTS) campaigns, constraints posed by the assay throughput and/or the reagent costs make it impractical to screen the entire deck. Therefore, it is desirable to effectively screen subsets of the collection based on a hypothesis or a diversity selection. How to select compound subsets is a subject of ongoing debate. The authors present an approach based on extended connectivity fingerprints to carry out diversity selection on a per plate basis (instead of a per compound basis). HTS data from 35 Novartis screens spanning 5 target classes were investigated to assess the performance of this approach. The analysis shows that selecting a fingerprint-diverse subset of 250K compounds, representing 20% of the screening deck, would have achieved significantly higher hit rates for 86% of the screens. This measure also outperforms the Murcko scaffold-based plate selection described previously, where only 49% of the screens showed similar improvements. Strikingly, the 2-fold improvement in average hit rates observed for 3 of 5 target classes in the data set indicates a target bias of the plate (and thus compound) selection method. Even though the diverse subset selection lacks any target hypothesis, its application shows significantly better results for some targets—namely, G-protein-coupled receptors, proteases, and protein-protein interactions—but not for kinase and pathway screens. The synthetic origin of the compounds in the diverse subset appears to influence the screening hit rates. Natural products were the most diverse compound class, with significantly higher hit rates compared to the compounds from the traditional synthetic and combinatorial libraries. These results offer empirical guidelines for plate-based diversity selection to enhance hit rates, based on target class and the library type being screened. (Journal of Biomolecular Screening 2009:690-699)

Cullin–RING ubiquitin E3 ligase regulation by the COP9 signalosome

The cullin–RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A–RBX1–DDB1–DDB2 complex (CRL4ADDB2) monitors the genome for ultraviolet-light-induced DNA damage. CRL4ADBB2 is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4ADDB2 and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN.

Reactive carbon species tamed for synthesis

A highly reactive form of carbon, known as a carbyne, holds great promise for organic synthesis, but has been difficult to prepare. Reactions that produce carbyne equivalents now unleash this synthetic potential. A highly reactive form of carbon, known as a carbyne, holds great promise for organic synthesis, but has been difficult to prepare. Reactions that produce carbyne equivalents now unleash this synthetic potential.

Abstract C54: Aryl-N-(1H-imidazol-2-yl)-acetamides: Nonpeptidic binders of Bir3 cIAP protein.

Inhibitor of Apoptosis Proteins (IAP) negatively regulate cell death through a caspase-3 and caspase-7 activation. IAP inhibitors (IAPi) were derived from the peptide sequence Alanine-Valine-Proline (AVP) based on Smac-protein binding to the BIR-3 domain of IAP protein. These peptidic compounds are low molecular weight and mimic Smac when binding to the BIR3 domain of XIAP, CIAP1, and CIAP2. However, peptides generally suffer from poor permeability that can potentially influence the amount of drugability largely due to the amide backbone and an alternate chemotype was sought. In an effort to find a replacement for AVP scaffolds, an FBS by NMR identified indole-carboxylic acid 1 as a hit in competitive binding studies. The indole scaffold was further refined through FEPOP, virtual screening, and SAR leading to the benzimidazole scaffold which upon further docking studies resulted in 2-aminobenzimidazole 2. In silico tools were utilized to propose a synthetic pharmacophore which resulted in a phenyl-hetero-imidazole tricyclic series peptidomimetic with only one amide moiety. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C54. Citation Format: Mark G. Palermo, Rohan Beckwith, Christopher S. Straub, Kara Herlihy, Yiping Shen, Xiaolu Zhang, Matthew Clapham, Brian Hurley. Aryl-N-(1H-imidazol-2-yl)-acetamides: Nonpeptidic binders of Bir3 cIAP protein. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C54.

Chapter Nineteen - Recent Advances in Small Molecule Target Identification Methods

Abstract In this chapter, four major target identification approaches including affinity-based proteomics, in silico target prediction, drug resistance-sequencing, and yeast-based approaches are discussed with demonstrative examples from the last few years.