Discovery of 2‑Acylaminothiophene-3-Carboxamides as Multitarget Inhibitors for BCR-ABL Kinase and Microtubules

The emergence of drug resistance of the BCR-ABL kinase inhibitor imatinib, especially toward the T315I gatekeeper mutation, poses a great challenge to targeted therapy in treating chronic myeloid leukemia (CML) patients. To discover novel inhibitors against drug-resistant CML bearing T315I mutation, we applied a physics-based hierarchical virtual screening approach to dock a large chemical library against ATP binding pockets of both wild-type (WT) and T315I mutant ABL kinases in a combinatorial fashion. This strategy automatically resulted in 87 compounds satisfying structural and energetic criteria of both WT and T315I mutant kinases. Among them, nine compounds, which share a common thiophene-based scaffold and adopt similar binding poses, were chosen for experimental testing and one of them was shown to have low micromolar inhibition activities against both WT and mutant ABL kinases. Structure–activity relationship analysis with a series of structural modifications based on 2-acylaminothiophene-3-carboxamide scaffold supports our predicted binding mode. Interestingly, the same chemical scaffold was also enriched in our previous virtual screening campaign against colchicine site of microtubules using the same computational protocol, which suggests our virtual screening strategy is capable of discovering small-molecule ligands targeting distinct protein binding sites without sharing any sequential and structural similarity. Furthermore, the multitarget inhibition activity of this class of compounds was assessed in cellular experiments. We expect that the 2-acylaminothiophene-3-carboxamide scaffold may serve as a promising starting point for developing multitarget inhibitors in cancer treatment by targeting both kinases and microtubules

Evaluation and Application of MD-PB/SA in Structure-Based Hierarchical Virtual Screening

Molecular dynamics (MD) based molecular mechanics Poisson–Boltzmann and surface area (MM-PB/SA) calculation (MD-PB/SA) has been widely used to estimate binding free energies for receptor–ligand complexes. While numerous reports have focused on assessing accuracy and efficiency, fewer studies have paid attention to performance in lead discovery. In the present study, we report a critical evaluation of MD-PB/SA in hierarchical virtual screening (HVS) both theoretically and practically. It is shown that based on native poses, MD-PB/SA could be well applied to predict the relative binding energy for both congeneric and diverse ligands for different protein targets. However, there is a limitation for MD-PB/SA to distinguish the native pose of one ligand from the artificial pose of another when a huge difference exists between two molecules. By combining a physics-based scoring function with a knowledge-based structural filter, we improve the predictability and validate the practical use of MD-PB/SA in lead discovery by identifying novel inhibitors of p38 MAP kinase. We also expand our study to other protein targets such as HIV-1 RT and NA to assess the general validity of MD-PB/SA

The schematic of hierarchical protein-protein docking procedure.

<p>The schematic of hierarchical protein-protein docking procedure.</p

The structural and energetic properties of six predetermined models through 10 ns MD simulation.

<p>The interaction energy is accounting the interaction energy between the kinase and pseudokinase domain. PK_RMSF is the RMSF of Cα atom of JH2 after superimposing on kinase domain in MD simulation. BSA is the buried surface areas and H/I are contacts between the hydrophobic residues within 5 Å and charged residues within 6 Å in the interfaces. KH_RMSF is the RMSF of Cα atom in kinase αC helix (residues 885 to 907) after superimposing on kinase domain in MD simulation.</p

The domain organization of full length JAK2.

<p>The domain organization of full length JAK2.</p

Starting models were generated by connecting sites identified by MutInf or by using dimer geometries from crystal structures.

<p>(A) Two predefined packing models were constructed by manually joining the αC helix in the JAK2 kinase domain with two highly coupled sites in the JH2 pseudokinase domain identified by MutInf. (B) Four additional packing models were built by alignment to crystal structures of kinase dimeric forms (FGFR1, FGFR2, BRAF and PKB).</p

Mutation of interfacial residues changed JAK2 kinase activity.

<p>(A) JAK2 mutants showed increased basal kinase activity. Activity of HA-tagged JAK2 mutants was measured by phospho-JAK2 antibodies. Total JAK2 level was measured by anti-HA antibodies. P-JAK2: phosphorylated JAK2. V: vector alone. WT: wild-type JAK2. (B) Hyperactive JAK2 mutants showed increased STAT5 activation. Activation of STAT5 was assessed using flow cytometry with Alexa647-conjugated antibodies to phospho-STAT5. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003022#s2" target="_blank">Results</a> were normalized to wild-type JAK2. (C) Hyperactive JAK2 mutants transformed BaF3/EpoR cells into factor-independent growth. Cell growth at each indicated day was measured by MTT assay. JAK2-V617F expressing cells became saturated on Day 5. WT: wild-type JAK2. (D) Mutations in JH1 reduced basal JAK2 kinase activity. Activation of STAT5 was determined as in (B). <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003022#s2" target="_blank">Results</a> were normalized to wild-type JAK2.</p

Structure-Based Discovery of Novel and Selective 5‑Hydroxytryptamine 2B Receptor Antagonists for the Treatment of Irritable Bowel Syndrome

Here we employed structure-based ligand discovery techniques to explore a recently determined crystal structure of the 5-hydroxytryptamine 2B (5-HT_2B) receptor. Ten compounds containing a novel chemical scaffold were identified; among them, seven molecules were active in cellular function assays with the most potent one exhibiting an IC₅₀ value of 27.3 nM. We then systematically probed the binding characteristics of this scaffold by designing, synthesizing, and testing a series of structural modifications. The structure–activity relationship studies strongly support our predicted binding model. The binding profiling across a panel of 11 5-HT receptors indicated that these compounds are highly selective for the 5-HT_2B receptor. Oral administration of compound <b>15</b> (30 mg/kg) produced significant attenuation of visceral hypersensitivity in a rat model of irritable bowel syndrome (IBS). We expect this novel scaffold will serve as the foundation for the development of 5-HT_2B antagonists for the treatment of IBS

Motions of the activation loop and αC-helix in JAK2-WT and JAK2-V617F in 30 ns MD simulation.

<p>(A) The JAK2-V617F mutant increases the flexibility of the activation loop in the kinase domain by restricting the movement of αC helix region in the JH2. The activation loop in the kinase domain of JAK2-WT (black) and JAK2-V617F (red) is shown alongside the crystal structure (green) in cartoon representation using PyMOL. Other residues are shown in surface representation. (B) RMSDs of Cα atoms during 30 ns of MD simulation show displacement of the kinase domain activation loop residues 994–1028 in the V617F mutant (black) but not the wildtpype (red). (C) Conformations of the αC helix in kinase domain after 30 ns MD for V617F mutant (black) and wild type (red). (D) RMSDs for the kinase αC helix (residues 586–606) in JAK2-WT and JAK2-V617F during 30 ns MD simulation show that the position of this helix is relatively stable in both cases. RMSDs are computed over Cα atoms of JAK2 with respect to the initial model after superposition of the kinase domain.</p

Correlated motions couple active to putative allosteric sites in the JAK2 JH1 and JH2 domains.

<p>(A) The pairwise matrix of the highly coupled residues in the inactive conformation of the JAK2 JH1 domain. (B) Strong correlations between the activation loop (green box) and the αC helix (red box) are observed in the JH1 domain (yellow in cartoon). (C) The pairwise matrix of the highly coupled residues in the active conformation of the JH2 domain. (D) The strong correlations of the loop between β7–β8 sheets near the hinge region (green box) with activation loop (red box) shown in the JH2 domain (blue in cartoon).</p