Identification of potent and selective non-covalent inhibitors of the Plasmodium falciparum proteasome.

We have identified short N,C-capped peptides that selectively inhibit the proteasome of the malaria-causing pathogen Plasmodium falciparum. These compounds are highly potent in culture with no toxicity in host cells. One cyclic biphenyl ether compound inhibited intraerythrocytic growth of P. falciparum with an IC50 of 35 nM, and we show that even a pulse treatment with this cyclic peptide induced parasite death due to proteasome inhibition. These compounds represent promising new antimalarial agents that target the essential proteasomal machinery of the parasite without toxicity toward the host.

Histone deacetylase inhibitors derived from 1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine and related heterocycles selective for the HDAC6 isoform.

Acyl derivatives of 4-(aminomethyl)-N-hydroxybenzamide are potent sub-type selective HDAC6 inhibitors. Constrained heterocyclic analogs based on 1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine show further enhanced HDAC6 selectivity and inhibitory activity in cells. Homology models suggest that the heterocyclic spacer can more effectively access the wider catalytic channel of HDAC6 compared to other HDAC sub-types.

N,C-Capped dipeptides with selectivity for mycobacterial proteasome over human proteasomes: role of S3 and S1 binding pockets.

We identified N,C-capped dipeptides that are selective for the Mycobacterium tuberculosis proteasome over human constitutive and immunoproteasomes. Differences in the S3 and S1 binding pockets appeared to account for the species selectivity. The inhibitors can penetrate mycobacteria and kill nonreplicating M. tuberculosis under nitrosative stress.

Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S beta5-subunit.

The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome based [corrected] on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin-proteasome system in cells. We show that these compounds are entirely selective for the beta5 (chymotrypsin-like) site over the beta1 (caspase-like) and beta2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC50 values for the human 20S beta5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin-luciferase reporter, activation of NFkappaB (nuclear factor kappaB) in response to TNF-alpha (tumour necrosis factor-alpha) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the beta5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the beta5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.

Discovery and optimization of N-acyl and N-aroylpyrazolines as B-Raf kinase inhibitors.

A high throughput screen identified N-aroylpyrazoline 1 as a selective inhibitor of the V600E mutant of B-Raf kinase. Parallel synthesis of acyl, aroyl, and sulfonyl derivatives led to the identification of several potent inhibitors in both enzymatic and cellular (pERK) assays such as compound 42.

Optimization of a series of dipeptides with a P3 threonine residue as non-covalent inhibitors of the chymotrypsin-like activity of the human 20S proteasome.

Starting from a tripeptide screening hit, a series of dipeptide inhibitors of the proteasome with Thr as the P3 residue has been optimized with the aid of crystal structures in complex with the ß-5/6 active site of y20S. Derivative 25, (ß5 IC(50)=7.4 nM) inhibits only the chymotryptic activity of the proteasome, shows cellular activity against targets in the UPS, and inhibits proliferation.

Discovery and optimization of pyrazoline compounds as B-Raf inhibitors.

The discovery of novel pyrazoline derivatives as B-Raf (V600E) inhibitors is described in this report. Chemical modification of the pyrazoline scaffold led to the development of SAR and identified potent and selective inhibitors of B-Raf (V600E). Determination of the pharmacokinetic properties of selected inhibitors is also reported.

Design, synthesis, and evaluation of naphthalene-sulfonamide antagonists of human CCR8.

The design, synthesis, and structure-activity relationship development of naphthalene-derived human CCR8 antagonists is described. In vitro binding assay results of these investigations are reported, critical interactions of the antagonists with CCR8 are defined, and preliminary physicochemical and pharmacokinetic data for the naphthalene scaffold are presented.

Discovery and characterization of aminopiperidinecoumarin melanin concentrating hormone receptor 1 antagonists.

4-(1-Benzo[1,3]dioxol-5-ylmethylpiperidine-4-ylmethyl)-6-chlorochromen-2-one (7) is a potent, orally bioavailable melanin concentrating hormone receptor 1 (MCHr1) antagonist that causes dose-dependent weight loss in diet-induced obese mice. Further evaluation of 7 in an anesthetized dog model of cardiovascular safety revealed adverse hemodynamic effects at a plasma concentration comparable to the minimally effective therapeutic concentration. These results highlight the need for scrutiny of the cardiovascular safety profile of MCHr1 antagonists.

Potent histone deacetylase inhibitors derived from 4-(aminomethyl)-N-hydroxybenzamide with high selectivity for the HDAC6 isoform.

A screen for HDAC6 inhibitors identified acyl derivatives of 4-(aminomethyl)-N-hydroxybenzamide as potent leads with unexpected selectivity over the other subtypes. We designed and synthesized constrained heterocyclic analogues such as tetrahydroisoquinolines that show further enhanced HDAC6 selectivity and inhibitory activity in cellular assays. Selectivity may be attributed to the benzylic spacer more effectively accessing the wider channel of HDAC6 compared to other HDAC subtypes as well as hydrophobic capping groups interacting with the protein surface near the rim of the active site.

Visual monitoring of solid-phase extraction using chromogenic fluorous synthesis supports.

Reductive aminations and further transformations of an azo dye and fluorous tagged aldehyde are described. The intensely colored 2,4-dialkoxybenzyl protected amines undergo Fmoc-based peptide coupling, Suzuki reactions, and sulfonamide formation with product isolation facilitated by visual monitoring of fluorous solid phase extraction. Target compounds are released from the supports in high yields and purities by treatment with trifluoroacetic acid (TFA).

Design and optimization of potent and orally bioavailable tetrahydronaphthalene Raf inhibitors.

Inhibition of mutant B-Raf signaling, through either direct inhibition of the enzyme or inhibition of MEK, the direct substrate of Raf, has been demonstrated preclinically to inhibit tumor growth. Very recently, treatment of B-Raf mutant melanoma patients with a selective B-Raf inhibitor has resulted in promising preliminary evidence of antitumor activity. This article describes the design and optimization of tetrahydronaphthalene-derived compounds as potent inhibitors of the Raf pathway in vitro and in vivo. These compounds possess good pharmacokinetic properties in rodents and inhibit B-Raf mutant tumor growth in mouse xenograft models.

Identification and structure-activity relationships of a new series of Melanocortin-4 receptor antagonists.

The identification and optimization of a series of acylguanidine-based melanocortin-4 receptor antagonists is discussed.

Identification and characterization of amino-piperidinequinolones and quinazolinones as MCHr1 antagonists.

Several potent, functionally active MCHr1 antagonists derived from quinolin-2(1H)-ones and quinazoline-2(1H)-ones have been synthesized and evaluated. Pyridylmethyl substitution at the quinolone 1-position results in derivatives with low-nM binding potency and good selectivity with respect to hERG binding.

Identification and characterization of 4-aryl-3,4-dihydropyrimidin-2(1H)-ones as inhibitors of the fatty acid transporter FATP4.

Several potent, cell permeable 4-aryl-dihydropyrimidinones have been identified as inhibitors of FATP4. Lipophilic ester substituents at the 5-position and substitution at the para-position (optimal groups being -NO(2) and CF(3)) of the 4-aryl group led to active compounds. In two cases racemates were resolved and the S enantiomers shown to have higher potencies.

Synthesis of 3-amino-1,2,4-benzothiadi- azine 1,1-dioxides via a tandem aza-Wittig/heterocumulene annulation.

[reaction: see text] Reaction of o-azidobenzenesulfonamides with polymer-supported triphenylphosphine affords the corresponding iminophosphoranes. Subsequent reaction with isocyanates gives 3-amino-1,2,4-benzothiadiazine 1,1-dioxides in high yields and purities. The reaction has been successfully applied to the synthesis of derivatives with various substituents at the 2- and 3-positions and in the benzenoid ring.

Identification of aminopiperidine benzamides as MCHr1 antagonists.

The identification of a novel series of benzamide-containing MCHr1 antagonists is described. Compound 22 displayed moderate efficacy in a diet induced obesity mice model.

Identification of ortho-amino benzamides and nicotinamides as MCHr1 antagonists.

Several potent and efficacious MCHr1 antagonists containing an ortho-amino benzamide or nicotinamide chemotype have been identified, exemplified by 28 and 50.

Synthesis and biological evaluation of imidazole-based small molecule antagonists of the melanocortin 4 receptor (MC4-R).

A novel series of imidazole-based small molecule antagonists of the melanocortin 4 receptor (MC4-R) is reported. Members of this series have been identified, which exhibit sub-micromolar binding affinity for the MC4-R, functional potency <100nM, and good oral exposure in rat. Antagonists of the MC4-R are potentially useful in the therapeutic treatment of involuntary weight loss due to advanced age or disease (e.g. cancer or AIDS), an area of large, unmet medical need.