Over 250 000 individual protein-protein interactions (PPIs) were identified in human proteome. Although many of these exhibit topologically complex and formidable surface areas, in many instances the successful binding is attained via a limited number of key residues ("hot spots“). There are multiple reports in the literature describing both computational and diversity-oriented approaches to developing small-molecule modulators of PPIs. Of these, non-peptidic a-helix and b-turn imetics are of particular importance due to their key role in multiple deregulated pathways leading to cancer, neurodegenerative disorders, inflammation and immunological disorders.

Library design

Traditional three residue approach

Alpha-helix Mimetics

Beta-turn Mimetics

Ligand-based approach

Target based approach. We carried out design of a focused compound set targeting selected alpha-helix domains and beta-turns such as mdm2-p53, mdm2-CK1α, HIF1-α , MEF2-HDAC4, HIV-1 gp41, CD81/CV, Bcl-2/Bcl-xL, etc. After the selection by virtual screening the preference was set to the structures bearing new chemotypes.

‘Traditional’ three-residue appraoch included design of compounds with unusual cores bearing regular key-residues that mimics i, i+3/i+4 and i+7-residues located on the one recognition face (‘hot spots’). In modeling a-helix interfaces, we accounted also two-face helix mimetics. For beta-turn mimetics we applied three query models with distinct types of interaction: π-π stacking, cation-π and H-bond interaction, S-π interaction. : The latter focused design exercise was exemplified by modeling the interaction between Bim BH3 domain with Mcl-1 and Bcl-2 and identifying fused polyhydrogenated aza-heterocycles with high Fsp³ character.

Ligand based approach. New chemotypes with close topology to known alpha-helix and beta-turns mimetics were selected. Shape based similarity and Pharmacophore screening were used as a main tools for the library design.

Unique Fsp³-enriched scaffolds exhibiting ladder-like cyclic skeletons were specially selected to enhance topological and pharmacophore interaction with the target alpha-helix and beta-turn motifs.

Examples of Selected Scaffolds in the Library

Until recently, covalent binders targeting Lysine residue attracted less attention as compared to those modifying Cysteine or Serine. Being among the essential amino acids, Lysine can be found both at the surface and in active sites of many enzymes (e.g. kinases, viral polymerases and integrases, aldolases, DOPA decarboxylase, P-glycoprotein), and it can also participate in catalytic reactions. Moreover, Lys residue is often involved as a key player in many important signaling and metabolic processes. An example can be protein ubiquitination, which mainly occurs through lysine residues on substrate proteins or itself. Selective modification of Lys residue becomes more and more attractive for the investigation of many signaling processes in live cells and, potentially, can play a key role in the discovery of next-generation drugs.

Lysine focused Library is plated at 20 mM concentration in DMSO and is available for fast supply in any custom format. Compounds pooling based on delta MW can be provided as a formatting option. Please request the list of expected molecular weight shifts for MS-based screenings.

Examples of compounds in the library

To address the increased interest and need for lysine-specific covalent binders, we have synthesized several libraries bearing warheads described as selective/preferred toward Lysine. Our renewed library contains only specially synthesized compounds with Lys-specific covalent warheads. The following types of covalent binders were used for the library construction:

Salicylic aldehydes

• Lys-specific, reversible
• The o-hydroxy group stabilizes covalent adducts
• Low reactivity

Vinyl Sulfonamides

• Irreversible binders
• Showed some selectivity toward Lys over Cys
• Highly reactive

α-Formyl Boronic acids

• Reversible and Lys specific
• Boronic residue in α-position dramatically enhances the stability of resulting adducts

Sulfonyl Fluorides

• Irreversible binders for Lys, pH-dependent
• Reactivity depends on structure
• Compounds selected to display moderate reactivity

Distribution by covalent warhead

The library can be screened directly at Enamine in our biology laboratories, which offer a wide range of screening techniques including MS-based screening, kinetics, and intrinsic reactivity measurements. In this case, we will be happy to offer you a discount on library costs and follow-up services depending on the collaboration scope.

Solubility is critically important for fragment-based screening; assured solubility of fragments at high concentrations can prevent a number of issues during the screening procedure. We have confirmed experimentally aqueous solubility for 7 500 fragments in standard phosphate buffer at 1 mM; measurements were performed using nephelometry-based method. Representative subset of 3 000 compounds was designed using multi-vectoral diversity selection.

Key features:

• High structural diversity was achieved via two approaches: diversity selection using fingerprint-based Tanimoto distance and molecular framework frequency analysis. Compounds bearing trivial and abundant chemotypes were removed to enhance novelty of the set.
• Guaranteed aqueous solubility at 1 mM in PBS buffer and at 200 mM in DMSO
• Soluble Fragment Diversity Set can be readily followed with analogues either from stock or from validated syntheses. All required building blocks are available from Enamine stock.

Enamine has been working on synthesis of new sp³-rich heterocycles and expanding of spirocyclic chemistry already over 16 years. Our own research in this area enabled synthesis of large variety of 3D shaped molecules that are well represented in stock screening collection. Synthesis of derivatized series of aliphatic and sterically hindered cores, often exclusive only for Enamine, led to a number of readily available analogs that is a crucial point in fragment hit optimization and follow-up stage.

Library design

• Ro3 compliant dataset was refined with strict MedChem filters (FAF-Drugs3) and Fsp³cut-off 0.35
• 3D-dimensionality criteria: NPR1≥ 0.15; NPR2≥ 1.15 – value of npr1 (PMI plot 1)
• K-mean clustering of preselected 8 000 3D fragments has been carried out using NPR1/2 values. Only centroid molecules were included in the library, PMI plot 2

PMI plot 1: green dots correspond to 8 000 compounds indicated as 3D Fragments Set from Enamine in-stock Ro3 compliant molecules (grey dots).

PMI plot 2: distribution of molecules in the library (centroids, blue spots) among of 8 000 of 3D-shaped initial

Examples of the molecules in the library

Z362565298

Z133805700

Z2261979776

Z1681343197

Z2111525185

Z2044981890

Z1262610408

Z2301534484