Ensemble-Based Docking Using Biased Molecular Dynamics

Proteins are dynamic molecules, and understanding their movements, especially as they relate to molecular recognition and protein–ligand interactions, poses a significant challenge to structure-based drug discovery. In most instances, protein flexibility is underrepresented in computer-aided drug design due to uncertainties on how it should be accurately modeled as well as the computational cost associated with attempting to incorporate flexibility in the calculations. One approach that aims to address these issues is ensemble-based docking. With this technique, ligands are docked to an ensemble of rigid protein conformations. Molecular dynamics (MD) simulations can be used to generate the ensemble of protein conformations for the subsequent docking. Here we present a novel approach that uses biased-MD simulations to generate the docking ensemble. The MD simulations are biased toward an initial protein–ligand X-ray complex structure. The biasing maintains some of the original crystallographic pocket-ligand information and thereby enhances sampling of the more relevant conformational space of the protein. Resulting trajectories are clustered to select a representative set of protein conformations, and ligands are docked to that reduced set of conformations. Cross-docking to this ensemble and then selecting the lowest scoring pose enables reliable identification of the correct binding mode. Various levels of biasing are investigated, and the method is validated for cyclin-dependent kinase 2 and factor Xa

Evaluating Free Energies of Binding and Conservation of Crystallographic Waters Using SZMAP

The SZMAP method computes binding free energies and the corresponding thermodynamic components for water molecules in the binding site of a protein structure [SZMAP, 1.0.0; OpenEye Scientific Software Inc.: Santa Fe, NM, USA, 2011]. In this work, the ability of SZMAP to predict water structure and thermodynamic stability is examined for the X-ray crystal structures of a series of protein–ligand complexes. SZMAP results correlate with higher-level replica exchange thermodynamic integration double decoupling calculations of the absolute free energy of bound waters in the test set complexes. In addition, SZMAP calculations show good agreement with experimental data in terms of water conservation (across multiple crystal structures) and B-factors over a subset of the test set. In particular, the SZMAP neutral entropy difference term calculated at crystallographic water positions within each of the complex structures correlates well with whether that crystallographic water is conserved or displaceable. Furthermore, the calculated entropy of the water probe relative to the continuum shows a significant degree of correlation with the B-factors associated with the oxygen atoms of the water molecules. Taken together, these results indicate that SZMAP is capable of quantitatively predicting water positions and their energetics and is potentially a useful tool for determining which waters to attempt to displace, maintain, or build in through water-mediated interactions when evolving a lead series during a drug discovery program

Discovery and Mechanistic Study of a Small Molecule Inhibitor for Motor Protein KIFC1

Centrosome amplification is observed in many human cancers and has been proposed to be a driver of both genetic instability and tumorigenesis. Cancer cells have evolved mechanisms to bundle multiple centrosomes into two spindle poles to avoid multipolar mitosis that can lead to chromosomal segregation defects and eventually cell death. KIFC1, a kinesin-14 family protein, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division, suggesting that KIFC1 is an attractive therapeutic target for human cancers. To this end, we have identified the first reported small molecule inhibitor AZ82 for KIFC1. AZ82 bound specifically to the KIFC1/microtubule (MT) binary complex and inhibited the MT-stimulated KIFC1 enzymatic activity in an ATP-competitive and MT-noncompetitive manner with a <i>K</i>_i of 0.043 μM. AZ82 effectively engaged with the minus end-directed KIFC1 motor inside cells to reverse the monopolar spindle phenotype induced by the inhibition of the plus end-directed kinesin Eg5. Treatment with AZ82 caused centrosome declustering in BT-549 breast cancer cells with amplified centrosomes. Consistent with genetic studies, our data confirmed that KIFC1 inhibition by a small molecule holds promise for targeting cancer cells with amplified centrosomes and provided evidence that functional suppression of KIFC1 by inhibiting its enzymatic activity could be an effective means for developing cancer therapeutics

Discovery of Potent KIFC1 Inhibitors Using a Method of Integrated High-Throughput Synthesis and Screening

KIFC1 (HSET), a member of the kinesin-14 family of motor proteins, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division. To explore the potential of KIFC1 as a therapeutic target for human cancers, a series of potent KIFC1 inhibitors featuring a phenylalanine scaffold was developed from hits identified through high-throughput screening (HTS). Optimization of the initial hits combined both design–synthesis–test cycles and an integrated high-throughput synthesis and biochemical screening method. An important aspect of this integrated method was the utilization of DMSO stock solutions of compounds registered in the corporate compound collection as synthetic reactants. Using this method, over 1500 compounds selected for structural diversity were quickly assembled in assay-ready 384-well plates and were directly tested after the necessary dilutions. Our efforts led to the discovery of a potent KIFC1 inhibitor, <b>AZ82</b>, which demonstrated the desired centrosome declustering mode of action in cell studies

Discovery of Disubstituted Imidazo[4,5‑<i>b</i>]pyridines and Purines as Potent TrkA Inhibitors

Trk receptor tyrosine kinases have been implicated in cancer and pain. A crystal structure of TrkA with AZ-23 (<b>1a</b>) was obtained, and scaffold hopping resulted in two 5/6-bicyclic series comprising either imidazo­[4,5-<i>b</i>]­pyridines or purines. Further optimization of these two fusion series led to compounds with subnanomolar potencies against TrkA kinase in cellular assays. Antitumor effects in a TrkA-driven mouse allograft model were demonstrated with compounds <b>2d</b> and <b>3a</b>

Pyrimidinone Nicotinamide Mimetics as Selective Tankyrase and Wnt Pathway Inhibitors Suitable for in Vivo Pharmacology

The canonical Wnt pathway plays an important role in embryonic development, adult tissue homeostasis, and cancer. Germline mutations of several Wnt pathway components, such as Axin, APC, and ß-catenin, can lead to oncogenesis. Inhibition of the poly­(ADP-ribose) polymerase (PARP) catalytic domain of the tankyrases (TNKS1 and TNKS2) is known to inhibit the Wnt pathway via increased stabilization of Axin. In order to explore the consequences of tankyrase and Wnt pathway inhibition in preclinical models of cancer and its impact on normal tissue, we sought a small molecule inhibitor of TNKS1/2 with suitable physicochemical properties and pharmacokinetics for hypothesis testing in vivo. Starting from a 2-phenyl quinazolinone hit (compound <b>1</b>), we discovered the pyrrolopyrimidinone compound <b>25</b> (AZ6102), which is a potent TNKS1/2 inhibitor that has 100-fold selectivity against other PARP family enzymes and shows 5 nM Wnt pathway inhibition in DLD-1 cells. Moreover, compound <b>25</b> can be formulated well in a clinically relevant intravenous solution at 20 mg/mL, has demonstrated good pharmacokinetics in preclinical species, and shows low Caco2 efflux to avoid possible tumor resistance mechanisms

Structure Based Design of Non-Natural Peptidic Macrocyclic Mcl‑1 Inhibitors

Mcl-1 is a pro-apoptotic BH3 protein family member similar to Bcl-2 and Bcl-xL. Overexpression of Mcl-1 is often seen in various tumors and allows cancer cells to evade apoptosis. Here we report the discovery and optimization of a series of non-natural peptide Mcl-1 inhibitors. Screening of DNA-encoded libraries resulted in hit compound <b>1</b>, a 1.5 μM Mcl-1 inhibitor. A subsequent crystal structure demonstrated that compound <b>1</b> bound to Mcl-1 in a β-turn conformation, such that the two ends of the peptide were close together. This proximity allowed for the linking of the two ends of the peptide to form a macrocycle. Macrocyclization resulted in an approximately 10-fold improvement in binding potency. Further exploration of a key hydrophobic interaction with Mcl-1 protein and also with the moiety that engages Arg256 led to additional potency improvements. The use of protein–ligand crystal structures and binding kinetics contributed to the design and understanding of the potency gains. Optimized compound <b>26</b> is a <3 nM Mcl-1 inhibitor, while inhibiting Bcl-2 at only 5 μM and Bcl-xL at >99 μM, and induces cleaved caspase-3 in MV4–11 cells with an IC₅₀ of 3 μM after 6 h

Structure Based Design of Non-Natural Peptidic Macrocyclic Mcl‑1 Inhibitors

Mcl-1 is a pro-apoptotic BH3 protein family member similar to Bcl-2 and Bcl-xL. Overexpression of Mcl-1 is often seen in various tumors and allows cancer cells to evade apoptosis. Here we report the discovery and optimization of a series of non-natural peptide Mcl-1 inhibitors. Screening of DNA-encoded libraries resulted in hit compound <b>1</b>, a 1.5 μM Mcl-1 inhibitor. A subsequent crystal structure demonstrated that compound <b>1</b> bound to Mcl-1 in a β-turn conformation, such that the two ends of the peptide were close together. This proximity allowed for the linking of the two ends of the peptide to form a macrocycle. Macrocyclization resulted in an approximately 10-fold improvement in binding potency. Further exploration of a key hydrophobic interaction with Mcl-1 protein and also with the moiety that engages Arg256 led to additional potency improvements. The use of protein–ligand crystal structures and binding kinetics contributed to the design and understanding of the potency gains. Optimized compound <b>26</b> is a <3 nM Mcl-1 inhibitor, while inhibiting Bcl-2 at only 5 μM and Bcl-xL at >99 μM, and induces cleaved caspase-3 in MV4–11 cells with an IC₅₀ of 3 μM after 6 h