Protein phosphorylation and signal transduction modulation: chemistry perspectives for small-molecule drug discovery.

Protein phosphorylation has been exploited by Nature in profound ways to control various aspects of cell proliferation, differentiation, metabolism, survival, motility and gene transcription. Cellular signal transduction pathways involve protein kinases, protein phosphatases, and phosphoprotein-interacting domain (e.g., SH2, PTB, WW, FHA, 14-3-3) containing cellular proteins to provide multidimensional, dynamic and reversible regulation of many biological activities. Knowledge of cellular signal transduction pathways has led to the identification of promising therapeutic targets amongst these superfamilies of enzymes and adapter proteins which have been linked to various cancers as well as inflammatory, immune, metabolic and bone diseases. This review focuses on protein kinase, protein phosphatase and phosphoprotein-interacting cellular protein therapeutic targets with an emphasis on small-molecule drug discovery from a chemistry perspective. Noteworthy studies related to molecular genetics, signal transduction pathways, structural biology, and drug design for several of these therapeutic targets are highlighted. Some exemplary proof-of-concept lead compounds, clinical candidates and/or breakthrough medicines are further detailed to illustrate achievements as well as challenges in the generation, optimization and development of small-molecule inhibitors of protein kinases, protein phosphatases or phosphoprotein-interacting domain containing cellular proteins.

Intravenous safety and pharmacokinetics of a novel dimerizer drug, AP1903, in healthy volunteers.

AP1903 is a novel gene-targeted drug that is being developed for use in drug-regulated cell therapies. An intravenous, single-blind, placebo- and saline-controlled, ascending-dose study was performed to evaluate the safety, tolerability, and pharmacokinetics of AP1903. Twenty-eight normal healthy male volunteers were randomized into five dosage groups of AP1903 (0.01, 0.05, 0.1, 0.5, and 1 mg/kg). Within each group, 4 volunteers received a single dose of AP1903, 1 volunteer received an equal volume of placebo, and 1 received an equal volume of normal saline. The only exception was in the 0.5 mg/kg group, in which 4 volunteers were dosed: 3 received AP1903 and 1 received normal saline. All dosages were administered as intravenous infusions over 2 hours. Clinical safety parameters were monitored, and serial blood and urine samples were collected for analysis of AP1903. No drug-related adverse events were observed at any of the dose levels with the possible exception of facial flushing in 1 volunteer at the 1.0 mg/kg dose level. AP1903 plasma levels were directly proportional to the administered dose, with mean Cmax values ranging from approximately 10 to 1,275 ng/mL over the 0.01 to 1.0 mg/kg dose range. Following the infusion period, blood concentrations revealed a rapid distribution phase, with plasma levels being reduced to approximately 18%, 7%, and 1% of the maximal concentration at 0.5, 2, and 10 hours postdose, respectively. AP1903 was shown to be safe and well tolerated at all dose levels and demonstrated a favorable pharmacokinetic profile at doses well above the anticipated therapeutic dose.

A novel phosphotyrosine mimetic 4'-carboxymethyloxy-3'-phosphonophenylalanine (Cpp): exploitation in the design of nonpeptide inhibitors of pp60(Src) SH2 domain.

The novel phosphotyrosine (pTyr) mimetic 4'-carboxymethyloxy-3'-phosphonophenylalanine (Cpp) has been designed and incorporated into a series of nonpeptide inhibitors of the SH2 domain of pp60(c-Src) (Src) tyrosine kinase. A 2.2 A X-ray crystal structure of 1a bound to a mutant form of Lck SH2 domain provides insight regarding the structure-activity relationships and supports the design concept of this new pTyr mimetic.

Selective inhibition of Src SH2 by a novel thiol-targeting tricarbonyl-modified inhibitor and mechanistic analysis by (1)H/(13)C NMR spectroscopy.

Detailed analysis of Src SH2 binding by peptides containing a novel tricarbonyl-modified pTyr moiety is described. We envisaged that Src SH2 selectivity might be obtained by exploiting the thiol group of Cys188 present in the pTyr binding pocket of the protein at the betaC3 position. Peptidyl as well as non-peptidyl compounds 1-4 possessing a 4-alpha,beta-diketoester-modified pTyr mimic exhibited micromolar affinity to Src SH2. Furthermore, these tricarbonyl compounds were selective for Src SH2 to the extent they showed no significant affinity for either Cys188Ser or Cys188Ala Src SH2 mutants. Upon closer examination of the binding of these tricarbonyls to Src SH2 using NMR of 13C-labeled compounds (6a, 6b, and 6c), we found that after the initial binding event the molecule disproportionated in a 'retro-Claisen' fashion to provide benzoic acid 16 and, following hydrolysis of the methyl ester 17, the hemiketal adduct of glyoxalic acid 18.

Bone-targeted Src SH2 inhibitors block Src cellular activity and osteoclast-mediated resorption.

Src, a nonreceptor tyrosine kinase, is an important regulator of osteoclast-mediated resorption. We have investigated whether compounds that bind to the Src SH2 domain inhibit Src activity in cells and decrease osteoclast-mediated resorption. Compounds were examined for binding to the Src SH2 domain in vitro using a fluorescence polarization binding assay. Experiments were carried out with compounds demonstrating in vitro binding activity (nmol/L range) to determine if they inhibit Src SH2 binding and Src function in cells, demonstrate blockade of Src signaling, and lack cellular toxicity. Cell-based assays included: (1) a mammalian two-hybrid assay; (2) morphological reversion and growth inhibition of cSrcY527F-transformed cells; and (3) inhibition of cortactin phosphorylation in csk-/- cells. The Src SH2 binding compounds inhibit Src activity in all three of these mechanism-based assays. The compounds described were synthesized to contain nonhydrolyzable phosphotyrosine mimics that bind to bone. These compounds were further tested and found to inhibit rabbit osteoclast-mediated resorption of dentine. These results indicate that compounds that bind to the Src SH2 domain can inhibit Src activity in cells and inhibit osteoclast-mediated resorption.

Src inhibitors: genomics to therapeutics.

Following the milestone discoveries that identified Src as the first known protein tyrosine kinase and as a prototype oncogene, as well as Src transgenic studies to validate it as a promising therapeutic target for osteoporosis, intense efforts are being made to create Src inhibitor drugs. Drug discovery strategies focused on both the non-catalytic and catalytic domains of Src have successfully resulted in promising Src inhibitor lead compounds with potential therapeutic applications for osteoporosis, cancer, and other diseases. Some noteworthy examples of Src inhibitors are described, and their chemical diversity, structure-based design, and biological activities in vitro and in vivo are illustrated. The potency, selectivity, and in vivo efficacy of key Src inhibitors are being investigated in molecular, cellular and animal models. Consequently, Src inhibitor drug development is imminent, and current studies are well-poised to achieve the ultimate milestone of a Src inhibitor therapeutic.

Structure-based design of an osteoclast-selective, nonpeptide src homology 2 inhibitor with in vivo antiresorptive activity.

Targeted disruption of the pp60(src) (Src) gene has implicated this tyrosine kinase in osteoclast-mediated bone resorption and as a therapeutic target for the treatment of osteoporosis and other bone-related diseases. Herein we describe the discovery of a nonpeptide inhibitor (AP22408) of Src that demonstrates in vivo antiresorptive activity. Based on a cocrystal structure of the noncatalytic Src homology 2 (SH2) domain of Src complexed with citrate [in the phosphotyrosine (pTyr) binding pocket], we designed 3',4'-diphosphonophenylalanine (Dpp) as a pTyr mimic. In addition to its design to bind Src SH2, the Dpp moiety exhibits bone-targeting properties that confer osteoclast selectivity, hence minimizing possible undesired effects on other cells that have Src-dependent activities. The chemical structure AP22408 also illustrates a bicyclic template to replace the post-pTyr sequence of cognate Src SH2 phosphopeptides such as Ac-pTyr-Glu-Glu-Ile (1). An x-ray structure of AP22408 complexed with Lck (S164C) SH2 confirmed molecular interactions of both the Dpp and bicyclic template of AP22408 as predicted from molecular modeling. Relative to the cognate phosphopeptide, AP22408 exhibits significantly increased Src SH2 binding affinity (IC(50) = 0.30 microM for AP22408 and 5.5 microM for 1). Furthermore, AP22408 inhibits rabbit osteoclast-mediated resorption of dentine in a cellular assay, exhibits bone-targeting properties based on a hydroxyapatite adsorption assay, and demonstrates in vivo antiresorptive activity in a parathyroid hormone-induced rat model.

Investigating protein-ligand interactions with a mutant FKBP possessing a designed specificity pocket.

Using structure-based design and protein mutagenesis we have remodeled the FKBP12 ligand binding site to include a sizable, hydrophobic specificity pocket. This mutant (F36V-FKBP) is capable of binding, with low or subnanomolar affinities, novel synthetic ligands possessing designed substituents that sterically prevent binding to the wild-type protein. Using binding and structural analysis of bumped compounds, we show here that the pocket is highly promiscuous-capable of binding a range of hydrophobic alkyl and aryl moieties with comparable affinity. Ligand affinity therefore appears largely insensitive to the degree of occupancy or quality of packing of the pocket. NMR spectroscopic analysis indicates that similar ligands can adopt radically different binding modes, thus complicating the interpretation of structure-activity relationships.

Signal transduction drug discovery: targets, mechanisms and structure-based design.

Signal transduction targets include catalytic and/or non-catalytic domains, which are critical to various aspects of cell growth, differentiation, metabolism and function, mitogenesis, motility and gene transcription. Specific examples of molecular targets include the catalytic domains of protein tyrosine kinases (PTKs) and of protein tyrosine phosphatases (PTPases), as well as related protein-protein interaction motifs (eg, SH2, PTB and SH3 domains). From the relationship of tyrosine phosphorylation to intracellular pathway regulation by PTKs and PTPases, the dynamic and reversible binding interactions of SH2 and PTB domain-containing proteins with their cognate phosphotyrosine (pTyr)-containing proteins, provide an additional dimension to the modulation of signal transduction pathways which exist as a result of pTyr formation, degradation and molecular recognition events. This review focuses on our current understanding of key relative to recent reports which have provided further insight into their three-dimensional structure and mechanism. This review also highlights recent progress in the design and optimization of molecular mechanism-based signal transduction inhibitors.

An efficient synthesis of a 4'-phosphonodifluoromethyl-3'-formyl-phenylalanine containing SRC SH2 ligand.

A CuBr-mediated, regioselective cross-coupling between methyl 2,5-diiodobenzoate (4) and [(diethoxyphosphinyl)difluoromethyl]zinc bromide is reported. Palladium-catalyzed incorporation of an amino acid side chain, followed by subsequent modifications resulted in the rapid construction of 2. Compound 2 was designed to engage Cys188 of the Src SH2 domain, however, this was not observed spectroscopically.

Structure-based design and synthesis of a novel class of Src SH2 inhibitors.

The structure-based design and synthesis of a novel class of 2,4-disubstituted thiazoles as Src SH2 inhibitors is described. Initial results are presented, including the X-ray and NMR analysis of one thiazole inhibitor bound to Lck and Src SH2.

Selective epimerization of rapamycin via a retroaldol/aldol mechanism mediated by titanium tetraisopropoxide.

We describe the efficient and selective epimerization of the immunosuppressant rapamycin to 28-epirapamycin under mild conditions. The mechanism of epimerization involves an equilibrium of the four C28/C29 diastereomers through a two-step retroaldol/aldol (macrocycle ring-opening/ring-closing) sequence. This retroaldol/aldol equilibration is not restricted to rapamycin but is also applicable to acyclic beta-hydroxyketones. A potentially useful extension of the method--the use of beta-hydroxyketones as enolate synthons for effecting inter- or intramolecular aldol reactions under neutral conditions--is demonstrated.

SH3 domains and drug design: ligands, structure, and biological function.

The ligand binding preferences, structural features, and biological function of SH3 (Src homology 3) domains are discussed. SH3 domains bind "core" Pro-rich peptide ligands (7-9 amino acids in length) in a polyproline II helical conformation in a highly conserved aromatic rich patch on the protein surface (approximately 390 A2). The ligands can interact with the protein in one of two orientations, depending on the position (N- vs C-terminal) of ligand residues binding to the SH3 selectivity pocket. Core SH3 ligands are characterized by relatively weak interactions (KD = 5-100 microM) that show little binding selectivity within SH3 families. Higher affinity, more selective contiguous ligands require additional flanking residues that bind to less conserved portions of the SH3 surface, with corresponding increase in ligand size and complexity. In contrast to peptide ligands, protein ligands of SH3 domains can exploit multiple discontiguous interactions to enhance affinity and selectivity. A protein-SH3 interaction that utilizes unique interactions may permit the design of small high affinity SH3 ligands. At present, the extended nature of the binding site and homologous nature of the core binding region among SH3 domains present key challenges for structure-based drug design.

Structural basis for the binding of proline-rich peptides to SH3 domains.

A common RXL motif was found in proline-rich ligands that were selected from a biased combinatorial peptide library on the basis of their ability to bind specifically to the SH3 domains from phosphatidylinositol 3-kinase (PI3K) or c-Src. The solution structure of the PI3K SH3 domain complexed to one of these ligands, RKLPPRPSK (RLP1), was determined. Structure-based mutations were introduced into the PI3K SH3 domain and the RLP1 ligand, and the influence of these mutations on binding was evaluated. We conclude that SH3 domains recognize proline-rich motifs possessing the left-handed type II polyproline (PPII) helix conformation. Two proline residues directly contact the receptor. Other prolines in the ligands appear to function as a molecular scaffold, promoting the formation of the PPII helix. Three nonproline residues consisting of combinations of arginine and leucine interact extensively with the SH3 domain and appear to confer ligand specificity.

1H and 15N assignments and secondary structure of the PI3K SH3 domain.

The sequential 1H and 15N assignments of the SH3 domain of human phosphatidyl inositol 3'-kinase (PI3K) were determined by a combination of homonuclear and heteronuclear NMR experiments. With the exception of several protons belonging to lysine and proline residues, all proton and proton-bearing amide nitrogen resonances were assigned. Based on the sequential nuclear Overhauser effects (NOEs), 3JNH-C alpha H coupling constants and locations of slowly exchanging amide protons, we determined that the secondary structures of the protein consists of six beta-strands, two beta-turns and four short helices. Additional long range NOEs indicate that these beta-strands form two antiparallel beta-sheets. The topology of secondary structural elements of the PI3K SH3 domain is similar to those of the SH3 domains from c-Src and alpha-spectrin, suggesting that the SH3 family has a common tertiary structural motif.

Structure of the PI3K SH3 domain and analysis of the SH3 family.

Src homology 3 (SH3) domains, which are found in many proteins involved in intracellular signal transduction, mediate specific protein-protein interactions. The three-dimensional structure of the SH3 domain in the p85 subunit of the phosphatidylinositol 3-kinase (PI3K) has been determined by multidimensional NMR methods. The molecule consists of four short helices, two beta turns, and two antiparallel beta sheets. The beta sheets are highly similar to corresponding regions in the SH3 domain of the tyrosine kinase Src, even though the sequence identity of the two domains is low. There is a unique 15 amino acid insert in PI3K that contains three short helices. There are substantial differences in the identity of the amino acids that make up the receptor site of SH3 domains. The results suggest that while the overall structures of the binding sites in the PI3K and Src SH3 domains are similar, their ligand binding properties may differ.

Solution conformation of endothelin and point mutants by nuclear magnetic resonance spectroscopy.

Two-dimensional NMR techniques were utilized to determine the secondary structural elements of endothelin-1 (ET-1), a potent vasoconstrictor peptide, and two of its point mutants, Met-7 to Ala-7 (ETM7A), and Asp-8 to Ala-8 (ETD8A) in acetic acid-d3/water solution. Sequence specific NMR assignments were determined for all three peptides, as well as chemical shifts and NOE connectivity patterns. The chemical shifts of ET-1 and ETM7A are identical (+/- 0.05 ppm) except for the site of substitution, whereas marked shift changes were detected between ET-1 and ETD8A. These chemical shift differences imply that the Asp-8 to Ala-8 mutation has induced a conformational change relative to the parent conformation. All three molecules show the same basic nuclear Overhauser effect (NOE) pattern, which suggests that the gross conformation of all three molecules is the same. Small changes in sequential NOE intensities and changes in medium-range NOE patterns indicate that there are subtle conformational differences between ET-1 and ETD8A.

An active covalently linked dimer of human interferon-gamma. Subunit orientation in the native protein.

We have constructed and expressed a covalently linked head to tail dimer of human interferon-gamma (IFN-gamma) in which two monomers are joined head to tail via a rigid peptide hinge using genetic engineering techniques. The hinge was derived from the human immunoglobin IgA1 sequence (Hallewell, R.A., Laria, I., Tabrizi, A., Carlin, G., Getzoff, E.D., Tainer, J.A., Cousens, L.S., and Mullenbach, G.T. (1989) J. Biol. Chem. 264, 5260-5268). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the polypeptide produced by this construction migrates as a 30,000 polypeptide species. The protein elutes as a single species by molecular sieve chromatography under native conditions. The covalently linked dimer exhibits one-half the antiviral activity of native dimeric IFN-gamma; receptor binding assays show the covalently linked dimer binds to the IFN-gamma receptor with one-half the avidity of native IFN-gamma. This difference is not due to conformational differences between the two molecules, as the aromatic region of the NMR spectrum of the purified covalently linked dimer is identical with that of the wild type protein. From these data, we suggest that human IFN-gamma associates in a head to tail dimer in its active configuration. Regions of IFN-gamma are contiguous with the amino and carboxyl termini and are obscured by the hinge peptide in the covalently linked dimer. Our studies demonstrate that these regions may be important for receptor-ligand interaction.