Anti-Trypanosomal Proteasome Inhibitors Cure Hemolymphatic and Meningoencephalic Murine Infection Models of African Trypanosomiasis

Current anti-trypanosomal therapies suffer from problems of longer treatment duration, toxicity and inadequate efficacy, hence there is a need for safer, more efficacious and 'easy to use' oral drugs. Previously, we reported the discovery of the triazolopyrimidine (TP) class as selective kinetoplastid proteasome inhibitors with in vivo efficacy in mouse models of leishmaniasis, Chagas Disease and African trypanosomiasis (HAT). For the treatment of HAT, development compounds need to have excellent penetration to the brain to cure the meningoencephalic stage of the disease. Here we describe detailed biological and pharmacological characterization of triazolopyrimidine compounds in HAT specific assays. The TP class of compounds showed single digit nanomolar potency against Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense strains. These compounds are trypanocidal with concentration-time dependent kill and achieved relapse-free cure in vitro. Two compounds, GNF6702 and a new analog NITD689, showed favorable in vivo pharmacokinetics and significant brain penetration, which enabled oral dosing. They also achieved complete cure in both hemolymphatic (blood) and meningoencephalic (brain) infection of human African trypanosomiasis mouse models. Mode of action studies on this series confirmed the 20S proteasome as the target in T. brucei. These proteasome inhibitors have the potential for further development into promising new treatment for human African trypanosomiasis

A Monoselective Sphingosine-1-Phosphate Receptor-1 Agonist Prevents Allograft Rejection in a Stringent Rat Heart Transplantation Model

SummaryFTY720 is an immunomodulator with demonstrated efficacy in a phase II trial of relapsing multiple sclerosis. FTY720-phosphate, the active metabolite generated upon phosphorylation in vivo, acts as a potent agonist on four of the five known sphingosine-1-phosphate (S1P1) receptors. AUY954, an aminocarboxylate analog of FTY720, is a low nanomolar, monoselective agonist of the S1P1 receptor. Due to its selectivity and pharmacokinetic profile, AUY954 is an excellent pharmacological probe of S1P1-dependent phenomena. Oral administration of AUY954 induces a profound and reversible reduction of circulating lymphocytes and, in combination with RAD001 (Certican/Everolimus, an mTOR inhibitor), is capable of prolonging the survival of cardiac allografts in a stringent rat transplantation model. This demonstrates that a selective agonist of the S1P1 receptor is sufficient to achieve efficacy in an animal model of transplantation

Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness

Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases

Evaluation of the GastroPlus™ Advanced Compartmental and Transit (ACAT) Model in Early Discovery

Purpose: The aim of this study was to evaluate the oral exposure predictions obtained early in drug discovery with a generic GastroPlus Advanced Compartmental And Transit (ACAT) model based on the in vivo intravenous blood concentration-time profile, in silico properties (lipophilicity, pKa) and in vitro high-throughput absorption-distribution-metabolism-excretion (ADME) data (as determined by PAMPA, solubility, liver microsomal stability assays). Methods: The model was applied to a total of 623 discovery molecules and their oral exposure was predicted in rats and/or dogs. The predictions of Cmax, AUClast and Tmax were compared against the observations. Results: The generic model proved to make predictions of oral Cmax, AUClast and Tmax within 3-fold of the observations for rats in respectively 65%, 68% and 57% of the 537 cases. For dogs, it was respectively 77%, 79% and 85% of the 124 cases. Statistically, the model was most successful at predicting oral exposure of Biopharmaceutical Classification System (BCS) class 1 compounds compared to classes 2 and 3, and was worst at predicting class 4 compounds oral exposure. Conclusion: The generic GastroPlus ACAT model provided reasonable predictions especially for BCS class 1 compounds. For compounds of other classes, the model may be refined by obtaining more information on solubility and permeability in secondary assays. This increases confidence that such a model can be used in discovery projects to understand the parameters limiting absorption and extrapolate predictions across species. Also, when predictions disagree with the observations, the model can be updated to test hypotheses and understand oral absorption

Snapshot PK: a rapid rodent in vivo preclinical screening approach.

Described in this article are strategies implemented to increase the throughput of in vivo rodent pharmacokinetic (PK) studies using the snapshot PK study design and automated methods for compound submission, sample processing, data analysis and reporting. Applying snapshot PK studies to categorize the oral exposure of >1300 discovery compounds as low, moderate or high resulted in an attrition rate of 86%. The follow up full PK studies on the remaining compounds found that 98% of the compounds were predicted in the correct (69%) or adjacent (29%) oral exposure category by the snapshot PK studies. These results demonstrate that the snapshot PK screen in rodents can serve as an effective and efficient in vivo tool in the compound selection process in drug discovery

TrkB inhibition by GNF-4256 slows growth and enhances chemotherapeutic efficacy in neuroblastoma xenografts

Purpose: Neuroblastoma (NB) is one of the most common and deadly pediatric solid tumors. NB is characterized by clinical heterogeneity, from spontaneous regression to relentless progression despite intensive multimodality therapy. There is compelling evidence that members of the tropomyosin receptor kinase (Trk) family play important roles in these disparate clinical behaviors. Indeed, TrkB and its ligand, brain-derived neurotrophic factor (BDNF), are expressed in 50-60 % of high-risk NBs. The BDNF/TrkB autocrine pathway enhances survival, invasion, metastasis, angiogenesis and drug resistance. Methods: We tested a novel pan-Trk inhibitor, GNF-4256 (Genomics Institute of the Novartis Research Foundation), in vitro and in vivo in a nu/nu athymic xenograft mouse model to determine its efficacy in inhibiting the growth of TrkB-expressing human NB cells (SY5Y-TrkB). Additionally, we assessed the ability of GNF-4256 to enhance NB cell growth inhibition in vitro and in vivo, when combined with conventional chemotherapeutic agents, irinotecan and temozolomide (Irino-TMZ). Results: GNF-4256 inhibits TrkB phosphorylation and the in vitro growth of TrkB-expressing NBs in a dose-dependent manner, with an IC50 around 7 and 50 nM, respectively. Furthermore, GNF-4256 inhibits the growth of NB xenografts as a single agent (p < 0.0001 for mice treated at 40 or 100 mg/kg BID, compared to controls), and it significantly enhances the antitumor efficacy of irinotecan plus temozolomide (Irino-TMZ, p < 0.0071 compared to Irino-TMZ alone). Conclusions: Our data suggest that GNF-4256 is a potent and specific Trk inhibitor capable of significantly slowing SY5Y-TrkB growth, both in vitro and in vivo. More importantly, the addition of GNF-4256 significantly enhanced the antitumor efficacy of Irino-TMZ, as measured by in vitro and in vivo growth inhibition and increased event-free survival in a mouse xenograft model, without additional toxicity. These data strongly suggest that inhibition of TrkB with GNF-4256 can enhance the efficacy of current chemotherapeutic treatment for recurrent/refractory high-risk NBs with minimal or no additional toxicity

Design, synthesis and biological evaluations of novel oxindoles as HIV-1 non-nucleoside reverse transcriptase inhibitors. Part I.

A novel oxindole was discovered as an HIV non-nucleoside reverse transcriptase inhibitor via HTS using a cell-based assay. Systematic structural modifications were carried out to establish its SAR. These modifications led to the identification of oxindoles with low nanomolar potency for inhibiting HIV replication. These novel and potent oxindoles could serve as advanced leads for further optimizations

Design, synthesis, and biological evaluations of novel oxindoles as HIV-1 non-nucleoside reverse transcriptase inhibitors. Part 2.

A series of heterocycle-containing oxindoles was synthesized and their HIV antiviral activities were assessed. Some of these analogs exhibited potent inhibitory activities against both wild-type virus and a number of drug-resistant mutant viruses. In addition, oxindole 9z also showed promising pharmacokinetics

Arylaminoethyl amides as noncovalent inhibitors of cathepsin S. Part 2: Optimization of P1 and N-aryl.

A systematic study of anilines led to the discovery of a metabolically robust fluoroindoline replacement for the alkoxy aniline toxicophore in 1. Investigations of the P1 pocket resulted in the discovery of a wide tolerance of functionality leading to the discovery of 11 as a potent and selective inhibitor of cathepsin S

Synthesis and SAR of succinamide peptidomimetic inhibitors of cathepsin S.

Peptidic, non-covalent inhibitors of lysosomal cysteine protease cathepsin S (1 and 2) were investigated due to low oral bioavailability, leading to an improved series of peptidomimetic inhibitors. Utilizing phenyl succinamides as the P2 residue increased the oral exposure of this lead series of compounds, while retaining selective inhibition of the cathepsin S isoform. Concurrent investigation of the P1 and P2 subsites resulted in the discovery of several potent and selective inhibitors of cathepsin S with good pharmacokinetic properties due to the elimination of saturated aliphatic P2 residues