Discovery of selective small-molecule HDAC6 inhibitor for overcoming proteasome inhibitor resistance in multiple myeloma.

Multiple myeloma (MM) has proven clinically susceptible to modulation of pathways of protein homeostasis. Blockade of proteasomal degradation of polyubiquitinated misfolded proteins by the proteasome inhibitor bortezomib (BTZ) achieves responses and prolongs survival in MM, but long-term treatment with BTZ leads to drug-resistant relapse in most patients. In a proof-of-concept study, we previously demonstrated that blocking aggresomal breakdown of polyubiquitinated misfolded proteins with the histone deacetylase 6 (HDAC6) inhibitor tubacin enhances BTZ-induced cytotoxicity in MM cells in vitro. However, these foundational studies were limited by the pharmacologic liabilities of tubacin as a chemical probe with only in vitro utility. Emerging from a focused library synthesis, a potent, selective, and bioavailable HDAC6 inhibitor, WT161, was created to study the mechanism of action of HDAC6 inhibition in MM alone and in combination with BTZ. WT161 in combination with BTZ triggers significant accumulation of polyubiquitinated proteins and cell stress, followed by caspase activation and apoptosis. More importantly, this combination treatment was effective in BTZ-resistant cells and in the presence of bone marrow stromal cells, which have been shown to mediate MM cell drug resistance. The activity of WT161 was confirmed in our human MM cell xenograft mouse model and established the framework for clinical trials of the combination treatment to improve patient outcomes in MM.

The cytoplasmic prolyl-tRNA synthetase of the malaria parasite is a dual-stage target of febrifugine and its analogs.

The emergence of drug resistance is a major limitation of current antimalarials. The discovery of new druggable targets and pathways including those that are critical for multiple life cycle stages of the malaria parasite is a major goal for developing next-generation antimalarial drugs. Using an integrated chemogenomics approach that combined drug resistance selection, whole-genome sequencing, and an orthogonal yeast model, we demonstrate that the cytoplasmic prolyl-tRNA (transfer RNA) synthetase (PfcPRS) of the malaria parasite Plasmodium falciparum is a biochemical and functional target of febrifugine and its synthetic derivative halofuginone. Febrifugine is the active principle of a traditional Chinese herbal remedy for malaria. We show that treatment with febrifugine derivatives activated the amino acid starvation response in both P. falciparum and a transgenic yeast strain expressing PfcPRS. We further demonstrate in the Plasmodium berghei mouse model of malaria that halofuginol, a new halofuginone analog that we developed, is active against both liver and asexual blood stages of the malaria parasite. Halofuginol, unlike halofuginone and febrifugine, is well tolerated at efficacious doses and represents a promising lead for the development of dual-stage next-generation antimalarials.

A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria.

Artemisinins are the cornerstone of anti-malarial drugs. Emergence and spread of resistance to them raises risk of wiping out recent gains achieved in reducing worldwide malaria burden and threatens future malaria control and elimination on a global level. Genome-wide association studies (GWAS) have revealed parasite genetic loci associated with artemisinin resistance. However, there is no consensus on biochemical targets of artemisinin. Whether and how these targets interact with genes identified by GWAS, remains unknown. Here we provide biochemical and cellular evidence that artemisinins are potent inhibitors of Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), revealing an unexpected mechanism of action. In resistant clinical strains, increased PfPI3K was associated with the C580Y mutation in P. falciparum Kelch13 (PfKelch13), a primary marker of artemisinin resistance. Polyubiquitination of PfPI3K and its binding to PfKelch13 were reduced by the PfKelch13 mutation, which limited proteolysis of PfPI3K and thus increased levels of the kinase, as well as its lipid product phosphatidylinositol-3-phosphate (PI3P). We find PI3P levels to be predictive of artemisinin resistance in both clinical and engineered laboratory parasites as well as across non-isogenic strains. Elevated PI3P induced artemisinin resistance in absence of PfKelch13 mutations, but remained responsive to regulation by PfKelch13. Evidence is presented for PI3P-dependent signalling in which transgenic expression of an additional kinase confers resistance. Together these data present PI3P as the key mediator of artemisinin resistance and the sole PfPI3K as an important target for malaria elimination.

Design and synthesis of a crosslinker for studying intracellular steroid trafficking pathways.

A crosslinker was designed and synthesized as a molecular tool for potential use in probing the intracellular trafficking pathways of steroids. The design was guided by computational modeling based upon a model for the transfer of cholesterol between two proteins, NPC1 and NPC2. These proteins play critical roles in the transport of low-density lipoprotein-derived cholesterol from the lumen of lysosomes to other subcellular compartments. Two modified cholesterol residues were covalently joined by a tether based on molecular modeling of the transient interaction of NPC1 and NPC2 during the transfer of cholesterol from the binding site of one of these proteins to the other. With two cholesterol molecules appropriately connected, we hypothesize that the cholesterol binding sites of both proteins will be simultaneously occupied in a manner that will stabilize the protein-protein interaction to permit detailed structural analysis of the resulting complex. A photoaffinity label has also been introduced into one of the cholesterol cores to permit covalent attachment of one of the units into its respective protein-binding pocket. The basic design of these crosslinkers should render them useful for examining interactions of the NPC1/NPC2 pair as well as other sterol transport proteins.

Characterization of the Anopheles gambiae octopamine receptor and discovery of potential agonists and antagonists using a combined computational-experimental approach.

BACKGROUND: Octopamine receptors (OARs) perform key functions in the biological pathways of primarily invertebrates, making this class of G-protein coupled receptors (GPCRs) a potentially good target for insecticides. However, the lack of structural and experimental data for this insect-essential GPCR family has promoted the development of homology models that are good representations of their biological equivalents for in silico screening of small molecules. METHODS: Two Anopheles gambiae OARs were cloned, analysed and functionally characterized using a heterologous cell reporter system. Four antagonist- and four agonist-binding homology models were generated and virtually screened by docking against compounds obtained from the ZINC database. Resulting compounds from the virtual screen were tested experimentally using an in vitro reporter assay and in a mosquito larvicide bioassay. RESULTS: Six An. gambiae OAR/tyramine receptor genes were identified. Phylogenetic analysis revealed that the OAR (AGAP000045) that encodes two open reading frames is an α-adrenergic-like receptor. Both splice variants signal through cAMP and calcium. Mutagenesis analysis revealed that D100 in the TM3 region and S206 and S210 in the TM5 region are important to the activation of the GPCR. Some 2,150 compounds from the virtual screen were structurally analysed and 70 compounds were experimentally tested against AgOAR45B expressed in the GloResponse™CRE-luc2P HEK293 reporter cell line, revealing 21 antagonists, 17 weak antagonists, 2 agonists, and 5 weak agonists. CONCLUSION: Reported here is the functional characterization of two An. gambiae OARs and the discovery of new OAR agonists and antagonists based on virtual screening and molecular dynamics simulations. Four compounds were identified that had activity in a mosquito larva bioassay, three of which are imidazole derivatives. This combined computational and experimental approach is appropriate for the discovery of new and effective insecticides.

Docking applied to the prediction of the affinity of compounds to P-glycoprotein.

P-glycoprotein (P-gp) is involved in the transport of xenobiotic compounds and responsible for the decrease of the drug accumulation in multi-drug-resistant cells. In this investigation we compare several docking algorithms in order to find the conditions that are able to discriminate between P-gp binders and nonbinders. We built a comprehensive dataset of binders and nonbinders based on a careful analysis of the experimental data available in the literature, trying to overcome the discrepancy noticeable in the experimental results. We found that Autodock Vina flexible docking is the best choice for the tested options. The results will be useful to filter virtual screening results in the rational design of new drugs that are not expected to be expelled by P-gp.

Synthesis and HDAC inhibitory activity of isosteric thiazoline-oxazole largazole analogs.

The synthesis of an isosteric analog of the natural product and HDAC inhibitor largazole is described. The sulfur atom in the thizaole ring of the natural product has been replaced with an oxygen atom, constituting an oxazole ring. The biochemical activity and cytotoxicity of this species is described.

Computational studies of the cholesterol transport between NPC2 and the N-terminal domain of NPC1 (NPC1(NTD)).

The transport of cholesterol from NPC2 to NPC1 is essential for the maintenance of cholesterol homeostasis in late endosomes. On the basis of a rigid docking model of the crystal structures of the N-terminal cholesterol binding domain of NPC1(NTD) and the soluble NPC2 protein, models of the NPC1(NTD)-NPC2-cholesterol complexes at the beginning and the end of the transport as well as the unligated NPC1(NTD)-NPC2 complex were studied using 86 ns MD simulations. Significant differences in the cholesterol binding mode and the overall structure of the two proteins compared to the crystal structures of the cholesterol binding separate units were obtained. Relevant residues for the binding are identified using MM/GBSA calculations and the influence of the mutations analyzed by modeling them in silico, rationalizing the results of previous mutagenesis experiments. From the calculated energies and the NEB (nudged elastic band) evaluation of the cholesterol transfer mechanism, an atomistic model is proposed of the transfer of cholesterol from NPC2 to NPC1(NTD) through the formation of an intermediate NPC1(NTD)-NPC2 complex.

Inhibition pattern of sulfamide-related compounds in binding to carbonic anhydrase isoforms I, II, VII, XII and XIV.

A set of sulfamides and sulfamates were synthesized and tested against several isoforms of carbonic anhydrase: CA I, CA II, CA VII, CA XII and CA XIV. The biological assays showed a broad range of inhibitory activity, and interesting results were found for several compounds in terms of activity (Ki <1µm) and selectivity: some aromatic sulfamides are active against CA I, CA II and/or CA VII; while they are less active in CA XII and CA XIV. On the other hand, bulky sulfamides are selective to CA VII. To understand the origin of the different inhibitory activity against each isozyme we used molecular modeling techniques such as docking and molecular dynamic simulations.

Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors.

Selective inhibition of protein methyltransferases is a promising new approach to drug discovery. An attractive strategy towards this goal is the development of compounds that selectively inhibit binding of the cofactor, S-adenosylmethionine, within specific protein methyltransferases. Here we report the three-dimensional structure of the protein methyltransferase DOT1L bound to EPZ004777, the first S-adenosylmethionine-competitive inhibitor of a protein methyltransferase with in vivo efficacy. This structure and those of four new analogues reveal remodelling of the catalytic site. EPZ004777 and a brominated analogue, SGC0946, inhibit DOT1L in vitro and selectively kill mixed lineage leukaemia cells, in which DOT1L is aberrantly localized via interaction with an oncogenic MLL fusion protein. These data provide important new insight into mechanisms of cell-active S-adenosylmethionine-competitive protein methyltransferase inhibitors, and establish a foundation for the further development of drug-like inhibitors of DOT1L for cancer therapy.

Identifying ligand binding conformations of the ß2-adrenergic receptor by using its agonists as computational probes.

Recently available G-protein coupled receptor (GPCR) structures and biophysical studies suggest that the difference between the effects of various agonists and antagonists cannot be explained by single structures alone, but rather that the conformational ensembles of the proteins need to be considered. Here we use an elastic network model-guided molecular dynamics simulation protocol to generate an ensemble of conformers of a prototypical GPCR, ß(2)-adrenergic receptor (ß(2)AR). The resulting conformers are clustered into groups based on the conformations of the ligand binding site, and distinct conformers from each group are assessed for their binding to known agonists of ß(2)AR. We show that the select ligands bind preferentially to different predicted conformers of ß(2)AR, and identify a role of ß(2)AR extracellular region as an allosteric binding site for larger drugs such as salmeterol. Thus, drugs and ligands can be used as "computational probes" to systematically identify protein conformers with likely biological significance.

On the inhibition of histone deacetylase 8.

Histone deacetylases are key regulators of gene expression and have recently emerged as important therapeutic targets for cancer and a growing number of non-malignant diseases. Many widely studied inhibitors of HDACs such as SAHA are thought to have low selectivity within or between the human HDAC isoform classes. Using an isoform-selective assay, we have shown that a number of the known inhibitors have in fact a low activity against HDAC8. Based on the wealth of structural information available for human HDAC8, we use a combination of docking and molecular dynamics simulations to determine the structural origin of the experimental results. A close relationship is found between the activity and the high surface malleability of HDAC8. These results provide a rationale for the recently described 'linkerless' HDAC8 selective inhibitors and design criteria for HDAC8 selective inhibitors.

Affinity of sulfamates and sulfamides to carbonic anhydrase II isoform: experimental and molecular modeling approaches.

Sixteen aromatic and aliphatic sulfamides and sulfamates were synthesized and tested in their inhibition to carbonic anhydrase CAII activity. The weaker inhibition pattern shown by sulfamides as compared to sulfamates is interpreted in this research by means of molecular modeling techniques, including known inhibitors (topiramate and its sulfamide cognate) in the analysis. The results nicely explain the origin of the inhibitory activity, which is not only related to positive interactions of the ligand with the active site residues but also to the solvation pattern characteristic of each ligand.

Synthesis and modeling of new benzofuranone histone deacetylase inhibitors that stimulate tumor suppressor gene expression.

New benzofuranones were synthesized and evaluated toward NCI-H661 non-small cell lung cancer cells. Benzamide derivatives possessed micromolar antiproliferative and histone deacetylase inhibitory activities and modulate histone H4 acetylation. Hydroxamic acids were found to be potent nanomolar antiproliferative agents and HDAC inhibitors. Computational analysis presented a rationale for the activities of the hydroxamate derivatives. Impact of the HDAC inhibition on the expression of E-cadherin and the SEMA3F tumor suppressor genes revealed new promising compounds for lung cancer treatments.

Synthesis and anticonvulsant activity of amino acid-derived sulfamides.

Sulfamides are promising functions for the design of new antiepileptic drugs ( Bioorg. Med. Chem. 2007, 15, 1556-1567; 5604-5614 ). Following previous research in this line, a set of amino acid-derived sulfamides has been designed, synthesized, and tested as new anticonvulsant compounds. The experimental data confirmed the ability of some of the structures to suppress the convulsions originated by the electrical seizure (MES test) at low doses (100 mg/kg).

Synthesis and conformation-activity relationships of the peptide isosteres of FK228 and largazole.

The peptide isosteres (10 and 11) of the naturally occurring and potent histone deacetylase (HDAC) inhibitors FK228 and largazole have been synthesized and evaluated side-by-side with FK228, largazole, and SAHA for inhibition of the class I HDACs 1, 2, 3, and 6.

Residues in the 11 A channel of histone deacetylase 1 promote catalytic activity: implications for designing isoform-selective histone deacetylase inhibitors.

Histone deacetylase 1 (HDAC1) has been linked to cell growth and cell cycle regulation, which makes it a widely recognized target for anticancer drugs. Whereas variations of the metal-binding and capping groups of HDAC inhibitors have been studied extensively, the role of the linker region is less well known, despite the potency of inhibitors with diverse linkers, such as MS-275. To facilitate a drug design that targets HDAC1, we assessed the influence of residues in the 11 A channel of the HDAC1 active site on activity by using an alanine scan. The mutation of eight channel residues to alanine resulted in a substantial reduction in deacetylase activity. Molecular dynamics simulations indicated that alanine mutation results in significant movement of the active-site channel, which suggests that channel residues promote HDAC1 activity by influencing substrate interactions. With little characterization of HDAC1 available, the combined experimental and computational results define the active-site residues of HDAC1 that are critical for substrate/inhibitor binding and provide important insight into drug design.

Structural origin of selectivity in class II-selective histone deacetylase inhibitors.

The development of class- and isoform-selective histone deacetylase (HDAC) inhibitors is highly desirable for the study of the complex interactions of these proteins central to transcription regulation as well as for the development of selective HDAC inhibitors as drugs in epigenetics. To provide a structural basis for the rational design of such inhibitors, a combined computational and experimental study of inhibition of three different histone deacetylase isoforms, HDAC1, -6, and -8, with three different hydroxamate inhibitors is reported. While SAHA was found to be unselective for the inhibition of class I and class II HDACs, the other inhibitors were found to be selective toward class II HDACs. Molecular dynamics simulations indicate that this selectivity is caused by both the overall shape of the protein surface leading to the active site and specific interactions of an aspartate residue in a polar loop and two phenylalanines and a methionine in a nonpolar loop. Monitoring the specific interactions as a function of the simulation time identifies a key sulfur-pi interaction. The implications of the structural motifs for the design of class II-selective HDAC inhibitors are discussed.

Nucleophilic activity of a linked bis{guanidine} leading to formation of a dicationic C4N4-heterocycle.

The methylene-linked bis{guanidine}, H(2)C{hpp}(2) (hppH = 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine), displays nucleophilic activity towards organic halides, including the activation of dichloromethane under ambient conditions affording the cyclic dication, [H(2)C{hpp}(2)CH(2)](2+)[Cl](2).

Competitive hydrolytic and elimination mechanisms in the urease catalyzed decomposition of urea.

We present a high-level quantum chemical study of possible elimination reaction mechanisms associated with the catalytic decomposition of urea at the binuclear nickel active site cluster of urease. Stable intermediates and transition state structures have been identified along several possible reaction pathways. The computed results are compared with those reported by Suarez et al. for the hydrolytic catalyzed decomposition. On the basis of these comparative studies, we propose a monodentate coordination of urea in the active site from which both the elimination and hydrolytic pathways can decompose urea into CO2 and NH3. This observation is counter to what has been experimentally suggested based on the exogenous observation of carbamic acid (the reaction product from the hydrolysis pathway). However, this does not address what has occurred at the active site of urease prior to product release. On the basis of our computed results, the observation that urea prefers the elimination channel in aqueous solution and on the observation of Lippard and co-workers of an elimination reaction channel in a urease biomimetic model, we propose that the elimination channel needs to be re-examined as a viable reaction channel in urease.