Applications of Biophysics in High Throughput Screening Hit Validation.

For approximately a decade, biophysical methods have been utilized to validate positive hits selected from high throughput screening campaigns with the goal to verify binding interactions using label-free assays. By applying label-free readouts, screen artifacts created by compound interference and fluorescence are discovered, enabling further characterization of the hits for their target specificity and selectivity. The use of several biophysical methods to extract this type of high content information is required in order to prevent the promotion of false positives to the next level of hit validation and to select the best candidates for further chemical optimization. The typical technologies applied in this arena include DLS (Dynamic Light Scattering), Turbidometry, RWG (Resonance Waveguide Grating), SPR (Surface Plasmon Resonance), DSF (Differential Scanning Fluorimetry), MS (Mass Spectrometry), and others. Each technology can provide different types of information of the binding interaction of interest. Thus, these technologies can be incorporated in a hit validation strategy according to the profile of chemical matter that is desired by the medicinal chemists and naturally to the amenability of the target protein to the technology’s screening format. Here we present the results of several screening strategies using biophysics with the objective to compare their approaches, discuss their advantages and challenges, and summarize their benefits in reference to lead discovery

Identification of a 5-[3-phenyl-(2-cyclic-ether)-methylether]-4-aminopyrrolo[2,3-d]pyrimidine series of IGF-1R inhibitors

We report structure-guided modifications of the benzyloxy substituent of the Insulin-like Growth Factor-1 Receptor (IGF-1R) inhibitor NVP-AEW541. This chemical group has been shown to confer selectivity against other protein kinases but at the expense of a metabolism liability. X-ray crystallography has revealed that the benzyloxy moiety interacts with a lysine cation of the IGF-1R kinase domain via its ether function and its aromatic pi-system and is nicely embedded in an induced hydrophobic pocket. We show that 1,4-diethers displaying an adequate hydrophobic and constrained shape are advantageous benzyloxy replacements. A single digit nanomolar inhibitor (compound 20, IC50 = 8.9 nM) was identified following this approach

Structure of TPR Domain–Peptide Complexes: Critical Elements in the Assembly of theHsp70–Hsp90 Multichaperone Machine

AbstractThe adaptor protein Hop mediates the association of the molecular chaperones Hsp70 and Hsp90. The TPR1 domain of Hop specifically recognizes the C-terminal heptapeptide of Hsp70 while the TPR2A domain binds the C-terminal pentapeptide of Hsp90. Both sequences end with the motif EEVD. The crystal structures of the TPR–peptide complexes show the peptides in an extended conformation, spanning a groove in the TPR domains. Peptide binding is mediated by electrostatic interactions with the EEVD motif, with the C-terminal aspartate acting as a two-carboxylate anchor, and by hydrophobic interactions with residues upstream of EEVD. The hydrophobic contacts with the peptide are critical for specificity. These results explain how TPR domains participate in the ordered assembly of Hsp70–Hsp90 multichaperone complexes

Beyond Consensus: Leveraging Inherent Biases in Fragment Based Screening Technologies

Fragment-based drug discovery (FBDD) has emerged as a strategy with great potential to recast the modern drug discovery process. Central to this method is the identification of small and simple molecular building blocks that bind a target of interest, and the subsequent incorporation of salient features of these fragments into larger and more complex compounds with enhanced target affinity and selectivity. A first step in FBDD often entails a fragment-based screen (FBS) to identify fragment “hits.” While there are theoretical advantages of using FBDD at the earliest stages of a drug discovery program, hurdles such as the integration of conflicting results from orthogonal screens have hindered its success. We present the meta-analysis of 35 fragment based campaigns at Novartis, which employed a generic 1,400 fragment library against diverse target families using various biophysical and biochemical techniques. By statistically interrogating the multidimensional FBS data, we aim to answer three questions: 1) What makes a fragment amenable for FBS? 2) How do different fragment screening technologies compare with each other? 3) What is the best way to pair FBS assay technologies? In addition to identifying properties that render fragments amenable for FBS, we compare in an unprecedented scale various screening technologies. We propose a quantitative way to compare the bias in various screening technologies, and demonstrate how this approach can be used to select technologies in order to ensure the greatest coverage of fragment hits

Optimization of a fragment-based screening hit towards potent DOT1L inhibitors interacting in an induced binding pocket

Mixed lineage leukemia (MLL) gene rearrangement induces leukemic transformation by ectopic recruitment of disruptor of telomeric silencing 1-like protein (DOT1L), a lysine histone methyltransferase, leading to local hypermeth-ylation of H3K79 and misexpression of genes (including HoxA) which drive the leukemic phenotype. A weak fragment-based screening hit identified by SPR was co-crystallized with DOT1L and optimized using structure-based ligand optimization to yield compound 8 (IC50 = 14 nM). This series of inhibitors is structurally not related to cofactor SAM and is not interacting with the SAM binding pocket but induces a pocket adjacent to the SAM binding site

Ligand discrimination by TPR domains. Relevance and selectivity of EEVD-recognition in Hsp70·Hop·Hsp90 complexes

Protein-protein interaction modules containing so-called tetratricopeptide repeats (TPRs) mediate the assembly of Hsp70/Hsp90 multi-chaperone complexes. The TPR1 and TPR2A domains of the Hsp70/Hsp90 adapter protein p60/Hop specifically bind to short peptides corresponding to the C-terminal tails of Hsp70 and Hsp90, respectively, both of which contain the highly conserved sequence motif EEVD-COOH. Here, we quantitatively assessed the contribution of TPR-mediated peptide recognition to Hsp70.Hop.Hsp90 complex formation. The interaction of TPR2A with the C-terminal pentapeptide of Hsp90 (AMEVD) is identified as the core contact for Hop binding to Hsp90. (In peptide sequences, italics are used to highlight residues specific for Hsp70 or Hsp90.) In contrast, formation of the Hsp70.Hop complex depends not only on recognition of the C-terminal Hsp70 heptapeptide (PTIEEVD) by TPR1 but also on additional contacts between Hsp70 and Hop. The sequence motifs for TPR1 and TPR2A binding were defined by alanine scanning of the C-terminal octapeptides of Hsp70 and Hsp90 and by screening of combinatorial peptide libraries. Asp0 and Val-1 of the EEVD motif are identified as general anchor residues, but the highly conserved glutamates of the EEVD sequence, which are critical in Hsp90 binding by TPR2A, do not contribute appreciably to the interaction of Hsp70 with TPR1. Rather, TPR1 prefers hydrophobic amino acids in these positions. Moreover, the TPR domains display a pronounced tendency to interact preferentially with hydrophobic aliphatic and aromatic side chains in positions -4 and -6 of their respective peptide ligands. Ile-4 in Hsp70 and Met-4 in Hsp90 are most important in determining the specific binding of TPR1 and TPR2A, respect

Pyrrolo-pyrimidones: a novel class of MK2 inhibitors with potent cellular activity.

Pyrrolo-pyrimidones of the general structure 1 were synthesized and evaluated for their potential as MK2 inhibitors. Potent derivatives were discovered which inhibit MK2 in the nanomolar range and show potent inhibition of cytokine release from LPS-stimulated monocytes. These derivatives were shown to inhibit phosphorylation of hsp27, a downstream target of MK2 and are modestly selective in a panel of 28 kinases

Novel 3-Aminopyrazole Inhibitors of MK-2 Discovered by Scaffold Hopping Strategy

New, selective 3-aminopyrazole based MK2-inhibitors were discovered by scaffold hopping strategy. The new derivatives proved to inhibit intracellular phosphorylation of hsp27 as well as LPS-induced TNFa release in cells. In addition, selected derivative 14e also inhibited LPS-induced TNFa release in vivo

New potent DOT1L inhibitors for in vivo evaluation in mouse

In MLL-rearranged cancer cells, disruptor of telomeric silencing 1-like protein (DOT1L) is aberrantly recruited to ectop-ic loci leading to local hypermethylation of H3K79 and consequently misexpression of leukemogenic genes. A struc-ture-guided optimization of a HTS hit led to the discovery of DOT1L inhibitors with subnanomolar potency, allowing to test the therapeutic principle of DOT1L inhibition in a preclinical mouse tumor xenograft model. Compounds dis-playing good exposure in mouse and nanomolar inhibition of target gene expression in cell were obtained and tested in viv

Optimization of a Fragment-Based Screening Hit toward Potent DOT1L Inhibitors Interacting in an Induced Binding Pocket

Mixed lineage leukemia (MLL) gene rearrangement induces leukemic transformation by ectopic recruitment of disruptor of telomeric silencing 1-like protein (DOT1L), a lysine histone methyltransferase, leading to local hypermethylation of H3K79 and misexpression of genes (including HoxA), which drive the leukemic phenotype. A weak fragment-based screening hit identified by SPR was cocrystallized with DOT1L and optimized using structure-based ligand optimization to yield compound <b>8</b> (IC₅₀ = 14 nM). This series of inhibitors is structurally not related to cofactor SAM and is not interacting within the SAM binding pocket but induces a pocket adjacent to the SAM binding site