Structural Impact of the Leukemia Drug 1-β-d-Arabinofuranosylcytosine (Ara-C) on the Covalent Human Topoisomerase I-DNA Complex

1-beta-d-Arabinofuranosylcytosine (Ara-C) is a potent antineoplastic drug used in the treatment of acute leukemia. Previous biochemical studies indicated the incorporation of Ara-C into DNA reduced the catalytic activity of human topoisomerase I by decreasing the rate of single DNA strand religation by the enzyme by 2-3-fold. We present the 3.1 A crystal structure of human topoisomerase I in covalent complex with an oligonucleotide containing Ara-C at the +1 position of the non-scissile DNA strand. The structure reveals that a hydrogen bond formed between the 2'-hydroxyl of Ara-C and the O4' of the adjacent -1 base 5' to the damage site stabilizes a C3'-endo pucker in the Ara-C arabinose ring. The structural distortions at the site of damage are translated across the DNA double helix to the active site of human topoisomerase I. The free sulfhydryl at the 5'-end of the nicked DNA strand in this trapped covalent complex is shifted out of alignment with the 3'-phosphotyrosine linkage at the catalytic tyrosine 723 residue, producing a geometry not optimal for religation. The subtle structural changes caused by the presence of Ara-C in the DNA duplex may contribute to the cytotoxicity of this leukemia drug by prolonging the lifetime of the covalent human topoisomerase I-DNA complex

Design and Synthesis of a Pan-Janus Kinase Inhibitor Clinical Candidate (PF-06263276) Suitable for Inhaled and Topical Delivery for the Treatment of Inflammatory Diseases of the Lungs and Skin

By use of a structure-based computational method for identification of structurally novel Janus kinase (JAK) inhibitors predicted to bind beyond the ATP binding site, a potent series of indazoles was identified as selective pan-JAK inhibitors with a type 1.5 binding mode. Optimization of the series for potency and increased duration of action commensurate with inhaled or topical delivery resulted in potent pan-JAK inhibitor 2 (PF-06263276), which was advanced into clinical studies

Discovery of a Potent and Selective Sphingosine Kinase 1 Inhibitor through the Molecular Combination of Chemotype-Distinct Screening Hits

Sphingosine kinase (SphK) is the major source of the lipid mediator and G protein-coupled receptor agonist sphingosine-1-phosphate (S1P). S1P promotes cell growth, survival, and migration and is a key regulator of lymphocyte trafficking. Inhibition of S1P signaling has been proposed as a strategy for treatment of inflammatory diseases and cancer. Two different formats of an enzyme-based high-throughput screen yielded two attractive chemotypes capable of inhibiting S1P formation in cells. The molecular combination of these screening hits led to compound <b>22a</b> (PF-543) with 2 orders of magnitude improved potency. Compound <b>22a</b> inhibited SphK1 with an IC₅₀ of 2 nM and was more than 100-fold selective for SphK1 over the SphK2 isoform. Through the modification of tail-region substituents, the specificity of inhibition for SphK1 and SphK2 could be modulated, yielding SphK1-selective, potent SphK1/2 dual, or SphK2-preferential inhibitors

Design of a Janus Kinase 3 (JAK3) Specific Inhibitor 1‑((2<i>S</i>,5<i>R</i>)‑5-((7<i>H</i>‑Pyrrolo[2,3‑<i>d</i>]pyrimidin-4-yl)­amino)-2-methylpiperidin-1-yl)­prop-2-en-1-one (PF-06651600) Allowing for the Interrogation of JAK3 Signaling in Humans

Significant work has been dedicated to the discovery of JAK kinase inhibitors resulting in several compounds entering clinical development and two FDA approved NMEs. However, despite significant effort during the past 2 decades, identification of highly selective JAK3 inhibitors has eluded the scientific community. A significant effort within our research organization has resulted in the identification of the first orally active JAK3 specific inhibitor, which achieves JAK isoform specificity through covalent interaction with a unique JAK3 residue Cys-909. The relatively rapid resynthesis rate of the JAK3 enzyme presented a unique challenge in the design of covalent inhibitors with appropriate pharmacodynamics properties coupled with limited unwanted off-target reactivity. This effort resulted in the identification of <b>11</b> (PF-06651600), a potent and low clearance compound with demonstrated in vivo efficacy. The favorable efficacy and safety profile of this JAK3-specific inhibitor <b>11</b> led to its evaluation in several human clinical studies

Design of a Janus Kinase 3 (JAK3) Specific Inhibitor 1‑((2<i>S</i>,5<i>R</i>)‑5-((7<i>H</i>‑Pyrrolo[2,3‑<i>d</i>]pyrimidin-4-yl)­amino)-2-methylpiperidin-1-yl)­prop-2-en-1-one (PF-06651600) Allowing for the Interrogation of JAK3 Signaling in Humans

Significant work has been dedicated to the discovery of JAK kinase inhibitors resulting in several compounds entering clinical development and two FDA approved NMEs. However, despite significant effort during the past 2 decades, identification of highly selective JAK3 inhibitors has eluded the scientific community. A significant effort within our research organization has resulted in the identification of the first orally active JAK3 specific inhibitor, which achieves JAK isoform specificity through covalent interaction with a unique JAK3 residue Cys-909. The relatively rapid resynthesis rate of the JAK3 enzyme presented a unique challenge in the design of covalent inhibitors with appropriate pharmacodynamics properties coupled with limited unwanted off-target reactivity. This effort resulted in the identification of <b>11</b> (PF-06651600), a potent and low clearance compound with demonstrated in vivo efficacy. The favorable efficacy and safety profile of this JAK3-specific inhibitor <b>11</b> led to its evaluation in several human clinical studies