Discovery of 5‑Azaindazole (GNE-955) as a Potent Pan-Pim Inhibitor with Optimized Bioavailability

Pim kinases have been identified as promising therapeutic targets for hematologic–oncology indications, including multiple myeloma and certain leukemia. Here, we describe our continued efforts in optimizing a lead series by improving bioavailability while maintaining high inhibitory potency against all three Pim kinase isoforms. The discovery of extensive intestinal metabolism and major metabolites helped refine our design strategy, and we observed that optimizing the pharmacokinetic properties first and potency second was a more successful approach than the reverse. In the resulting work, novel analogs such as <b>20</b> (GNE-955) were discovered bearing 5-azaindazole core with noncanonical hydrogen bonding to the hinge

Mechanistic and Structural Understanding of Uncompetitive Inhibitors of Caspase-6

<div><p>Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound’s inhibitory activity is also dependent on the amino acid sequence and P1’ character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.</p> </div

Inhibitor potency and selectivity against caspase family members.

<p>(A) Schematic of divalent tetrapeptide substrate proteolysis to release R110 fluorophore. Removal of both tetrapeptides by caspases is required for signal generation at 535 nm. Concentration-response analysis of compound <b>3</b> (B) and VEID-CHO (C) against caspase-6 (green), caspase-3 (black or red) or caspase-7 (blue). The particular divalent R110 peptide substrate used with each enzyme is indicated in the figure key and assay specifics can be found in Experimental Procedures. Potency values for (B–C) can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050864#pone.0050864.s005" target="_blank">Table S2</a>. Concentration response curves were generated in duplicate and represent 1 of at least 2 experiments with similar results. Each curve is normalized to zero and 100% based on no enzyme or DMSO, respectively. Data represent mean ± standard error of the mean.</p

Structure of the <i>N</i>-furoyl-phenylalanine screening hit (2) and the optimized analog 3.

<p>Potency values represent the inhibition of caspase-6 cleavage of (VEID)₂R110 substrate.</p

Crystal structure of caspase-6 ternary complex with 3 and covalently bound VEID inhibitor reveals the uncompetitive mechanism of this series of compounds.

<p>(A) Crystal structure of the ternary complex of caspase-6 with zVEID and compound <b>3</b> (PDB-ID 4HVA). The caspase-6 dimer is represented as cartoon with the A and B chains colored light blue and grey, respectively, and the L4 loop colored purple. The zVEID inhibitors are represented as sticks and are colored pink. Each inhibitor is covalently bound to the catalytic cysteine (Cys163) in both chain A and B. Two molecules of <b>3</b> are shown as ball and stick representation and colored orange. (B) Close up of the active site of chain A colored according to (A) with hydrogen bonds shown as black dashes. (C) Structural comparison of caspase-6 ternary complex with <b>3</b> bound (light blue) and caspase-6 binary complex with bound VEID-CHO (wheat) (PDB-ID 3OD5) illustrating the difference in the conformation of the tip of the L4 loop in the two crystal structures (residues 261–271).</p

SPR detection of 3 binding to multiple caspase-6 surfaces confirms uncompetitive binding mode.

<p>(A) Catalytically inactive caspase-6 (green), apo-caspase-6 (blue) and caspase-6 saturated with VEID-FMK inhibitor (purple) were captured to chip surfaces and exposed to VEID-AMC, (VEID)₂R110 and/or <b>3</b> to qualitatively monitor binding. Cooperative binding of <b>3</b> and (VEID)₂R110 to C163 caspase-6 illustrate formation of the Michaelis-Menten complex. (B) Sensograms representing injections of escalating concentrations of <b>3</b> over VEID-FMK inhibitor-blocked caspase-6 surface (black). The inset represents similar injections of <b>3</b> over an unblocked apo-caspase-6 surface (blue). (C) Concentration-response analysis of data from (B) when compound <b>3</b> was injected over VEID-blocked caspase-6 surface (black) and apo-caspase-6 (blue) surfaces.</p

Docking models of caspase-6/VEID-R110/3 ternary complex explains fluorophore-dependent potency of this series of compounds.

<p>(A) Docking model of the Michaelis-Menten complex formed between caspase-6 (light blue), VEID-R110 (green sticks) and <b>3</b> (wheat sticks). (B) Docking model of the tetrahedral intermediate between caspase-6, VEID-R110 (green sticks) and <b>3</b> (wheat sticks) with substrate covalently bound to Cys163. (C) Depiction of monovalent VEID substrates with R110 or AMC fluorophores.</p

Kinetic caspase-6 enzymatic studies with compound 3 show uncompetitive mechanism of inhibition with (VEID)₂R110 substrate.

<p>(A) The initial enzyme velocity of caspase-6 was plotted against the indicated concentration of (VEID)₂R110 substrate in the presence of 0 nM (DMSO-black), 3 nM (red), 10 nM (orange), 30 nM (green) or 100 nM (blue) compound <b>3</b>. Double reciprocal plot of this data can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050864#pone.0050864.s001" target="_blank">Figure S1</a> and Michaelis-Menten constants can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050864#pone.0050864.s006" target="_blank">Table S3</a>. (B) Concentration-response analysis of compound <b>3</b> when tested in the presence of 0.5 µM (red), 5 µM (black) or 20 µM (blue) (VEID)₂R110 substrate. Michaelis-Menten kinetic experiments were performed with single points while concentration-response curves were performed in duplicate. Each data set represents 1 of at least 3 experiments with similar results.</p

Discovery of Potent and Selective Pyrazolopyrimidine Janus Kinase 2 Inhibitors

The discovery of somatic Jak2 mutations in patients with chronic myeloproliferative neoplasms has led to significant interest in discovering selective Jak2 inhibitors for use in treating these disorders. A high-throughput screening effort identified the pyrazolo­[1,5-<i>a</i>]­pyrimidine scaffold as a potent inhibitor of Jak2. Optimization of lead compounds <b>7a</b>–<b>b</b> and <b>8</b> in this chemical series for activity against Jak2, selectivity against other Jak family kinases, and good in vivo pharmacokinetic properties led to the discovery of <b>7j</b>. In a SET2 xenograft model that is dependent on Jak2 for growth, <b>7j</b> demonstrated a time-dependent knock-down of pSTAT5, a downstream target of Jak2

A Unique Approach to Design Potent and Selective Cyclic Adenosine Monophosphate Response Element Binding Protein, Binding Protein (CBP) Inhibitors

The epigenetic regulator CBP/P300 presents a novel therapeutic target for oncology. Previously, we disclosed the development of potent and selective CBP bromodomain inhibitors by first identifying pharmacophores that bind the KAc region and then building into the LPF shelf. Herein, we report the “hybridization” of a variety of KAc-binding fragments with a tetrahydroquinoline scaffold that makes optimal interactions with the LPF shelf, imparting enhanced potency and selectivity to the hybridized ligand. To demonstrate the utility of our hybridization approach, two analogues containing unique Asn binders and the optimized tetrahydroquinoline moiety were rapidly optimized to yield single-digit nanomolar inhibitors of CBP with exquisite selectivity over BRD4(1) and the broader bromodomain family