Predictability of the covalent binding of acidic drugs in man

Although metabolism via glucuronide conjugation has generally been considered a detoxification route for carboxylic acids, the newly discovered chemical reactivity of these conjugates, leading to covalent binding with proteins, is consistent with the toxicity observed for drugs containing the carboxylic acid moiety. Here we report that degradation rates (intramolecular rearrangement and hydrolysis) for 9 drug glucuronide metabolites show an excellent correlation (r²=0.995) with the extents of drug covalent binding to albumin in vitro. Furthermore, this binding capacity is predictable based on chemical structure of the acid and depends on the degree of substitution at the carbon alpha to the carboxylic acid. The in vivo covalent binding in humans for these drugs is also predictable (r²=0.873) when the extent of adduct formation is corrected for the measured plasma glucuronide concentrations. These results suggest that the structure of a carboxylic acid drug may predict the degree to which the corresponding acyl glucuronides will form covalent adducts that probably/possibly lead to toxicity. This information could be a useful adjunct in drug design.     

Targeted albumin-based nanoparticles for delivery of amphipathic drugs

We report the preparation and physical and biological characterization of human serum albumin-based micelles of approximately 30 nm diameter for the delivery of amphipathic drugs, represented by doxorubicin. The micelles were surface conjugated with cyclic RGD peptides to guide selective delivery to cells expressing the α(v)β(3) integrin. Multiple poly(ethylene glycol)s (PEGs) with molecular weight of 3400 Da were used to form a hydrophilic outer layer, with the inner core formed by albumin conjugated with doxorubicin via disulfide bonds. Additional doxorubicin was physically adsorbed into this core to attain a high drug loading capacity, where each albumin was associated with about 50 doxorubicin molecules. The formed micelles were stable in serum but continuously released doxorubicin when incubated with free thiols at concentrations mimicking the intracellular environment. When incubated with human melanoma cells (M21+) that express the α(v)β(3) integrin, higher uptake and longer retention of doxorubicin was observed with the RGD-targeted micelles than in the case of untargeted control micelles or free doxorubicin. Consequently, the RGD-targeted micelles manifested cytotoxicity at lower doses of drug than control micelles or free drug.     

Two kinase family dramas

In this issue, Lietha and colleagues (2007) report the structure of focal adhesion kinase (FAK) and reveal how FAK maintains an autoinhibited state. Together with the structure of another tyrosine kinase, ZAP-70 (Deindl et al., 2007), this work highlights the diversity of mechanisms that nature has evolved within the kinase superfamily to regulate their activity through autoinhibition.     

Mechanisms of regulating the Raf kinase family

The MAP Kinase pathway is a key signalling mechanism that regulates many cellular functions such as cell growth, transformation and apoptosis. One of the essential components of this pathway is the serine/threonine kinase, Raf. Raf (MAPKK kinase, MAPKKK) relays the extracellular signal from the receptor/Ras complex to a cascade of cytosolic kinases by phosphorylating and activating MAPK/ERK kinase (MEK; MAPK kinase, MAPKK) that phosphorylates and activates extracellular signal regulated kinase (ERK; mitogen-activated protein kinase, MAPK), which phosphorylates various cytoplasmic and nuclear proteins. Regulation of both Ras and Raf is crucial in the proper maintenance of cell growth as oncogenic mutations in these genes lead to high transforming activity. Ras is mutated in 30% of all human cancers and B-Raf is mutated in 60% of malignant melanomas. The mechanisms that regulate the small GTPase Ras as well as the downstream kinases MEK and extracellular signal regulated kinase (ERK) are well understood. However, the regulation of Raf is complex and involves the integration of other signalling pathways as well as intramolecular interactions, phosphorylation, dephosphorylation and protein-protein interactions. From studies using mammalian isoforms of Raf, as well as C. elegans lin45-Raf, common patterns and unique differences of regulation have emerged. This review will summarize recent findings on the regulation of Raf kinase.     

Genetically encoding latent bioreactive amino acids and the development of covalent protein drugs

As natural proteins generally do not bind targets in a covalent mode, the therapeutic potential of covalent protein drugs remains largely unexplored. Recently, latent bioreactive amino acids have been incorporated into proteins through genetic code expansion, which selectively react with nearby natural residues via proximity-enabled reactivity, generating diverse covalent linkages for proteins in vitro and in cells. These new covalent linkages provide novel avenues for protein research and engineering. In addition, a general platform technology, proximity-enabled reactive therapeutics (PERx), has been established for the development of covalent protein drugs. The first covalent protein drug demonstrates advantageous features in cancer immunotherapy in mice. Selective introduction of covalent bonds into proteins will advance biological studies, synthetic biology, and biotherapeutics with the power of biocompatible covalent chemistries.     

Targeted disruption of the gene for the PAK5 kinase in mice

PAK5 is a member of the group B family of PAK serine/threonine kinases and is an effector for the Rho GTPase Cdc42. PAK5 is highly expressed in the brain and is expressed at lower levels in several other tissues. In cell lines, PAK5 has been shown to play a role in filopodia formation and neurite outgrowth. To examine the biological function of PAK5, we deleted the PAK5 gene in mice. The phenotypes of the PAK5-null mice are completely different from those of mice null for PAK4, another member of the group B PAK family. Unlike PAK4-null mice, which are embryonic lethal, PAK5-null mice develop normally and are fertile. The nervous system appears normal in the absence of PAK5, as do other tissues in which PAK5 is normally expressed. Our results suggest functional redundancy between PAK5 and other Rho GTPase targets.     

Isolation of two members of the rat MAP kinase kinase gene family

Mitogen-activated protein (MAP) kinase kinase (MAPKK) is a recently characterized activator of MAP kinase (MAPK), and is considered to be regulated by a protooncogene product c-Raf-1. It is, however, unclear whether the signals originating from c-Raf-1 utilize this phosphorylation cascade to lead to oncogenesis. To clarify this point, we isolated rat MAPKK cDNAs, and identified two distinct cDNAs encoding MAPKK and a highly related kinase, both with molecular weights of 5 kDa (MEK1 and MEK2). Genomic Southern blot analyses suggested that MAPKK. may form a large gene family.     

Structural relationships in the adenylate kinase family

The sequences of five distantly related adenylate kinases have been aligned. The local conservation of amino acids is discussed in the light of the known three-dimensional structure of one of the enzymes, the cytosolic isoenzyme 1 (AK1) from porcine muscle. The similarity profile outlines clearly the active site in the cleft of the spatial structure of AK1. The alignment reveals further that the enzyme family can be subdivided into small and large variants according to the presence or absence of a particular segment of about 30 residues in the middle of the chain. The extra segments of the large variants are strongly conserved.     

A chemical genetic approach for covalent inhibition of analogue-sensitive aurora kinase

The perturbation of protein kinases with small organic molecules is a powerful approach to dissect kinase function in complex biological systems. Covalent kinase inhibitors that target thiols in the ATP binding pocket of the kinase domain proved to be ideal reagents for the investigation of highly dynamic cellular processes. However, due to the covalent inhibitors' possible off-target reactivities, it is required that the overall shape of the inhibitor as well as the intrinsic reactivity of the electrophile are precisely tuned to favor the reaction with only the desired cysteine. Here we report on the design and biological characterization of covalent anilinoquinazolines as potent inhibitors of genetically engineered Aurora kinase in fission yeast.     

Comparative surface geometry of the protein kinase family

Identifying conserved pockets on the surfaces of a family of proteins can provide insight into conserved geometric features and sites of protein–protein interaction. Here we describe mapping and comparison of the surfaces of aligned crystallographic structures, using the protein kinase family as a model. Pockets are rapidly computed using two computer programs, FADE and Crevasse. FADE uses gradients of atomic density to locate grooves and pockets on the molecular surface. Crevasse, a new piece of software, splits the FADE output into distinct pockets. The computation was run on 10 kinase catalytic cores aligned on the αF‐helix, and the resulting pockets spatially clustered. The active site cleft appears as a large, contiguous site that can be subdivided into nucleotide and substrate docking sites. Substrate specificity determinants in the active site cleft between serine/threonine and tyrosine kinases are visible and distinct. The active site clefts cluster tightly, showing a conserved spatial relationship between the active site and αF‐helix in the C‐lobe. When the αC‐helix is examined, there are multiple mechanisms for anchoring the helix using spatially conserved docking sites. A novel site at the top of the N‐lobe is present in all the kinases, and there is a large conserved pocket over the hinge and the αC‐β4 loop. Other pockets on the kinase core are strongly conserved but have not yet been mapped to a protein–protein interaction. Sites identified by this algorithm have revealed structural and spatially conserved features of the kinase family and potential conserved intermolecular and intramolecular binding sites.     

Targeted liposomes for delivery of protein-based drugs into the cytoplasm of tumor cells

Our goal was to deliver therapeutically active macromolecules into the cytosol of target cells. First, attempts were made to prepare virosomes that specifically interact with OVCAR-3 cells (human ovarian cancer cells). Detergent solubilized influenza virus envelopes were reconstituted forming virosomes. Cell specificity was introduced by incorporating PEG-derivatized lipids with mAB 323/A3 (Fab' fragments) connected to their distal PEG end. These cell-specific, modified virosomes maintained their fusogenic activity when lowering the pH. Most importantly, antibody-mediated binding was a prerequisite for low-pH induced membrane fusion. However, basically, there are two problems with this approach: (1) these virosomes are quite leaky and (2) virosomes can be expected to be immunogenic. A solution to tackle leakage and potential immunogenicity of these site-specific liposomal structures is to use immuno-PEG-liposomes with a pH-dependent fusogen inside the liposome. The system that we designed to test this concept consisted of (1) the fusogenic di-peptide dINF-7, (2) the monoclonal antibody 425 connected to the distal end of PEG-PE (for site specific binding and endosomal uptake), (3) diphtheria toxin chain A (DTA, as carrier-dependent active compound) and phosphatidylcholine/cholesterol as 'bilayer backbone'. A series of tests were performed to show that selective binding and pH-dependent destabilization of (endosomal) membranes indeed occurred. To test the cytotoxic activity of these DTA loaded liposomes, OVCAR-3 cells were used for testing. OVCAR-3 cells express the epidermal growth factor receptor, which is the ligand for antibody 425. In vitro, these site specific and fusogenic liposomes showed a remarkable, cell specific cytotoxic effect.     

Conserved spatial patterns across the protein kinase family

Protein kinases are a large family of enzymes heavily involved in signal transduction, regulation of metabolism, and control of cell growth and differentiation. These functions require precise recognition of widely diverse signals and substrates, and very detailed control of protein kinase activity. Large molecules interact primarily through recognition of surface features. Comparison of surfaces is complicated by both sequence diversity and conformational variability, including multiple possible rotameric states of side chains. We used a recently developed method of protein surface comparison to compare different serine/threonine and tyrosine kinases. As we have shown, two hydrophobic cores inside a protein kinase molecule are connected by a unique formation, called the "spine". It exists only in the active conformation of protein kinases and is dynamically disassembled during the inactivation process. Detection of such structures by any other method was not possible as the residues which comprise the spine do not form any sequence or 3D motifs in a traditional sense.     

Autocatalytic mechanism and consequences of covalent heme attachment in the cytochrome P4504A family

The prosthetic heme group in the CYP4A family of cytochrome P450 enzymes is covalently attached to an I-helix glutamic acid residue. This glutamic acid is conserved in the CYP4 family but is absent in other P450 families. As shown here, the glutamic acid is linked, presumably via an ester bond, to a hydroxyl group on the heme 5-methyl group. Mutation of the glutamic acid to an alanine in CYP4A1, CYP4A3, and CYP4A11 suppresses covalent heme binding. In wild-type CYP4A3 68% of the heme is covalently bound to the heterologously expressed protein, but in the CYP4A3/E318D mutant, 47% of the heme is unchanged, 47% is present as noncovalently bound 5-hydroxymethylheme, and only 6% is covalently bound to the protein. In the CYP4A3/E318Q mutant, the majority of the heme is unaltered, and <2% is covalently linked. The proportion of covalently bound heme in the recombinant CYP4A proteins increases with time under turnover conditions. The catalytic activity is sensitive in some, but not all, CYP4A enzymes to the extent of covalent heme binding. Mutations of Glu(318) in CYP4A3 decrease the apparent k(cat) values for lauric acid hydroxylation. The key conclusions are that (a) covalent heme binding occurs via an ester bond to the heme 5-methyl group, (b) covalent binding of the heme is mediated by an autocatalytic process, and (c) fatty acid oxidation is sensitive in some CYP4A enzymes to the presence or absence of the heme covalent link.