Fragment-Based Discovery of Novel VE-PTP Inhibitors Using Orthogonal Biophysical Techniques.

Tyrosine phosphorylation is an essential post-translational modification that regulates various biological events and is implicated in many diseases including cancer and atherosclerosis. Vascular endothelial protein tyrosine phosphatase (VE-PTP), which plays an important role in vascular homeostasis and angiogenesis, is therefore an attractive drug target for these diseases. However, there are still no drugs targeting PTP including VE-PTP. In this paper, we report the discovery of a novel VE-PTP inhibitor, Cpd-2, by fragment-based screening combining various biophysical techniques. Cpd-2 is the first VE-PTP inhibitor with a weakly acidic structure and high selectivity, unlike known strongly acidic inhibitors. We believe that this compound represents a new possibility for the development of bioavailable VE-PTP inhibitors.

System truncation accelerates binding affinity calculations with the fragment molecular orbital method: A benchmark study.

The fragment molecular orbital (FMO) method is a fast quantum-mechanical method that divides systems into pieces of fragments and performs ab initio calculations. The system truncation enables further speed improvement. In this article, we systematically study the effects of system truncations on binding affinity calculations obtained with FMO in combination with either the polarizable continuum model (FMO/PCM) or in combination with the Møller-Plesset method (FMO-MP2). We have used five protein complexes with ligands of several charged states. The calculated binding energies of the size variants of the truncated system, including only a restricted number of atoms around the ligand, are compared to the energy obtained from a full system. The result shows that the systems could be truncated to a radius of 8 Å from neutral ligands within an error of 0.7 kcal/mol, and 12 Å from charged ligands within an error of 1.1 kcal/mol for calculating the binding energy in solution.

Antiviral Activity and Resistance Profile of the Novel HIV-1 Non-Catalytic Site Integrase Inhibitor JTP-0157602.

HIV-1 integrase (IN) is an essential enzyme for viral replication. Non-catalytic site integrase inhibitors (NCINIs) are allosteric HIV-1 IN inhibitors and a potential new class of antiretrovirals. In this report, we identified a novel NCINI, JTP-0157602, with an original scaffold. JTP-0157602 exhibited potent antiviral activity against HIV-1 and showed a serum-shifted 90% effective concentration (EC90) of 138 nM, which is comparable to those of the FDA-approved IN strand transfer inhibitors (INSTIs). This compound was fully potent against a wide range of recombinant viruses with IN polymorphisms, including amino acids 124/125, a hot spot of IN polymorphisms. In addition, JTP-0157602 retained potent antiviral activity against a broad panel of recombinant viruses with INSTI-related resistance mutations, including multiple substitutions that emerged in clinical studies of INSTIs. Resistance selection experiments of JTP-0157602 led to the emergence of A128T and T174I mutations, which are located at the lens epithelium-derived growth factor/p75 binding pocket of IN. JTP-0157602 inhibited HIV-1 replication mainly during the late phase of the replication cycle, and HIV-1 virions produced by reactivation from HIV-1 latently infected Jurkat cells in the presence of JTP-0157602 were noninfectious. These results suggest that JTP-0157602 and analog compounds can be used to treat HIV-1 infectious diseases. IMPORTANCE Non-catalytic site integrase inhibitors (NCINIs) are allosteric HIV-1 integrase (IN) inhibitors that bind to the lens epithelium-derived growth factor (LEDGF)/p75 binding pocket of IN. NCINIs are expected to be a new class of anti-HIV-1 agents. In this study, we present a novel NCINI, JTP-0157602, which has potent activity against a broad range of HIV-1 strains with IN polymorphisms. Furthermore, JTP-0157602 shows strong antiviral activity against IN strand transfer inhibitor-resistant mutations, suggesting that JTP-0157602 and its analogs are potential agents for treating HIV-1 infections. Structural modeling indicated that JTP-0157602 binds to the LEDGF/p75 binding pocket of IN, and the results of in vitro resistance induction revealed the JTP-0157602 resistance mechanism of HIV-1. These data shed light on developing novel NCINIs that exhibit potent activity against HIV-1 with broad IN polymorphisms and multidrug-resistant HIV-1 variants.

The discovery of 3,3-dimethyl-1,2,3,4-tetrahydroquinoxaline-1-carboxamides as AMPD2 inhibitors with a novel mechanism of action.

AMP deaminase 2 (AMPD2) has been thought to play an important role in energy homeostasis and immuno-oncology, while selective AMPD2 inhibitors are highly demanded to clarify the physiological function of AMPD2. In this report, we describe selective AMPD2 inhibitors inducing allosteric modulation. Based on hypothesis that compounds that exhibit increased inhibition by preincubation would cause conformational change of the enzyme, starting from HTS hit compound 4, we discovered compound 8 through the SAR study. From X-ray structural information of 8, this chemical series has a novel mechanism of action that changes the substrate pocket to prevent AMP from binding. Further elaboration of compound 8 led to the tool compound 21 which exhibited potent inhibitory activity of AMPD2 in ex vivo evaluation of mouse liver.

Fragment Molecular Orbital Based Affinity Prediction toward Pyruvate Dehydrogenase Kinases: Insights into the Charge Transfer in Hydrogen Bond Networks.

The fragment molecular orbital (FMO) method is a fast quantum-mechanics method that divides systems into pieces of fragments and performs ab initio calculations. The method has been expected to improve the accuracy of describing protein-ligand interactions by incorporating electronic effects. In this article, FMO calculation with solvation methods were applied to the affinity prediction at the ATP-binding site of PDHK4. As the ionized aspartic acid lies at the center and is involved in the complex hydrogen bond networks, this system has turned out to be a difficult target to describe by traditional molecular-mechanics method. In the FMO calculation with the polarizable continuum model (PCM) solvation method, a considerable amount of charge (-0.27e) was transferred from the ionized aspartate to the surrounding residues. We found that using FMO with the PCM solvation method was important to increase the correlation, and by incorporating the ligand deformation energy, the correlation was improved to R = 0.81 for whole twelve compounds and R = 0.91 without one outlier compound.

Fragment-based lead discovery to identify novel inhibitors that target the ATP binding site of pyruvate dehydrogenase kinases.

A fragment-based lead discovery approach was applied to Pyruvate Dehydrogenase Kinases (PDHKs) to discover inhibitors against the ATP binding site with novel chemotypes. X-ray fragment screening toward PDHK4 provided a fragment hit 1 with a characteristic interaction in a deep pocket of the ATP binding site. While known inhibitors utilize several water molecules in a deep pocket to form water-mediated hydrogen bond interactions, the fragment hit binds deeper in the pocket with a hydrophobic group. Displacement of a remaining water molecule in the pocket led to the identification of lead compound 7 with a notable improvement in inhibition potency. This lead compound possessed high ligand efficiency (LE) and showed decent selectivity profile. Two additional lead compounds 10 and 13 with new scaffolds with tricyclic and bicyclic cores were generated by merging structural information of another fragment hit 2. The characteristic interaction of these novel inhibitors in a deep pocket provides new structural insights about PDHKs ATP binding site and opens a novel direction for the development of PDHKs inhibitors.

Structure-based drug design of novel and highly potent pyruvate dehydrogenase kinase inhibitors.

Pyruvate dehydrogenase kinases (PDHKs) are fascinating drug targets for numerous diseases, including diabetes and cancers. In this report, we describe the result of our structure-based drug design from tricyclic lead compounds that led to the discovery of highly potent PDHK2 and PDHK4 dual inhibitors in enzymatic assay. The C3-position of the tricyclic core was explored, and the PDHK2 X-ray structure with a representative compound revealed a novel ATP lid conformation in which the phenyl ring of Phe326 mediated the interaction of the Arg258 sidechain and the compound. Compounds with amide linkers were designed to release the ATP lid by forming an intramolecular pi-pi interaction, and these compounds showed single-digit nM IC50 values in an enzymatic assay. We also explored the C4-position of the tricyclic core to reproduce the interaction observed with the C3-position substitution, and the pyrrolidine compound showed the same level of IC50 values. By optimizing an interaction with the Asn255 sidechain through a docking simulation, compounds with 2-carboxy pyrrole moiety also showed single-digit nM IC50 values without having a cation-pi interaction with the Arg258 sidechain.

Generation and cycloaddition of polymer-supported azomethine ylide via a 1,2-silatropic shift of alpha-silylimines: traceless synthesis of pyrrolidine derivatives.

The 1,3-dipolar cycloaddition of polymer-supported azomethine ylides to dipolarophiles gave pyrrolidine derivatives in good yields. The azomethine ylides were generated from resin-bound alpha-silylimines via a 1,2-silatropic shift. The features of this method are not only a traceless synthesis but also a unique solid-phase route to pyrrolidines with extensive diversity. [reaction: see text]