Computational Models of the Gastrointestinal Environment. 1. The Effect of Digestion on the Phase Behavior of Intestinal Fluids

Improved models of the gastrointestinal environment have great potential to assist the complex process of drug formulation. Molecular dynamics (MD) is a powerful method for investigating phase behavior at a molecular level. In this study we use multiple MD simulations to calculate phase diagrams for bile before and after digestion. In these computational models, undigested bile is represented by mixtures of palmitoyl-oleoylphosphatidylcholine (POPC), sodium glycodeoxycholate (GDX), and water. Digested bile is modeled using a 1:1 mixture of oleic acid and palmitoylphosphatidylcholine (lysophosphatidylcholine, LPC), GDX, and water. The computational phase diagrams of undigested and digested bile are compared, and we describe the typical intermolecular interactions that occur between phospholipids and bile salts. The diffusion coefficients measured from MD simulation are compared to experimental diffusion data measured by DOSY-NMR, where we observe good qualitative agreement. In an additional set of simulations, the effect of different ionization states of oleic acid on micelle formation is investigated

Rapid Elaboration of Fragments into Leads Applied to Bromodomain‑3 Extra-Terminal Domain

The development of low-affinity fragment hits into higher-affinity leads is a major hurdle in fragment-based drug design. Here, we demonstrate the Rapid Elaboration of Fragments into Leads (REFiL) by applying an integrated workflow that provides a systematic approach to generate higher-affinity binders without the need for structural information. The workflow involves the selection of commercial analogues of fragment hits to generate preliminary structure–activity relationships. This is followed by parallel microscale chemistry using chemoinformatically designed reagent libraries to rapidly explore chemical diversity. After a fragment screen against bromodomain-3 extra-terminal (BRD3-ET) domain, we applied the REFiL workflow, which allowed us to develop a series of ligands that bind to BRD3-ET. With REFiL, we were able to rapidly improve binding affinity > 30-fold. REFiL can be applied readily to a broad range of proteins without the need for a structure, allowing the efficient evolution of low-affinity fragments into higher-affinity leads and chemical probes

Rapid Elaboration of Fragments into Leads Applied to Bromodomain‑3 Extra-Terminal Domain

The development of low-affinity fragment hits into higher-affinity leads is a major hurdle in fragment-based drug design. Here, we demonstrate the Rapid Elaboration of Fragments into Leads (REFiL) by applying an integrated workflow that provides a systematic approach to generate higher-affinity binders without the need for structural information. The workflow involves the selection of commercial analogues of fragment hits to generate preliminary structure–activity relationships. This is followed by parallel microscale chemistry using chemoinformatically designed reagent libraries to rapidly explore chemical diversity. After a fragment screen against bromodomain-3 extra-terminal (BRD3-ET) domain, we applied the REFiL workflow, which allowed us to develop a series of ligands that bind to BRD3-ET. With REFiL, we were able to rapidly improve binding affinity > 30-fold. REFiL can be applied readily to a broad range of proteins without the need for a structure, allowing the efficient evolution of low-affinity fragments into higher-affinity leads and chemical probes

NMR data driven-HADDOCK docking model of PaDsbA—Fragment 1 complex.

<p><b>(A)</b> Overlay of the two hundred best scoring HADDOCK model structures after water refinement showing the conformation of Fragment <b>1</b> in the complex. <b>(B)</b> The lowest energy conformer from the HADDOCK calculation is shown as a representative model of the PaDsbA—Fragment <b>1</b> complex. PaDsbA1 is shown in cartoon. Methyl containing residues for which intermolecular NOEs were detected, are shown in blue-white sticks. Val19 and Val21 methyls are present in close proximity of the Fragment <b>1</b> binding site (shown in magenta sticks), however no NOE cross peaks were observed from these methyls to ligand protons.</p

Validated fragment hits with K_D values < 5 mM.

<p>Validated fragment hits with K_D values < 5 mM.</p

X-ray crystal structure of PaDsbA1-Fragment 1 complex.

<p>The structure of PaDsbA1 in complex with Fragment <b>1</b> was solved by X-ray crystallography. <b>(A)</b> Residues from both the helical (H2, H6) and TRX (B2, B1-B2, H6) domains contribute to the binding of Fragment <b>1</b>. Selected side chains, which make contact with Fragment <b>1</b> are shown as sticks, and hydrogen bonds identified in the complex as dashed black lines. <b>(B)</b> 2Fo-Fc (blue) electron density map for Fragment <b>1</b>, was generated from a simulated annealing omit map and is shown contoured at 1.0 σ. The maps are shown within a 2 Å radius of each atom of Fragment <b>1</b>. <b>(C)</b> Stereo representation highlighting the subset of side chain residues involved in either hydrogen bond or hydrophobic contacts with Fragment <b>1</b> in the complex. Hydrogen bonds are shown as black dashed lines.</p

Structural statistics for the bundle of 20 energy-minimized NMR conformers of apo oxidised PaDsbA1 (3–192).

<p>Structural statistics for the bundle of 20 energy-minimized NMR conformers of apo oxidised PaDsbA1 (3–192).</p

Assignment of methyl resonances of oxidized PaDsbA1 in the presence of Fragment 1.

<p><b>(A)</b> Overlay of the methyl region of the ¹³C-HSQC spectra of PaDsbA1 in the absence (blue) and presence (red) of Fragment <b>1</b>. A subset of residues undergoes significant chemical shift perturbations. <b>(B)</b> Overlay of the ¹³C-HSQC spectra of PaDsbA1 in the absence (blue) and presence increasing concentrations of Fragment <b>1</b> (0.2–3.3 mM). Selected methyl resonance assignments are shown. The resonances for Leu63 and Leu144 undergo the largest chemical shift perturbations as indicated by arrows.</p

NMR data driven HADDOCK docking model and X-ray crystal structure of PaDsbA1–Fragment 1 complex.

<p>The representative HADDOCK model and the crystal structure are shown in cartoon representation coloured green and magenta, respectively. The binding mode of Fragment <b>1</b> (shown as sticks) is similar in both models.</p

X-ray crystal structure of PaDsbA1 and Fragment 1: Refinement and model quality.

<p>X-ray crystal structure of PaDsbA1 and Fragment 1: Refinement and model quality.</p