Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excellent target for clinical immunosuppression. We report the development of a specific, orally active inhibitor of JAK3, CP-690,550, that significantly prolonged survival in a murine model of heart transplantation and in cynomolgus monkeys receiving kidney transplants. CP-690,550 treatment was not associated with hypertension, hyperlipidemia, or lymphoproliferative disease. On the basis of these preclinical results, we believe JAK3 blockade by CP-690,550 has potential for therapeutically desirable immunosuppression in human organ transplantation and in other clinical settings.

There is a critical need for safer and more convenient treatments for organ transplant rejection and autoimmune disorders such as rheumatoid arthritis. Janus tyrosine kinases (JAK1, JAK3) are expressed in lymphoid cells and are involved in the signaling of multiple cytokines important for various T cell functions. Blockade of the JAK1/JAK3-STAT pathway with a small molecule was anticipated to provide therapeutic immunosuppression/immunomodulation. The Pfizer compound library was screened against the catalytic domain of JAK3 resulting in the identification of a pyrrolopyrimidine-based series of inhibitors represented by CP-352,664 (2a). Synthetic analogues of 2a were screened against the JAK enzymes and evaluated in an IL-2 induced T cell blast proliferation assay. Select compounds were evaluated in rodent efficacy models of allograft rejection and destructive inflammatory arthritis. Optimization within this chemical series led to identification of CP-690,550 1, a potential first-in-class JAK inhibitor for treatment of autoimmune diseases and organ transplant rejection.

Technological advancements including an open-cylindrical Penning trap with capacitively coupled ICR cell, selective ion accumulation with a resolving quadrupole, and a voltage gradient used during ion extraction from an octopole ion trap, have individually improved dynamic range and sensitivity in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS). Documented here is a new instrument utilizing these technologies toward the robust detection and fragmentation of biomolecules Ͼ10 kDa. Up to 55-fold enhancement in ion population by selective ion accumulation combined with 10-to 20-fold signal-to-noise improvement by application of a DC voltage gradient to an accumulation octopole during the ion transfer event offers improved signal-to-noise (or speed) of MS/MS experiments, for proteins from Methanococcus jannaschii and Saccharomyces cerevisiae whole cell lysates. After external quadrupole filtering with a 40 m/z window, three proteins were fragmented (and identified) in parallel from the database of Methanococcus jannaschii. Electron capture dissociation (ECD) of an intact yeast protein provides extensive sequence information resulting in a high degree of localization for an N-terminal acetylation. Hybrid fragmentation, infrared multiphoton dissociation (IRMPD) followed by low energy electrons (ECD), with the electron source located laterally off the z-axis and external to the magnet bore, presents a strategy for identification of proteins by means of the sequence tag approach. Automated implementation of diverse MS n approaches in a Q-FTMS instrument promises to help realize "top-down" proteomics in the future. (J Am Soc Mass Spectrom 2004, 15, 1099 -1108

A genetic algorithms (GA) based strategy is described for the identification or optimization of active leads. This approach does not require the synthesis and evaluation of huge libraries. Instead it involves iterative generations of smaller sample sets, which are assayed, and the “experimentally” determined biological response is used as an input for GA to rapidly find better leads. The GA described here has been applied to the identification of potent and selective stromelysin substrates from a combinatorial-based population of 206 or 64 000 000 possible hexapeptides. Using GA, we have synthesized less then 300 unique immobilized peptides in a total of five generations to achieve this end. The results show that each successive generation provided better and unique substrates. An additional strategy of utilizing the knowledge gained in each generation in a spin-off SAR activity is described here. Sequences from the first generations were evaluated for stromelysin and collagenase activity to identify stromelysin-selective substrates. GlyProSerThr-TyrThr with Tyr as the P1‘ residue is such an example. A number of peptides replacing Tyr with unusual monomers were synthesized and evaluated as stromelysin substrates. This led to the identification of Ser(OBn) as the best and most selective P1‘ residue for stromelysin.