Keykavous Parang

Mechanism-based design of a protein kinase inhibitor.

Protein kinase inhibitors have applications as anticancer therapeutic agents and biological tools in cell signaling. Based on a phosphoryl transfer mechanism involving a dissociative transition state, a potent and selective bisubstrate inhibitor for the insulin receptor tyrosine kinase was synthesized by linking ATPγS to a peptide substrate analog via a two-carbon spacer. The compound was a high affinity competitive inhibitor against both nucleotide and peptide substrates and showed a slow off-rate. A crystal structure of this inhibitor bound to the tyrosine kinase domain of the insulin receptor confirmed the key design features inspired by a dissociative transition state, and revealed that the linker takes part in the octahedral coordination of an active site Mg2+. These studies suggest a general strategy for the development of selective protein kinase inhibitors.

Protein pyrophosphorylation by inositol pyrophosphates is a posttranslational event

In a previous study, we showed that the inositol pyrophosphate diphosphoinositol pentakisphosphate (IP7) physiologically phosphorylates mammalian and yeast proteins. We now report that this phosphate transfer reflects pyrophosphorylation. Thus, proteins must be prephosphorylated by ATP to prime them for IP7 phosphorylation. IP7 phosphorylates synthetic phosphopeptides but not if their phosphates have been masked by methylation or pyrophosphorylation. Moreover, IP7 phosphorylated peptides are more acid-labile and more resistant to phosphatases than ATP phosphorylated peptides, indicating a different type of phosphate bond. Pyrophosphorylation may represent a novel mode of signaling to proteins.

Inhibitors of Protein Kinase Signaling Pathways Emerging Therapies for Cardiovascular Disease

Abstract—Protein kinases are enzymes that covalently modify proteins by attaching phosphate groups (from ATP) to serine, threonine, and/or tyrosine residues. In so doing, the functional properties of the protein kinase’s substrates are modified. Protein kinases transduce signals from the cell membrane into the interior of the cell. Such signals include not only those arising from ligand-receptor interactions but also environmental perturbations such as when the membrane undergoes mechanical deformation (ie, cell stretch or shear stress). Ultimately, the activation of signaling pathways that use protein kinases often culminates in the reprogramming of gene expression through the direct regulation of transcription factors or through the regulation of mRNA stability or protein translation. Protein kinases regulate most aspects of normal cellular function. The pathophysiological dysfunction of protein kinase signaling pathways underlies the molecular basis of many cancers and of several manifestations of cardiovascular disease, such as hypertrophy and other types of left ventricular remodeling, ischemia/reperfusion injury, angiogenesis, and atherogenesis. Given their roles in such a wide variety of disease states, protein kinases are rapidly becoming extremely attractive targets for drug discovery, probably second only to heterotrimeric G protein-coupled receptors (eg, angiotensin II). Here, we will review the reasons for this explosion in interest in inhibitors of protein kinases and will describe the process of identifying novel drugs directed against kinases. We will specifically focus on disease states for which drug development has proceeded to the point of clinical or advanced preclinical studies.

Impairment of TrkB-PSD-95 signaling in Angelman syndrome.

Brain-derived neurotrophic factor signaling is defective in Angelman syndrome and can be rescued by disruption of Arc/PSD95 binding.

Designing bisubstrate analog inhibitors for protein kinases

Protein kinases play critical roles in signal transduction pathways by transmitting extracellular signals across the cell membrane to distant locations in the cytoplasm and the nucleus. The development of protein kinase inhibitors has been hindered by the broad overlapping substrate specificities exhibited by these enzymes. The design of bisubstrate analog inhibitors could provide for the enhancement of specificity and potency in protein kinase inhibition. Bisubstrate analog inhibitors form a special group of protein kinase inhibitors that mimic two natural substrates/ligands and that simultaneously associate with two regions of given kinases. Most bisubstrate analogs have been designed to mimic the phosphate donor (ATP) and the acceptor components (Ser-, Thr-, or Tyr-containing peptides). Recent studies have emphasized the importance of maintaining a specific distance between these two components to achieve potent inhibition. In this review, we present a discussion of the methods for designing protein kinase inhibitors by mechanism-based approaches. Emphasis is given to bivalent approaches, with an interpretation of what has been learned from more and less successful examples. Future challenges in this area are also highlighted.

Cell-Penetrating Homochiral Cyclic Peptides as Nuclear-Targeting Molecular Transporters†

The intracellular delivery of biologically active cargos by employing linear cell-penetrating peptides (CPPs) has been previously reported. Conjugation to linear cationic CPPs, such as TAT (trans-acting activator of transcription; a peptide derived from the HIV-1 transactivator protein), 3] penetratin, antennapedia, or oligoarginine, efficiently enhances the cellular uptake through different mechanisms. The cellular uptake and internalization of many CPPs along with the conjugated cargo occurs predominantly by an endocytic pathway that involves macropinocytosis, a caveolae pathway, clathrin-mediated endocytosis, or lipid-raft dependent endocytosis. Endosomal uptake represents a major challenge in targeted intracellular drug delivery since some compounds are trapped in endosomes and cannot reach the biological targets in the cytoplasm or nucleus. Thus, strategies that promote endosomal escape or avoid endosomal routes are required for improving bioavailability. Moreover, the nuclear delivery of cell-impermeable and water-insoluble molecules remains a major challenge. The nucleus is a desirable target because the genetic information of the cell and transcription machinery resides there. To date, most approaches for nuclear delivery of compounds have taken advantage of covalent conjugation, which requires release of the cargo from the conjugate and/or endosomal escape. There is therefore a need to develop alternative stable peptide carriers that avoid endosomal pathways and/or covalent conjugation. Compared to linear peptides that are susceptible to hydrolysis by endogenous peptidases, cyclic peptides are enzymatically more stable. The cell-penetrating properties and application of homochiral l-cyclic peptides in drug delivery remain unexplored. Previous studies on linear CPPs by our research group and others indicated that an optimal balance of positive charge and hydrophobicity is required for interactions with the cell membrane and deep penetration into the lipid bilayer. 4,8–10] Herein, we report the design and evaluation of amphipathic homochiral l-cyclic peptides for potential applications as CPPs and/or as molecular transporters of bioactive compounds. Eleven cyclic peptides, namely [WR]4, [FK]4, [AK]4, [EL]4, [RFEF]2, [EK]4, [ER]4, [FR]4, [RFE]3, [WR]3, and [WR]5 (Scheme 1), which contain l-amino acids, were synthesized by employing 9-fluorenylmethyloxycarbonyl (Fmoc) based peptide chemistry. The selection of the cyclic peptides was based on the presence of hydrophobic residues (e.g., W, F, L) and charged residues (e.g., K, R, E). We hypothesized that an optimal amphipathic cyclic peptide that contains appropriate residues, and that undergoes intermolecular and intramolecular interactions can act as a CPP and/or entrap and deliver a bioactive compound intracellularly. To examine the potential application of the cyclic peptides as molecular transporters, a model experiment was performed with lamivudine (( )-2’,3’-dideoxy-3’-thiacytidine, 3TC). 3TC is a nucleoside reverse transcriptase inhibitor that blocks HIV-1 and hepatitis B virus replication. The efficient cellular uptake of 3TC is critical for effective antiviral activity. To monitor the molecular transport ability of the cyclic peptides, a carboxyfluorescein derivative of 3TC (F-3TC) was synthesized. The cellular uptake of the fluorescently labeled 3TC (F3TC) was examined in the leukemia CCRF-CEM cell line in the presence or absence of cyclic peptides. After 1 h incubation at 37 8C, the cells were treated with trypsin to remove the cell-surface-bound drug. The cellular uptake of F3TC was monitored by fluorescence-activated cell sorting (FACS; Figure 1a) and fluorescence microscopy (Figure 1b). The cyclic peptides did not exhibit any cytotoxicity by using an MTT assay at an experimental concentration of 50 mm in four different cell lines, namely CCRF-CEM, HT-29, MDAMD-468, and SK-OV-3, thus showing consistent results (Figure S12). FACS and fluorescence microscopy showed significantly higher fluorescence signals in the cells treated with F-3TCloaded [WR]4 and [WR]5 compared to those treated with other F-3TC-loaded cyclic peptides and with F-3TC alone, thus suggesting that the uptake of F-3TC is facilitated by [WR]n (n = 4, 5) and is dependent on nature of amino acids. F3TC-loaded [WR]5 exhibited a cellular uptake that was approximately five times higher than that of F-3TC alone (Figure 1a). Phosphopeptides are valuable probes for studying phosphoprotein–protein interactions because these peptides mimic the interactions between the negatively charged phosphate group of phosphoproteins and positively charged amino acids in the binding pockets of a number of proteins. Studying negatively charged phosphopeptides in cellular systems is challenging because these peptides do not readily [*] Dr. D. Mandal, A. Nasrolahi Shirazi, Prof. K. Parang Department of Biomedical and Pharmaceutical Sciences University of Rhode Island 41 Lower College Road, Kingston, RI 02881 (USA) E-mail: kparang@uri.edu

Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase

Protein tyrosine kinases (PTK) are key enzymes of mammalian signal transduction. For the fidelity of signal transduction, each PTK phosphorylates only one or a few proteins on specific Tyr residues. Substrate specificity is thought to be mediated by PTK–substrate docking interactions and recognition of the phosphorylation site sequence by the kinase active site. However, a substrate-docking site has not been determined on any PTK. C-terminal Src kinase (Csk) is a PTK that specifically phosphorylates Src family kinases on a C-terminal Tyr. In this study, by sequence alignment and site-specific mutagenesis, we located a substrate-docking site on Csk. Mutations in the docking site disabled Csk to phosphorylate, regulate, and complex with Src but only moderately affected its general kinase activity. A peptide mimicking the docking site potently inhibited (IC50 = 21 μM) Csk phosphorylation of Src but only moderately inhibited (IC50 = 422 μM) its general kinase activity. Determination of the substrate-docking site provides the structural basis of substrate specificity in Csk and a model for understanding substrate specificity in other PTKs.

Synthesis of 3-phenylpyrazolopyrimidine-1,2,3-triazole conjugates and evaluation of their Src kinase inhibitory and anticancer activities.

A series of two classes of 3-phenylpyrazolopyrimidine-1,2,3-triazole conjugates were synthesized using click chemistry approach. All compounds were evaluated for inhibition of Src kinase and human ovarian adenocarcinoma (SK-Ov-3), breast carcinoma (MDA-MB-361), and colon adenocarcinoma (HT-29). Hexyl triazolyl-substituted 3-phenylpyrazolopyrimidine exhibited inhibition of Src kinase with an IC(50) value of 5.6 μM. 4-Methoxyphenyl triazolyl-substituted 3-phenylpyrazolopyrimidine inhibited the cell proliferation of HT-29 and SK-Ov-3 by 73% and 58%, respectively, at a concentration of 50 μM.

Current targets for anticancer drug discovery.

The call for the discovery of less toxic, more selective, and more effective agents to treat cancer has become more urgent. Inhibition of angiogenesis continues to be one of the main streams in the current cancer drug discovery activity. Insights into tumor angiogenesis biology have led to the identification of a number of molecules, which are important for the progression of these processes. Of particular interest is a group of growth factors including fibroblast growth factor, platelet-derived growth factor, and vascular endothelial growth factor. These growth factors and their corresponding receptor tyrosine kinases have become important targets for inhibition of the proliferation of endothelial cells, the main component of blood vessels. The validated targets for inhibition of angiogenesis also include a family of matrix metalloproteinases and cell adhesion molecules. In the closely related area, protein kinases have emerged as one of the most important targets for drug discovery. Besides growth factor receptor tyrosine kinases, numerous other protein kinases implicated in malignancies have been identified including non-receptor kinases such as Bcl-Abl and Src kinases. In addition, the cell cycle regulators (cyclin-dependent kinases, p21 gene) and apoptosis modulators (Bcl-2 oncoprotein, p53 tumor suppressor gene, survivin protein, etc) have also attracted renewed interest as potential targets for anticancer drug discovery. Other molecular targets include protein farnesyltransferase (FTase), histone deacetylase (HDAC), and telomerase, which have essential roles in cellular signal transduction pathways (FTase, HDAC) and cell life-span (telomerase). This review presents a comprehensive summary and discussion on the most important targets currently attracting a great deal of interest in contemporary anticancer drug design and discovery. Recent advances complementing these targets are also highlighted.

Development of cytarabine prodrugs and delivery systems for leukemia treatment

Importance of the field: Cytarabine is a polar nucleoside drug used for the treatment of myeloid leukemia and non-Hodgkins lymphoma. The drug has a short plasma half-life, low stability and limited bioavailability. Overdosing of patients with continuous infusions may lead to side effects. Thus, various prodrug strategies and delivery systems have been explored extensively to enhance the half-life, stability and delivery of cytarabine. Among the recent cytarabine prodrugs, amino acid conjugate ValCytarabine and fatty acid derivative CP-4055 (in Phase III trials) have been investigated for the treatment of leukemia and solid tumors, respectively. Alternatively, delivery systems of cytarabine have emerged for the treatment of different cancers. The liposomal-cytarabine formulation (DepoCyt, Pacira Pharmaceuticals Inc., New Jersey, USA) has been approved for the treatment of lymphomatous meningitis. Areas covered in this review: Various prodrug strategies evaluated for cytarabine are discussed. Then, the review summarizes the drug delivery systems that have been used for more effective cancer therapy. What the reader will gain: This review provides in-depth discussion of the prodrug strategy and delivery systems of cytarabine derivatives for the treatment of cancer. The design of cytarabine prodrugs and delivery systems provides insights for designing the next generation of more effective anticancer agents with enhanced delivery and stability. Take home message: Strategies on designing cytarabine prodrug and delivery formulations showed great promise in developing effective anticancer agents with better therapeutic profile. Similar studies with other anticancer nucleosides can be an alternative approach to gaining access to more effective anticancer agents.