David Limburg

The novel benzopyran class of selective cyclooxygenase-2 inhibitors. Part 2: The second clinical candidate having a shorter and favorable human half-life.

In this Letter, we provide the structure-activity relationships, optimization of design, testing criteria, and human half-life data for a series of selective COX-2 inhibitors. During the course of our structure-based drug design efforts, we discovered two distinct binding modes within the COX-2 active site for differently substituted members of this class. The challenge of a undesirably long human half-life for the first clinical candidate 1t(1/2)=360 h was addressed by multiple strategies, leading to the discovery of 29b-(S) (SC-75416) with t(1/2)=34 h.

Selective inducible microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors derived from an oxicam template

Here we describe the SAR of a series of potent and selective mPGES-1 inhibitors based on an oxicam template. Compound 13j demonstrated low nanomolar mPGES-1 inhibition in an enzyme assay. In addition, it displayed PGE(2) inhibition in a cell-based assay (0.42microM) and had over 238-fold selectivity for mPGES-1 over COX-2 and over 200-fold selectivity for mPGES-1 over 6-keto PGF(1alpha).

Covalent inhibitors of interleukin-2 inducible T cell kinase (itk) with nanomolar potency in a whole-blood assay.

We wish to report a strategy that targets interleukin-2 inducible T cell kinase (Itk) with covalent inhibitors. Thus far, covalent inhibition of Itk has not been disclosed in the literature. Structure-based drug design was utilized to achieve low nanomolar potency of the disclosed series even at high ATP concentrations. Kinetic measurements confirmed an irreversible binding mode with off-rate half-lives exceeding 24 h and moderate on-rates. The analogues are highly potent in a cellular IP1 assay as well as in a human whole-blood (hWB) assay. Despite a half-life of approximately 2 h in resting primary T cells, the covalent inhibition of Itk resulted in functional silencing of the TCR pathway for more than 24 h. This prolonged effect indicates that covalent inhibition is a viable strategy to target the inactivation of Itk.

The novel benzopyran class of selective cyclooxygenase-2 inhibitors. Part III: The three microdose candidates

In this manuscript, we report the discovery of the substituted 2-trifluoromethyl-2H-benzopyran-3-carboxylic acids as a novel series of potent and selective cyclooxygenase-2 (COX-2) inhibitors. We provide the structure-activity relationships, optimization of design, testing criteria, and human half-life data. The challenge of a surprisingly long half-life (t(1/2)=360 h) of the first clinical candidate 1 and human t(1/2) had been difficult to predict based on allometric scaling for this class of highly ppb compounds. We used a microdose strategy which led to the discovery of clinical agents 18c-(S), 29b-(S), and 34b-(S) with human half-life of 57, 13, and 11 h.

Neuroprotective Effects of Inhibitors of Dipeptidyl Peptidase-IV In Vitro and In Vivo

For the first time, we demonstrated both neuroprotective and neuroregeneratrive effects of common DPP-IV inhibitors in vitro and in vivo. DPP IV inhibitors protect motor neurons from excitotoxic cell death. They are systemically active and protect striatal innervation of dopaminergic neurons, when administered concurrently with MPTP. Furthermore, DPP-IV inhibitors promote recovery of striatal innervation density when given in a therapeutic manner following MPTP treatment. These data suggest that DPP IV inhibitors may provide protective effects on neurons and promote their use as therapies for treatment of neurodegenerative disorders.

Synthesis, molecular modeling and biological evaluation of aza-proline and aza-pipecolic derivatives as FKBP12 ligands and their in vivo neuroprotective effects

Nonimmunosuppressant ligands, exemplified by GPI 1046 (1), for the peptidyl-prolyl isomerase FKBP12 have been found to unexpectedly possess powerful neuroprotective and neuroregenerative effects in vitro and in vivo. We have extensively explored the therapeutic utility of FKBP12 ligands based on analogues of proline and pipecolic acid. As part of our ongoing program to explore novel structural classes of FKBP12 ligands, we herein wish to report a new class of FKBP12 ligands containing aza-proline and aza-pipecolic acid analogues. Details of the synthetic studies, together with biological activity will be presented.

Use of parallel-synthesis combinatorial libraries for rapid identification of potent FKBP12 inhibitors

Using simple, inexpensive equipment, we have used solution-phase parallel synthesis to rapidly prepare hundreds of sulfonamide- and urea-containing FKBP inhibitors, resulting in rapid identification of extremely potent compounds in these series.

Design and chemoproteomic functional characterization of a chemical probe targeted to bromodomains of BET family proteins

Bromodomain-containing proteins form the signal-reading element of a principal system for the control of gene expression in eukaryotes. Their potential as targets for selective drug action is increasingly being assessed and exploited. Deep characterization of the specificity, potency and other attributes of prototypical agents is an essential element of this process. Continuing studies of a dihydroquinazolinone-based series (prototype: PFI-1) with specificity for members of the BET (bromodomain and extra terminal) family led to the discovery of quinolin-2(1H)-one inhibitors with similar potency and selectivity, but increased chemical stability. Structure-guided design then led to the elaboration of a desthiobiotinylated analog retaining a high fraction of the potency of its parent compound and therefore suitable for chemoproteomic affinity capture experiments. These experiments, conducted using nuclear extracts of THP-1 cells, extended confidence in the selectivity of the series as first proposed. An additional and subsequent evaluation of specificity performed with a panel of recombinant bromodomains (BROMOscan™, DiscoveRx) supported the BET family specificity of the dihydroquinazolinone and quinolin-2(1H)-one series while adding appreciation of weaker effects shown at other bromodomains.

Selectively targeting an inactive conformation of interleukin-2-inducible T-cell kinase by allosteric inhibitors

ITK (interleukin-2-inducible T-cell kinase) is a critical component of signal transduction in T-cells and has a well-validated role in their proliferation, cytokine release and chemotaxis. ITK is an attractive target for the treatment of T-cell-mediated inflammatory diseases. In the present study we describe the discovery of kinase inhibitors that preferentially bind to an allosteric pocket of ITK. The novel ITK allosteric site was characterized by NMR, surface plasmon resonance, isothermal titration calorimetry, enzymology and X-ray crystallography. Initial screening hits bound to both the allosteric pocket and the ATP site. Successful lead optimization was achieved by improving the contribution of the allosteric component to the overall inhibition. NMR competition experiments demonstrated that the dual-site binders showed higher affinity for the allosteric site compared with the ATP site. Moreover, an optimized inhibitor displayed non-competitive inhibition with respect to ATP as shown by steady-state enzyme kinetics. The activity of the isolated kinase domain and auto-activation of the full-length enzyme were inhibited with similar potency. However, inhibition of the activated full-length enzyme was weaker, presumably because the allosteric site is altered when ITK becomes activated. An optimized lead showed exquisite kinome selectivity and is efficacious in human whole blood and proximal cell-based assays.

Synthesis and evaluation of chiral bicyclic proline FKBP12 ligands

As part of our ongoing program to explore novel structural classes of FKBP12 ligands, we herein wish to report a new class of FKBP12 ligands containing chiral bicyclic proline analogues. Details of the synthetic routes, together with preliminary biological activity, will be presented.