Design, synthesis and biological evaluation of novel hybrids targeting mTOR and HDACs for potential treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major contributor to global cancer incidence and mortality. Many pathways are involved in the development of HCC and various proteins including mTOR and HDACs have been identified as potential drug targets for HCC treatment. In the present study, two series of novel hybrid molecules targeting mTOR and HDACs were designed and synthesized based on parent inhibitors (MLN0128 and PP121 for mTOR, SAHA for HDACs) by using a fusion-type molecular hybridization strategy. In vitro antiproliferative assays demonstrated that these novel hybrids with suitable linker lengths exhibited broad cytotoxicity against various cancer cell lines, with significant activity against HepG2 cells. Notably, DI06, an MLN0128-based hybrid, exhibited antiproliferative activity against HepG2 cells with an IC50 value of 1.61 µM, which was comparable to those of both parent drugs (MLN0128, IC50 = 2.13 µM and SAHA, IC50 = 2.26 µM). In vitro enzyme inhibition assays indicated that DI06, DI07 and DI17 (PP121-based hybrid) exhibited nanomolar inhibitory activity against mTOR kinase and HDACs (e.g., HDAC1, HDAC2, HDAC3, HADC6 and HADC8). Cellular studies and western blot analyses uncovered that in HepG2 cells, DI06 and DI17 induced cell apoptosis by targeting mTOR and HDACs, blocked the cell cycle at the G0/G1 phase and suppressed cell migration. The potential binding modes of the hybrids (DI06 and DI17) with mTOR and HDACs were investigated by molecular docking. DI06 displayed better stability in rat liver microsomes than DI07 and DI17. Collectively, DI06 as a novel mTOR and HDACs inhibitor presented here warrants further investigation as a potential treatment of HCC.

Discovery, Structure-Activity Relationships, and In Vivo Evaluation of Novel Aryl Amides as Brain Penetrant Adaptor Protein 2-Associated Kinase 1 (AAK1) Inhibitors for the Treatment of Neuropathic Pain.

Effective treatment of chronic pain, in particular neuropathic pain, without the side effects that often accompany currently available treatment options is an area of significant unmet medical need. A phenotypic screen of mouse gene knockouts led to the discovery that adaptor protein 2-associated kinase 1 (AAK1) is a potential therapeutic target for neuropathic pain. The synthesis and optimization of structure-activity relationships of a series of aryl amide-based AAK1 inhibitors led to the identification of 59, a brain penetrant, AAK1-selective inhibitor that proved to be a valuable tool compound. Compound 59 was evaluated in mice for the inhibition of µ2 phosphorylation. Studies conducted with 59 in pain models demonstrated that this compound was efficacious in the phase II formalin model for persistent pain and the chronic-constriction-injury-induced model for neuropathic pain in rats. These results suggest that AAK1 inhibition is a promising approach for the treatment of neuropathic pain.

Design, synthesis and biological evaluation of dual mTOR/HDAC6 inhibitors in MDA-MB-231 cells.

The excessive activation of histone deacetylase (HDAC) and mammalian target of rapamycin (mTOR) signaling promotes tumor growth and progression. We proposed that dual targeting mTOR and HDAC inhibitors is a promising strategy for triple negative breast cancer (TNBC) treatment. In this study, a series of dual mTOR/HDAC6 inhibitors were designed and synthesized by structure-based strategy. 10g was documented to be a potent dual mTOR/HDAC6 inhibitor with IC50 value of 133.7 nM against mTOR and 56 nM against HDAC6, presenting mediate antiproliferative activity in TNBC cells. Furthermore, we predicted the binding mode of 10g and mTOR/HDAC6 by molecule docking. In addition, 10g was documented to induce significant autophagy, apoptosis and suppress migration in MDA-MB-231 cells. Collectively, these findings revealed that 10g is a novel potent dual mTOR/HDAC6 inhibitor, which provides promising rationale for the combination of dual mTOR/HDAC6 inhibitors for TNBC treatment.

Exploration of benzofuran-based compounds as potent and selective Plasmodium falciparum glycogen synthase kinase-3 (PfGSK-3) inhibitors.

Plasmodium falciparum glycogen synthase kinase-3 (PfGSK-3) has been identified as a potential target for the development of novel drugs against multi-drug resistant malaria. A series of benzofuran-based compounds was synthesised and evaluated as inhibitors of recombinantly expressed and purified PfGSK-3 and human glycogen synthase kinase-3 beta (HsGSK-3ß). Of this series, five compounds (5k, 5m, 5p, 5r, 5s) preferentially inhibited PfGSK-3, with four of these compounds exhibiting IC50 values in the sub-micromolar range (0.00048-0.440 µM). Evaluation of the structure-activity relationships required for PfGSK-3 selective inhibition indicated that a C6-OCH3 substitution on ring A is preferred, while the effect of the ring B substituent on activity, in decreasing order is: C4'-CN > C4'-F > C3'-OCH3 > C3',4'-diCl. To date, development of PfGSK-3 inhibitors has been limited to the 4-phenylthieno[2,3-b]pyridine class. Chalcone-based scaffolds, such as the benzofurans described herein, are promising new hits which can be explored for future design of PfGSK-3 selective inhibitors.

Calcium-dependent protein kinase 21 phosphorylates 14-3-3 proteins in response to ABA signaling and salt stress in rice.

The calcium-dependent protein kinases (CDPKs) are a class of plant-specific kinase that directly bind Ca2+ and mediate the calcium-signaling pathways to play important physiological roles in growth and development. The rice genome contains 31 CDPK genes, one of which, OsCPK21, is known to modulate the abscisic acid (ABA) and salt stress responses in this crop; however, the molecular mechanisms underlying this regulation are largely unknown. In the present study, we performed yeast two-hybrid screening, glutathione S-transferase pull-down, co-immunoprecipitation, and bimolecular fluorescence complementation assays to confirm the interaction between OsCPK21 and one of its putative targets, Os14-3-3 (OsGF14e). We used an in vitro kinase assay and site-directed mutagenesis to verify that OsCPK21 phosphorylates OsGF14e at Tyr-138. We used real-time PCR to reveal that several ABA and salt inducible genes were more highly expressed in the OsCPK21-OE and OsGF14e WT-OE plants than in the mutant OsGF14e Y138A-OE and wild-type plants. These results suggest that OsCPK21 phosphorylates OsGF14e to facilitate the response to ABA and salt stress.

Synthesis and biological evaluation of imidazo[1,5-a]pyridine-benzimidazole hybrids as inhibitors of both tubulin polymerization and PI3K/Akt pathway.

A series of imidazo[1,5-a]pyridine-benzimidazole hybrids (5a­aa) were prepared and evaluated for their cytotoxic activity against a panel of sixty human tumor cell lines. Among them compounds 5d and 5l showed significant cytotoxic activity with GI50 values ranging from 1.06 to 14.9 µM and 0.43 to 7.73 µM, respectively. Flow cytometric analysis revealed that these compounds arrest the cell cycle at G2/M phase and induced cell death by apoptosis. The tubulin polymerization assay (IC50 of 5d is 3.25 µM and 5l is 1.71 µM) and immunofluorescence analysis showed that these compounds effectively inhibited the microtubule assembly in human breast cancer cells (MCF-7). Further, the apoptotic effects of compounds were confirmed by Hoechst staining, mitochondrial membrane potential, cytochrome c release, ROS generation, caspase 9 activation and DNA fragmentation analysis. After treatment with these compounds for 48 h, p-PTEN and p-AKT levels were markedly decreased. Moreover, these compounds did not significantly inhibit the normal human embryonic kidney cells, HEK-293. The molecular docking simulations predicted the binding interactions of 5d and 5l with colchicine binding site of the tubulin, which is in compliance with the antiproliferative activity data.

New targets for pharmacological intervention in the glutamatergic synapse.

Excitotoxicity is thought to be a major mechanism in many human disease states such as ischemia, trauma, epilepsy and chronic neurodegenerative disorders. Briefly, synaptic overactivity leads to the excessive release of glutamate that activates postsynaptic cell membrane receptors, which upon activation open their associated ion channel pore to produce ion influx. To date, although molecular basis of glutamate toxicity remain uncertain, there is general agreement that N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptors plays a key role in mediating at least some aspects of glutamate neurotoxicity. On this view, research has focused in the discovery of new compounds able to either reduce glutamate release or activation of postsynaptic NMDA receptors. Although NMDA receptor antagonists prevent excitotoxicity in cellular and animal models, these drugs have limited usefulness clinically. Side effects such as psychosis, nausea, vomiting, memory impairment, and neuronal cell death accompany complete NMDA receptor blockade, dramatizing the crucial role of the NMDA receptor in normal neuronal processes. Recently, however, well-tolerated compounds such as memantine has been shown to be able to block excitotoxic cell death in a clinically tolerated manner. Understanding the biochemical properties of the multitude of NMDA receptor subtypes offers the possibility of developing more effective and clinically useful drugs. The increasing knowledge of the structure and function of this postsynaptic NMDA complex may improve the identification of specific molecular targets whose pharmacological or genetic manipulation might lead to innovative therapies for brain disorders.

A synthetic miniprotein that binds specific DNA sequences by contacting both the major and the minor groove.

Attachment of a slightly modified basic region of a bZIP protein (GCN4) to a distamycin-related tripyrrole provides a bivalent system capable of binding with high affinity to specific DNA sequences. Appropriate adjustment of the linker between the two units has led to a hybrid that binds a 9 base-pair-long DNA site (TTTTATGAC) with low nanomolar affinity at 4 degrees C. Circular dichroism and gel retardation studies indicate that the binding occurs by simultaneous insertion of the bZIP basic region into the DNA major groove and the tripyrrole moiety into the minor groove of the flanking sequence. Analysis of hybrids bearing alternative linkers revealed that tight, specific binding is strongly dependent on the length and nature of the connecting unit.

Probing backbone hydrogen bonds in the hydrophobic core of GCN4.

Backbone amide hydrogen bonds play a central role in protein secondary and tertiary structure. Previous studies have shown that substitution of a backbone ester (-COO-) in place of a backbone amide (-CONH-) can selectively destabilize backbone hydrogen bonds in a protein while maintaining a similar conformation to the native backbone structure. The majority of these studies have focused on backbone substitutions that were accessible to solvent. The GCN4 coiled coil domain is an example of a stable alpha-helical dimer that possesses a well-packed hydrophobic core. Amino acids in the a and d positions of the GCN4 helix, which pack the hydrophobic core, were replaced with the corresponding alpha-hydroxy acids in the context of a chemoselectively ligated heterodimer. While the overall structure and oligomerization state of the heterodimer were maintained, the overall destabilization of the ester analogues was greater (average DeltaDeltaG of 3+ kcal mol(-1)) and more variable than previous studies. Since burial of the more hydrophobic ester should stabilize the backbone and reduce the DeltaDeltaG, the increased destabilization must come from another source. However, the observed destabilization is correlated with the protection factors for individual amide hydrogens from previous hydrogen exchange experiments. Therefore, our results suggest that backbone engineering through ester substitution is a useful approach for probing the relative strength of backbone hydrogen bonds.

Structure-based design of a leucine zipper protein with new DNA contacting region.

We have employed a structure-based design to construct a small folding domain from the F-actin bundling protein villin that contains the amino acids necessary for the DNA binding of the basic leucine zipper protein GCN4 and have compared its DNA binding with GCN4. The monomeric motif folds into a stable domain and binds DNA in a rigid-body mechanism, while its affinity is not higher than that of the basic region peptide. The addition of the leucine zipper region to the folded domain restored its sequence-specific DNA binding comparable to that of GCN4. Unlike the monomeric folded domain, its leucine zipper derivative undergoes a conformational change upon DNA binding. CD spectral and thermodynamic studies indicate that the DNA-contacting region is folded in the presence or absence of DNA and suggest that the junction between the DNA-contacting and the leucine zipper regions transits to a helix in the presence of DNA. These results demonstrate that the structural transition outside the direct-contacting region, which adjusts the precise location of the DNA-contacting region, plays a critical role in the specific complex formation of basic leucine zipper proteins.

A GCN4 variant with a C-terminal basic region binds to DNA with wild-type affinity.

Basic-region leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a leucine zipper dimerization domain and a basic region that directly contacts DNA. In all naturally occurring bZip proteins, the basic region is positioned N-terminal to the leucine zipper. We have designed a series of model bZip peptides in which the basic region of the yeast transcriptional activator GCN4 is placed C-terminal to its leucine zipper. DNA-binding studies demonstrate that the optimal reverse GCN4 (rGCN4) peptide is able to bind specifically and with wild-type affinity to DNA despite this unnatural arrangement of the two subdomains. These results suggest that a thermodynamic basis for the observed N-terminal positioning of the basic region relative to the dimerization domain is unlikely.

De novo design, synthesis and characterization of membrane-active peptides.

Our current level of understanding of membrane-protein folding is primitive, but it is beginning to advance. Previously [Choma, Gratkowski, Lear and DeGrado (2000) Nat. Struct. Biol. 7, 161-166], we described studies of the association in detergent micelles of short, simple-sequence hydrophobic peptides modified from the sequence of the water-soluble, homodimeric coiled-coil GCN4-P1 peptide using the principle that the interiors of membrane proteins are similar to those of water-soluble proteins. Here, we discuss more quantitative aspects of the association equilibrium and compare the free energies of association of a number of mutant peptides designed to explore specific features responsible for the association.

Buried polar residues in coiled-coil interfaces.

Coiled coils, estimated to constitute 3-5% of the encoded residues in most genomes, are characterized by a heptad repeat, (abcdefg)(n), where the buried a and d positions form the interface between multiple alpha-helices. Although generally hydrophobic, a substantial fraction ( approximately 20%) of these a- and d-position residues are polar or charged. We constructed variants of the well-characterized coiled coil GCN4-p1 with a single polar residue (Asn, Gln, Ser, or Thr) at either an a or a d position. The stability and oligomeric specificity of each variant were measured, and crystal structures of coiled-coil trimers with threonine or serine at either an a or a d position were determined. The structures show how single polar residues in the interface affect not only local packing, but also overall coiled-coil geometry as seen by changes in the Crick supercoil parameters and core cavity volumes.

Conformational characterization of a helix-nucleated bicyclic GCN4 decapeptide by proton NMR.

A bicyclic decapeptide, GCN4brM1, which was designed to be a helix-locked analog of the DNA-binding basic region from the yeast transcription factor GCN4, was synthesized and characterized using circular dichroism (CD) spectropolarimetry and 1H-NMR. This peptide has two Lys(i), Asp(i+4) side chain lactam bridges incorporated into its structure in overlapping positions in the peptide chain, linking residues 3 and 7 and residues 4 and 8. CD spectra of GCN4brM1 in aqueous solution are consistent with the expected helical conformation, and indicate that this conformation is remarkably resistant to heat denaturation and is essentially unchanged by addition of 50% (v/v) trifluoroethanol (TFE) as cosolvent. NMR spectra measured in aqueous solution at -5 degrees C show long-range nuclear Overhauser effects (NOEs) that are consistent with an alpha-helical conformation throughout the peptide structure. The measured 3J(HN) coupling constants are also in agreement with an alpha-helical structure. Extremely slow proton-deuterium exchange rates measured for backbone amides in the middle of the peptide indicate that this helix is highly stabilized and rarely unfolds within the side chain bridged sequence. NOE-constrained molecular dynamics simulations gave rise to a single family of converged structures that are fully alpha-helical throughout the GCN4brM1 backbone, and show a single, well-defined conformation for the two side chain bridges. This study demonstrates that two overlapping Lys(i), Asp(i+4) lactam bridges, positioned in consecutive residue positions in a hexapeptide segment, form a rigid alpha-helical structure in aqueous solution that is propagated in both the N-terminal and C-terminal directions.

Rational design of a GCN4-derived mimetic of interleukin-4.

In this work we describe the rational design of two helix coiled coil peptide mimetics of interleukin-4 (IL-4) which are able to recognize and bind its high affinity receptor (IL-4R alpha). We have used the leucine-zipper domain of the yeast transcription factor GCN4 as a scaffold into which the putative binding epitope of IL-4 for IL-4R alpha was transferred in a stepwise manner, using computer-aided molecular modeling. The resulting molecules bind IL-4R alpha with affinities ranging from 2 mM to 5 microM, depending on the fraction of the IL-4 binding site incorporated and on their stability. To our knowledge this is the first time a molecule capable of binding a cytokine receptor has been successfully designed in a rational manner.

A natural motif approach to protein design: a synthetic leucine zipper peptide mimics the biological function of the platelet factor 4 protein.

The design of smaller functional mimics of large proteins has long been an important challenge. In this study we use the natural leucine zipper as a structural template to design a 31-residue peptide analog that mimics the function of the larger platelet factor 4 (PF4) protein. The heparin binding activity of PF4 has been introduced into an unrelated leucine zipper sequence only by virtue of incorporating four lysines of PF4. Circular dichroism and binding experiments have shown that the designed leucine zipper peptide adopts a stable helical conformation and shows significant PF4-like heparin binding activity. These results strongly suggest that the lysine residues play an important role in the binding of PF4 to heparin. The de novo generation of the PF4 function in a designed leucine zipper peptide demonstrates that the leucine zipper motif is a useful scaffold for the design of functional peptides and proteins.

Synthesis and properties of the peptide corresponding to the mutant form of the leucine zipper of the transcriptional activator GCN4 from yeast.

A 33 membered polypeptide corresponding to the leucine zipper region of the yeast transcriptional activator GCN4 was synthesized by solid phase chemical synthesis and characterized. Asparagine in the hydrophobic core of the molecule is replaced by valine in the synthetic variant. The correctness of amino acid sequence of the preparation is corroborated by direct sequencing. High-speed equilibrium ultracentrifugation, ultraviolet circular dichroism spectroscopy and scanning microcalorimetry have been employed to demonstrate that in solution the peptide forms a highly stable triple-stranded alpha-helical coiled coil. The stability of the mutant form is 40 degrees C higher than the dimeric form of natural peptide under similar conditions. It was proposed that location of some polar groups in the 'a' and 'd' positions of natural two-stranded coiled coils may be regarded as protection against alternative triple- and multistranded conformations.