Structure activity relationships of new inhibitors of mammalian 2,3-oxidosqualene cyclase designed from isoquinoline derivatives.

We have designed more potent inhibitors from the previously reported LF 05-0038, a 6-isoquinolinol based inhibitor of 2,3-oxidosqualene cyclase (IC50: 1.1 microM). Replacement of the 3-OH group by various 3-substituted amino groups, and modification of the alkyl chain borne by the endocyclic nitrogen led to inhibitors with IC50 in the range of 0.15 to 1 microM. In a second step, opening of the bicyclic ring system afforded the corresponding aminoalkylpiperidines which were slightly more potent. Finally, introduction of suitable aromatic containing moieties on the piperidine nitrogen yielded very potent inhibitors such as 20x (IC50 = 18 nM) easy to synthesize and achiral. The recent availability of the crystal structure of squalene-hopene cyclase allowed us to construct a three-dimensional (3D) model of the related 2,3-oxidosqualene cyclase (OSC) which was tentatively used to describe the possible mode of binding of our compounds and which can be useful for designing new inhibitors.

Longterm protection of mice against collagen-induced arthritis after short-term LF 15-0195 treatment: modulation of B and T lymphocyte activation.

OBJECTIVES: LF 15-0195 is an immunosuppressive agent obtained by organic synthesis, currently under clinical development for the treatment of vasculitis. We define the effects of LF 15-0195 in the murine collagen-induced arthritis (CIA) model, an experimental model of human rheumatoid arthritis. METHODS: In our model, CIA was elicited in DBA/1 mice by immunization with bovine type II collagen (CII) in Freund's complete adjuvant, followed by a repeat injection 21 days later. Disease onset was observed 6 days after booster injection. In these experiments, mice were treated with 5 daily LF 15-0195 injections starting after the booster injection (days 21-25). The mice were observed for 40 days after the start of treatment, during which time arthritis was scored using clinical score and paw swelling assessment. Modulation of humoral immunity was documented by measuring the serum level of anti-CII IgG1 and IgG2a and cellular immunity by cytokines production by lymph node cells (LNC) and their proliferation in vitro. RESULTS: Short-term treatment of LF 15-0195 after booster injection prevented longterm development of CIA. LF 15-0195 inhibited B cell differentiation with a marked suppression of anti-CII IgG1 and IgG2a synthesis. Functional analyses of T lymphocytes showed that LF 15-0195 treatment reduces cytokine production by LNC after CII, anti-CD3, lipopolysaccharide stimulation. CONCLUSION: LF 15-0195 treatment during a short time period prevented development of arthritis, inhibited humoral-specific response longterm, induced a decrease in the number of LNC, and decreased cytokine production of T LNC after ex vivo stimulation.

LF 15-0195 immunosuppressive agent enhances activation-induced T-cell death by facilitating caspase-8 and caspase-10 activation at the DISC level.

The deoxyspergualin derivative LF 15-0195 has demonstrated some efficacy in animal models of autoimmune and graft-versus-host diseases and is currently tested in clinics. The molecular mechanisms of LF 15-0195 immunosuppressive activity remained unknown. We show that exposure to LF 15-0195 sensitizes Jurkat T cells to apoptosis induced by an agonistic anti-CD95 antibody (CH11 clone) and by the cytokine TNF-related apoptosis-inducing ligand. LF 15-0195 does not demonstrate any significant effect on the postmitochondrial activation of caspases, nor does it modify overall expression of CD95, Fas-associated death domain, and procaspase-8. The compound facilitates the recruitment of these molecules to the death-inducing signaling complex (DISC) and enhances caspase-8 and -10 activation, thus increasing cytochrome c and direct IAP binding with low pI (DIABLO)/Smac mitochondrial release. LF 15-0195 also sensitizes Jurkat T cells to CD3-mediated apoptosis, an in vitro model for activation-induced T-cell death (AICD). LF 15-0195-mediated sensitization to AICD was further confirmed in human peripheral T cells exposed to anti-CD3 antibodies, then cultured in the presence of interleukin-2. In these cells, LF 15-0195 increased apoptosis triggered by either anti-CD95 antibodies or CD3 restimulation, whereas no effect was observed on "passive apoptosis." Finally, in bone marrow recipient mice, LF 15-0195 enhanced allogeneic donor T-cell death, which required a functional CD95 pathway. These results suggest that LF 15-0195 sensitizes T cells to AICD by increasing caspase activation at the DISC level in response to CD95 engagement. This original mechanism, together with LF 15-0195 efficacy in various disease models, makes this compound a promising immunosuppressive drug.