Selective inhibition of phosphoinositide 3-kinase p110α preserves lymphocyte function.

Class IA phosphoinositide 3-kinase (PI3K) is essential for clonal expansion, differentiation, and effector function of B and T lymphocytes. The p110δ catalytic isoform of PI3K is highly expressed in lymphocytes and plays a prominent role in B and T cell responses. Another class IA PI3K catalytic isoform, p110α, is a promising drug target in cancer but little is known about its function in lymphocytes. Here we used highly selective inhibitors to probe the function of p110α in lymphocyte responses in vitro and in vivo. p110α inhibition partially reduced B cell receptor (BCR)-dependent AKT activation and proliferation, and diminished survival supported by the cytokines BAFF and IL-4. Selective p110δ inhibition suppressed B cell responses much more strongly, yet maximal suppression was achieved by targeting multiple PI3K isoforms. In mouse and human T cells, inhibition of single class IA isoforms had little effect on proliferation, whereas pan-class I inhibition did suppress T cell expansion. In mice, selective p110α inhibition using the investigational agent MLN1117 (previously known as INK1117) did not disrupt the marginal zone B cell compartment and did not block T cell-dependent germinal center formation. In contrast, the selective p110δ inhibitor IC87114 strongly suppressed germinal center formation and reduced marginal zone B cell numbers, similar to a pan-class I inhibitor. These findings show that although acute p110α inhibition partially diminishes AKT activation, selective p110α inhibitors are likely to be less immunosuppressive in vivo compared with p110δ or pan-class I inhibitors.

Retinal vascular permeability suppression by topical application of a novel VEGFR2/Src kinase inhibitor in mice and rabbits.

Retinal and choroidal vascular diseases, with their associated abnormalities in vascular permeability, account for the majority of patients with vision loss in industrialized nations. VEGF is upregulated in ischemic retinopathies such as diabetes and is known to dramatically alter vascular permeability in a number of nonocular tissues via Src kinase-regulated signaling pathways. VEGF antagonists are currently in clinical use for treating the new blood vessels and retinal edema associated with neovascular eye diseases, but such therapies require repeated intraocular injections. We have found that vascular leakage following intravitreal administration of VEGF in mice was abolished by systemic or topical delivery of what we believe is a novel VEGFR2/Src kinase inhibitor; this was confirmed in rabbits. The relevance of Src inhibition to VEGF-associated alterations in vascular permeability was further substantiated by genetic studies in which VEGF injection or laser-induced vascular permeability failed to augment retinal vascular permeability in Src-/- and Yes-/- mice (Src and Yes are ubiquitously expressed Src kinase family members; Src-/- and Yes-/- mice lacking expression of these kinases show no vascular leak in response to VEGF). These findings establish a role for Src kinase in VEGF-mediated retinal vascular permeability and establish a potentially safe and painless topically applied therapeutic option for treating vision loss due to neovascular-associated retinal edema.

An investigation of bone resorption and Dictyostelium discoideum growth inhibition by bisphosphonate drugs.

We report the results of 3D-QSAR/CoMFA investigations of the activity of bisphosphonate drugs, farnesyl pyrophosphate synthase (FPPSase) inhibitors, in the inhibition of bone resorption as well as the growth of Dictyostelium discoideum. In the case of D. discoideum, we find an experimental versus QSAR predicted pIC(50) R(2) value of 0.94 for 16 bisphosphonates over the 9-1200 microM range of IC(50) values, a cross-validated R(2) = 0.90, and a bootstrapped R(2) = 0.94, and we demonstrate that this approach has predictive utility (a 0.18 pIC(50) rms error for three test sets of 3 predictions). In bone resorption, we find an experimental versus predicted pLED (lowest effective dose) R(2) = 0.79 for 35 bisphosphonates over the 0.0001-1 mg of P/kg LED range, a cross-validated R(2) = 0.75, and a bootstrapped R(2) = 0.79. Two sets of 31 compounds were used as training sets for the predicted pLED values for two sets of 4 compounds which have an rms error of 0.44, larger than that found with D. discoideum. However, this can be attributed to the rather large uncertainties in the experimental bone resorption data which are almost all reported in decade steps (DeltapLED = 1). The CoMFA predicted (rat) bone antiresorptive pLED values are in agreement with literature (human recombinant) FPPSase inhibition results with an rms error of 0.45 (a factor of 2.8 error in activity prediction). We also report the single-crystal X-ray crystallographic structure of the compound most active in D. discoideum growth inhibition, 2-(3-picolyl)-aminomethylene-1,1-bisphosphonic acid. The structure clearly shows the presence of bond length alternation in the picolyl ring and a planar amino group linked by a very short (1.346 A) bond to the picolyl group, an amidinium-like structure which is also expected to occur in other highly active species such as minodronate and zoledronate. Overall, these results show that it is now possible to predict the activity of bisphosphonates using 3D-QSAR/CoMFA methods, although bone resorption studies should benefit from additional, accurate information on enzyme inhibition.

Inhibition of geranylgeranyl diphosphate synthase by bisphosphonates and diphosphates: a potential route to new bone antiresorption and antiparasitic agents.

We report the inhibition of a human recombinant geranylgeranyl diphosphate synthase (GGPPSase) by 23 bisphosphonates and six azaprenyl diphosphates. The IC50 values range from 140 nM to 690 microM. None of the nitrogen-containing bisphosphonates that inhibit farnesyl diphosphate synthase were effective in inhibiting the GGPPSase enzyme. Using three-dimensional quantitative structure-activity relationship/comparative molecular field analysis (CoMFA) methods, we find a good correlation between experimental and predicted activity: R2 = 0.938, R(cv)2 = 0.900, R(bs)2 = 0.938, and F-test = 86.8. To test the predictive utility of the CoMFA approach, we used three training sets of 25 compounds each to generate models to predict three test sets of three compounds. The rms pIC50 error for the nine predictions was 0.39. We also investigated the pharmacophore of these GGPPSase inhibitors using the Catalyst method. The results demonstrated that Catalyst predicted the pIC50 values for the nine test set compounds with an rms error of 0.28 (R2 between experimental and predicted activity of 0.948).

Activity of bisphosphonates against Trypanosoma brucei rhodesiense.

We report the results of a comparative molecular field analysis (CoMFA) investigation of the growth inhibition of the bloodstream form of Trypanosoma brucei rhodesiense trypomastigotes by bisphosphonates. A quantitative three-dimensional structure-activity relationship CoMFA model for a set of 26 bisphosphonates having a range of activity spanning approximately 3 orders of magnitude (minimum IC(50) = 220 nM; maximum IC(50) = 102 microM) yielded an R(2) value of 0.87 with a cross-validated R(2) value of 0.79. The predictive utility of this approach was tested for three sets of three compounds: the average pIC(50) error was 0.23. For the nitrogen-containing bisphosphonates, in general, the activity was aromatic- >> aliphatic-containing side chains. The activity of aromatic species lacking an alkyl ring substitution decreased from ortho to meta to para substitution; halogen substitutions also reduced activity. For the aliphatic bisphosphonates, the IC(50) values decreased nearly monotonically with increasing chain length (down to IC(50) = 2.0 microM for the n-C(11) alkyl side chain species). We also show, using a "rescue" experiment, that the molecular target of the nitrogen-containing bisphosphonate, risedronate, in T. b. rhodesiense is the enzyme farnesyl pyrophosphate synthase. In addition, we report the LD(50) values of bisphosphonates in a mammalian cell general toxicity screen and present a comparison between the therapeutic indices and the IC(50) values in the T. b. rhodesiense growth inhibition assay. Several bisphosphonates were found to have large therapeutic indices (> or =200:1) as well as low IC(50) values, suggesting their further investigation as antiparasitic agents against T. b. rhodesiense.

Effective and selective targeting of leukemia cells using a TORC1/2 kinase inhibitor.

Targeting the mammalian target of rapamycin (mTOR) protein is a promising strategy for cancer therapy. The mTOR kinase functions in two complexes, TORC1 (target of rapamycin complex-1) and TORC2 (target of rapamycin complex-2); however, neither of these complexes is fully inhibited by the allosteric inhibitor rapamycin or its analogs. We compared rapamycin with PP242, an inhibitor of the active site of mTOR in both TORC1 and TORC2 (hereafter referred to as TORC1/2), in models of acute leukemia harboring the Philadelphia chromosome (Ph) translocation. We demonstrate that PP242, but not rapamycin, causes death of mouse and human leukemia cells. In vivo, PP242 delays leukemia onset and augments the effects of the current front-line tyrosine kinase inhibitors more effectively than does rapamycin. Unexpectedly, PP242 has much weaker effects than rapamycin on the proliferation and function of normal lymphocytes. PI-103, a less selective TORC1/2 inhibitor that also targets phosphoinositide 3-kinase (PI3K), is more immunosuppressive than PP242. These findings establish that Ph(+) transformed cells are more sensitive than normal lymphocytes to selective TORC1/2 inhibitors and support the development of such inhibitors for leukemia therapy.

Radical cure of experimental cutaneous leishmaniasis by the bisphosphonate pamidronate.

The effects in vivo of the bisphosphonate drug pamidronate, used in bone resorption therapy, were investigated in an experimental model of cutaneous leishmaniasis. Pamidronate at an intraperitoneal dose of 10 mg/kg/day for 5 days effects a radical cure of cutaneous leishmaniasis in Balb/c mice, as evidenced by long-term disappearance of lesions; disappearance of amastigotes in lesion sites, as determined by histopathological analysis and cultivation of material obtained from lesions; and polymerase chain reaction analysis of necropsy material, using probes specific for kinetoplast DNA. Pamidronate is, therefore, a new lead compound for the synthesis of drugs effective against cutaneous leishmaniasis.

In vivo activities of farnesyl pyrophosphate synthase inhibitors against Leishmania donovani and Toxoplasma gondii.

The in vivo activities of three bisphosphonates were determined against Leishmania donovani and Toxoplasma gondii. Alendronate was essentially inactive against both parasites. Pamidronate was active against L. donovani by intravenous administration. Risedronate had a 50% effective dosage of five 2.6-mg/kg of body weight intraperitoneal doses against L. donovani-infected mice but was less effective against T. gondii-infected mice.