Acquisition of high-affinity, SH2-targeted ligands via a spatially focused library.
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Benzene Derivatives↗
Biomedical subjects
D S Lawrence
Publications and source records attributed to D S Lawrence.
Light-activated proteins.
A wide assortment of caged compounds, which are species whose biological activity can be unleashed with light, have been synthesized and used to investigate a variety of biological phenomena. In contrast, the construction of caged proteins and their application to biological systems has lagged far behind. Recent advances in the synthesis of caged proteins, as well as the development of intracellular protein delivery systems, furnish a framework upon which light-activated proteins can be designed, synthesized and employed to address questions of biological significance.
Animals↗
Definition of optimal substrate recognition motifs of Ca2+-calmodulin-dependent protein kinases IV and II reveals shared and distinctive features.
The substrate recognition determinants of Ca2+-calmodulin-dependent protein kinase (CaMK) IV and CaMKIIalpha were investigated using peptide substrates modeled on the amino acid sequence encompassing Ser-9 of synapsin I. For both kinases, hydrophobic residues (Leu or Phe) at the -5 position, are well tolerated, whereas non-hydrophobic residues (Arg, Ala, or Asp) decrease Vmax/Km by 55- to >4000-fold. At the -3 position, substitution of Ala for Arg leads to decreases of 99- and 343- fold in Vmax/Km for CaMKIV and CaMKIIalpha, respectively. For both kinases, the nature of the residues occupying the -4, -1, and + 4 positions exerts relatively little influence on phosphorylation kinetics. CaMKIV and CaMKIIalpha respond differently to substitutions at the -2 and +1 positions. Substitution of Arg at the -2 position with non-basic residues (Gln or Ala) leads to 6-fold decreases in Vmax/Km for CaMKIV, but 17-28-fold increases for CaMKIIalpha. Additionally, peptides containing Leu, Asp, or Ala at the +1 position are phosphorylated with similar efficiencies by CaMKIV, whereas the Leu-substituted peptide is preferred by CaMKIIalpha (by a factor of 5.8-9.7-fold). Thus, CaMKIV and CaMKIIalpha preferentially phosphorylate substrates with the motifs: Hyd-X-Arg-X-X-Ser*/Thr*, and Hyd-X-Arg-NB-X-Ser*/Thr*-Hyd, respectively, where Hyd represents a hydrophobic, X any, and NB a non-basic amino acid residue. The different specificities of the two kinases may contribute to their targeting to distinct physiological substrates during Ca2+-dependent cellular events.
Amino Acid Sequence↗
Protein kinase inhibitors: the tyrosine-specific protein kinases.
Inhibitors for tyrosine-specific protein kinases ultimately may constitute a novel family of medicinally active agents. Unfortunately, the challenges associated with the acquisition of inhibitors for these enzyme targets are unlike any that have ever been encountered in medicinal chemistry. Protein kinases pose a variety of obstacles in regard to inhibitor design, nearly all of which deal with, in one fashion or another, the issue of specificity. The protein kinase family is extraordinarily large, with estimates that the human genome codes for as many as 2000 protein kinases. Furthermore, inhibitors that are directed to the ATP-binding sites of these enzymes must contend with the presence of a large number of other ATP-utilizing proteins and, in addition, must compete with the high intracellular concentrations of ATP. Although specificity ultimately may prove to be less of a concern with peptide-based inhibitors, these agents neither are readily bioavailable nor do they bind with the requisite affinity to the protein-binding domains of protein kinases. In the face of these challenges, an enormous number of inhibitors have been synthesized and evaluated for the tyrosine-specific protein kinases. The advantages and disadvantages associated with inhibitors that are targeted to the ATP-binding site, the protein-binding site, and nonactive site regions required for appropriate subcellular localization are discussed. The handful of tyrosine-specific protein kinases that have been selected as targets to date and their roles in various disease processes are described as well.
Adenosine Triphosphate↗
Identification of a second aryl phosphate-binding site in protein-tyrosine phosphatase 1B: a paradigm for inhibitor design.
The structure of the catalytically inactive mutant (C215S) of the human protein-tyrosine phosphatase 1B (PTP1B) has been solved to high resolution in two complexes. In the first, crystals were grown in the presence of bis-(para-phosphophenyl) methane (BPPM), a synthetic high-affinity low-molecular weight nonpeptidic substrate (Km = 16 microM), and the structure was refined to an R-factor of 18. 2% at 1.9 A resolution. In the second, crystals were grown in a saturating concentration of phosphotyrosine (pTyr), and the structure was refined to an R-factor of 18.1% at 1.85 A. Difference Fourier maps showed that BPPM binds PTP1B in two mutually exclusive modes, one in which it occupies the canonical pTyr-binding site (the active site), and another in which a phosphophenyl moiety interacts with a set of residues not previously observed to bind aryl phosphates. The identification of a second pTyr molecule at the same site in the PTP1B/C215S-pTyr complex confirms that these residues constitute a low-affinity noncatalytic aryl phosphate-binding site. Identification of a second aryl phosphate binding site adjacent to the active site provides a paradigm for the design of tight-binding, highly specific PTP1B inhibitors that can span both the active site and the adjacent noncatalytic site. This design can be achieved by tethering together two small ligands that are individually targeted to the active site and the proximal noncatalytic site.
Base Sequence↗
Nonphosphorylatable tyrosine surrogates. Implications for protein kinase inhibitor design.
Tyrosine-specific protein kinases are known to utilize short synthetic tyrosine-containing peptides as substrates and, as a consequence, a number of inhibitory peptides have been prepared by replacing the tyrosine moiety in these peptides with a nonphosphorylatable phenylalanine residue. Unfortunately, the inhibitory efficacy of these phenylalanine-based peptides is often disappointing. These results demonstrate the need for nonphosphorylatable tyrosine surrogates that enhance enzyme affinity. As a consequence, we prepared nearly two dozen different phenethylamine derivatives, attached them to the C terminus of an active site-directed peptide (Glu-Glu-Leu-Leu), and examined their effectiveness as inhibitors of pp60(c-)src. Three derivatives exhibit enhanced inhibitory activity (relative to phenethylamine), including para-substituted sulfonamide and guanidino analogs as well as a pentafluoro-containing species. The para-sulfonamide derivative was selected for further study and was found to function as a competitive inhibitor versus variable peptide substrate and as a noncompetitive inhibitor versus variable ATP. In short, the enhanced inhibitory activity of the sulfonamide derivative is not due to the association of this moiety with the ATP binding site. Furthermore, peptides containing the para-guanidino and pentafluoro derivatives of phenylalanine were prepared. These species also display enhanced inhibitory activity toward pp60(c-)src relative to the corresponding phenylalanine-based peptide.
Amino Acid Sequence↗
Vanadium oxoanions and cAMP-dependent protein kinase: an anti-substrate inhibitor.
Vanadium oxoions have been shown to elicit a wide range of effects in biological systems, including an increase in the quantity of phosphorylated proteins. This response has been attributed to the inhibition of protein phosphatases, the indirect activation of protein kinases via stimulation of enzymes at early steps in signal transduction pathways and/or the direct activation of protein kinases. We have evaluated the latter possibility by exploring the effects of vanadate, decavanadate and vanadyl cation species on the activity of the cAMP-dependent protein kinase (PKA), a serine/threonine kinase. Vanadate, in the form of monomer, dimer, tetramer and pentamer species, neither inhibits nor activates PKA. In marked contrast, decavandate is a competitive inhibitor (Ki = 1.8 +/- 0.1 mM) of kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), a peptide-based substrate. This inhibition pattern is especially surprising, since the negatively charged decavanadate would not be predicted to bind to the region of the active site of the enzyme that accommodates the positively charged kemptide substrate. Our studies suggest that decavanadate can associate with kemptide in solution, which would prevent kemptide from interacting with the enzyme. Vanadium(IV) also inhibits the PKA-catalysed phosphorylation of kemptide, but with an IC50 of 366 +/- 10 microM. However, in this case V4+ appears to bind to the Mg(2+)-binding site, since it can substitute for Mg2+. In the absence of Mg2+, the optimal concentration of vanadium(IV) for the PKA-catalysed phosphorylation of kemptide is 100 microM, with concentrations above 100 microM being markedly inhibitory. However, even at the optimal 100 microM V4+ concentration, the Vmax and K(m) values (for kemptide) are significantly less favourable than those obtained in the presence of 100 microM Mg2+. In summary, we have found that oxovanadium ions can directly alter the activity of the serine/threonine-specific PKA.
Animals↗
Synergies and coincidence requirements between NO, cGMP, and Ca2+ in the induction of cerebellar long-term depression.
Parallel fiber synapses onto Purkinje neurons in acute cerebellar slices undergo long-term depression (LTD) when presynaptic activity coincides with postsynaptic depolarization. These electrical inputs can be respectively replaced by nitric oxide (NO) and Ca2+ photolytically released inside the Purkinje neuron, showing that these two messengers are sufficient for LTD induction. NO acts via cGMP production because inhibitors of guanylate cyclase prevent LTD but can be circumvented by photoreleased cGMP combined with Ca2+ elevation. Three inhibitors of cGMP-dependent protein kinase, Rp-8Br-PET-cGMPS, KT5823, and a novel pseudosubstrate peptide, all block LTD. LTD induction permits <10 ms gap between NO release and Ca2+ elevation, whereas 200-300 ms is allowed between uncaged cGMP and Ca2+ increase. This surprising difference in timing precision can be explained either by tighter localization and faster decay of cGMP when generated by NO rather than uncaging, or by two independent coincidence detectors in series.
Animals↗
VHR and PTP1 protein phosphatases exhibit remarkably different active site specificities toward low molecular weight nonpeptidic substrates.
The dual-specificity protein phosphatases have recently been shown to act as key regulators of mitogenic signaling pathways as well as of the cell cycle process. The are unusual catalysts in that they can utilize protein substrates containing phosphotyrosine as well as phosphoserine/threonine. The dual-specificity phosphatases and the protein tyrosine phosphatases (PTPase) share the active site motif (H/V)C(X)5R(S/T) but display little amino acid sequence identity outside of the active site. Although the dual-specificity phosphatases and the PTPases appear to bring about phosphate monoester hydrolysis through a similar mechanism, there is very limited information about the structural features that control the substrate specificity for the two groups of enzymes. As a first step in the development of selective dual-specificity phosphatase inhibitors, we have examined the active site substrate specificity of the human dual-specificity phosphatase, VHR [for VH1-Related; Ishibashi et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12170-12174]. Like the tyrosine-specific PTP1, VHR also preferentially catalyzes the hydrolysis of aromatic phosphates. However, we demonstrate herein that relatively modest changes in the substitution patterns on the phosphorylated aromatic nucleus generates dramatic, and differential, swings in substrate selectivity for VHR and PTP1. For example, VHR appears to be significantly more accommodating than PTP1 toward sterically-demanding substrates. Thus, the active site specificity of these two protein phosphatases is decidedly dissimilar. In addition, we have also identified several low molecular weight compounds that are more efficient substrates than the most potent peptidic substrates ever reported for VHR. Finally, we have shown that the Michaelis constants exhibited by these substrates are accurate assessments of enzyme affinity. Consequently, it should be possible to develop phospatase-selective inhibitors based upon the distinct substrate specificities of these enzymes.
Amino Acid Sequence↗
Potent low molecular weight substrates for protein-tyrosine phosphatase.
The ability of protein-tyrosine phosphatases (PTPases) to catalyze the hydrolysis of simple aromatic phosphates has been recognized for some time. However, these compounds are significantly poorer substrates than their peptide-based counterparts containing phosphotyrosine. Consequently, the effort to create potent PTPase substrates has predominantly focused on the use of peptidic carriers to deliver the phosphotyrosine moiety to the enzyme active site. We now report the synthesis and evaluation of several low molecular weight aromatic phosphates that serve as robust substrates for the rat PTPase, PTP1. We initially surveyed the ability of PTP1 to catalyze the hydrolysis of a variety of phenyl phosphate structural variants. Sterically demanding substituents positioned ortho and (to a lesser extent) meta to the phosphate group severely compromise the ability of these species to serve as phosphatase substrates. However, both benzylic and negatively charged substituents para to the hydrolyzable phosphate dramatically promote hydrolytic efficiency, which appears to be augmented through a dramatic enhancement in the affinity of the substrate for PTP1. The best substrate examined in this study exhibits a Km of 16 +/- 3 microM. In addition, it serves as an inhibitor of the PTP1-catalyzed hydrolysis of p-nitrophenyl phosphate with a Ki of 4.9 +/- 0.7 microM. The extraordinary structural simplicity of this compound, as well as those of several others described herein, provides a promising starting point for the design of potent PTPase inhibitors.
Animals↗
Rate-determining steps for tyrosine phosphorylation by the kinase domain of v-fps.
The rate-determining steps in the phosphorylation of four tyrosine-containing peptides by the kinase domain of the nonreceptor tyrosine protein kinase v-fps were measured using viscosometric methods. The peptides were phosphorylated by a fusion protein of glutathione-S-transferase and the kinase domain of v-fps (GST-kin) and the initial velocities were determined by a coupled enzyme assay. Peptides I (EEEIYEEIE), II (EAEIYEAIE), and III (DADIYDAID) were phosphorylated by GST-kin with similar kinetic constants. The viscosogens, glycerol and sucrose, were found to have intermediate effects on kcat and no effect on kcat/Kpeptide for the phosphorylation of these three peptides. The data are interpreted according to the Stokes-Einstein equation and a simple three-step mechanism involving substrate binding, phosphoryl group transfer, and net product release. Two competitive inhibitors (EAEIFEAIE and DADIFDAID) exhibited K1 values that are 6-10-fold higher than the Kpeptide values for their analogous peptide substrates. The data imply that peptides I-III are in rapid equilibrium with the enzyme and that kcat is partially limited by both phosphoryl group transfer (40-100 s-1) and product release (17-22 s-1). GST-kin phosphorylates peptide IV (R5AENLEYamide) with a low Km (100 microM) and a kcat that is 40-fold lower than that for peptide I. No effect of solvent viscosity was observed for the phosphorylation of this peptide on either kcat or kcat/Kpeptide. This suggests that highly viscous solutions do not perturb structure and that the rate-determining step for this poor substrate is phosphoryl group transfer. The data indicate that the kinase domain of v-fps phosphorylates its best substrate with a chemical rate constant that is at least 5-fold lower than that for the serine-specific cAMP-dependent protein kinase and its best substrate LRRASLG (Adams & Taylor, 1992). Interestingly, both enzymes exhibit a similar affinity for their substrates and both enzymes release their products at a similar rate. This implies that the differences in catalytic efficiency between serine- and tyrosine-specific protein kinases lie exclusively in the rate constants for phosphoryl group transfer and not in substrate absorption or product desorption.
Amino Acid Sequence↗
Precision targeting of protein kinases. An affinity label that inactivates the cGMP- but not the cAMP-dependent protein kinase.
Although the cAMP-dependent (PKA) and cGMP-dependent protein kinases (PKG) usually participate in unrelated biological processes, their enzymological properties are decidedly similar. Based upon the multitude of comparative studies conducted to date, it appears that these two enzymes exhibit very similar peptide substrate specificities. Furthermore, most inhibitors that have been reported for PKG serve in a nearly equal capacity for PKA. Consequently, the task of distinguishing between these enzymes, especially under in vivo conditions, has proved to be daunting. However, we have recently found that PKA will only phosphorylate non-amino acid residues whose alpha-configuration corresponds to that found in L-amino acids, whereas PKG will catalyze the phosphorylation of residues corresponding to both L- and D-amino acids (Wood, J., Mendelow, M., Yan, X., Corbin, J.D., Francis, S.H., and Lawrence, D.S. (1996) J. Biol. Chem. 271, 174-179). Based on these results, we have designed a potent affinity label for PKG (KI = 21.1 +/- 4.7 microM), that has no measurable activity toward PKA. This represents the first example of an peptide-based inactivator that fully distinguishes between these two closely related enzymes. These results suggest that a similar strategy may provide highly specific inactivators for other protein kinases as well.
Adenosine Triphosphate↗
The active site specificity of the Yersinia protein-tyrosine phosphatase.
Yersinia protein-tyrosine phosphatase substrates have been synthesized employing an expedient methodology that incorporates phosphorylated non-amino acid residues into an active site-directed peptide. While the peptidic portion of these compounds serves an enzyme targeting role, the nonpeptidic component provides a critical assessment of the range of functionality that can be accommodated within the active site region. We have found that the Yersinia phosphatase hydrolyzes both L- and D-stereoisomers of phosphotyrosine in active site-directed peptides, with the former serving as a 10-fold more efficient substrate than the latter. In addition, this enzyme catalyzes the hydrolysis of a variety of aromatic and aliphatic phosphates. Indeed, a peptide bearing the achiral phosphotyrosine analog, phosphotyramine, is not only the most efficient substrate described in this study, it is also one of the most efficient substrates ever reported for the Yersinia phosphatase. Straight chain peptide-bound aliphatic phosphates of the general structure, (Glu)4-NH-(CH2)n-OPO3(2-) (n = 2-8), are also hydrolyzed, where the most efficient substrate contains seven methylene groups. Finally, a comparison of the substrate efficacy of the peptide-bound species with that of the corresponding non-peptidic analogs, reveals that the peptide component enhances kcat/Km by up to nearly 3 orders of magnitude.
Binding Sites↗
Precision substrate targeting of protein kinases. The cGMP- and cAMP-dependent protein kinases.
The cAMP-dependent (PKA) and cGMP-dependent protein kinases (PKG) share a strong primary sequence homology within their respective active site regions. Not surprisingly, these enzymes also exhibit overlapping substrate specificities, a feature that often interferes with efforts to elucidate their distinct biological roles. In this report, we demonstrate that PKA and PKG exhibit dramatically different behavior with respect to the phosphorylation of alpha-substituted alcohols. Although PKA will phosphorylate only residues that contain an alpha-center configuration analogous to that found in L-serine, PKG utilizes residues that correspond to both L- and D-serine as substrates. The PKG/PKA selectivity of these substrates is the highest ever reported.
Amino Acid Sequence↗
Is protein kinase substrate efficacy a reliable barometer for successful inhibitor design?
We have addressed the question of whether protein kinase substrate efficacy is a reliable barometer for successful inhibitor design by assessing the dependence of kcat and kcat/Km for eight separate alcohol-bearing residues on solvent viscosity. We have found that the Km for three structurally distinct primary alcohol-containing peptides overestimates the affinity that these species exhibit for the cAMP-dependent protein kinase. In all three cases, the rate-determining step is product release, and substrate binding is best described as rapid equilibrium. In contrast, peptides containing the following phosphorylatable residues all provide Km values that are accurate assessments of substrate affinity for the protein kinase: a secondary alcohol, a simple phenol, and a primary alcohol with a relatively long side chain. In the latter three instances, the rate-determining step is phosphoryl transfer. Finally, two aromatic alcohol-containing residues that possess lipophilic side chains exhibit Michaelis constants that underestimate enzyme affinity. These results demonstrate that while it may be tempting to employ structural elements from the most efficient substrates (e.g. primary alcohols) for inhibitor design, less effective substrates may serve as a more accurate assessment of inhibitory success.
Alcohols↗
Photochemistry and photophysical properties of novel, unsymmetrically substituted metallophthalocyanines.
A series of novel, unsymmetrically substituted metallo-phthalocyanines was synthesized, along with their symmetrically substituted analogs, and the effects of structure and metal substitution on their photophysical and photoredox properties were investigated. The macrocycles were synthesized using a mixed-condensation method followed by chromatographic separation of the resulting soluble products. They possess a catechol "active site" and three tert-butyl groups for enhanced solubility. The ground- and excited-state photophysical properties of the free-base, Zn(II) and Pd(II) macrocycles were measured and compared with their symmetrically substituted (tetra[tert-butyl]) analogs. The efficiency with which these macrocycles sensitize the formation of singlet oxygen was determined and discussed in the context of the excited-state photophysical properties. Several examples of photoinduced electron transfer reactions with one- and two-electron acceptors are demonstrated and discussed. These soluble molecules can be tuned to optimize their photochemical and redox properties by varying the central metal, axial ligands and other substituents, thereby providing a series of molecules for the investigation of photodynamic therapy and photoinduced electron transfer mechanisms.
Indoles↗
Precision substrate targeting of protein kinases v-Abl and c-Src.
The active site substrate specificities of v-Abl and c-Src are compared and contrasted. Both enzymes catalyze the phosphorylation of a broad assortment of peptide-bound aliphatic and aromatic alcohols, such as achiral and simple straight chain residues. In addition, both protein kinases exhibit a "dual specificity" with respect to the ability to utilize D- and L-configurational isomers as substrates. However, c-Src and v-Abl are extremely inefficient as catalysts for certain structural arrangements, including secondary alcohols and primary alcohols containing large substituents in close proximity to the hydroxyl moiety. In addition to these similarities, these enzymes also display noteworthy differences in catalytic behavior. Whereas c-Src exhibits a modest preference for aromatic versus aliphatic alcohols, v-Abl does not. Most dramatic is the ability of c-Src to utilize short chain alcohols as substrates, an activity virtually absent from the catalytic repertoire of v-Abl. The implications of these observations are 2-fold. First, because both enzymes are able to accommodate a wide variety of structural variants within their respective active site regions, there exists a substantial degree of flexibility with respect to inhibitor design. Second, because these enzymes exhibit disparate active site specificities, it is possible that other tyrosine-specific protein kinases will display unique substrate specificities as well. Consequently, it may ultimately be possible to exploit these differences to generate inhibitors that precisely target specific protein kinases.
Amino Acid Sequence↗
The design of peptide-based substrates for the cdc2 protein kinase.
The substrate sequence specificity of the cdc2 protein kinase from Pisaster ochraceus has been evaluated. The peptide, Ac-Ser-Pro-Gly-Arg-Arg-Arg-Arg-Lys-amide, serves as an efficient cdc2 kinase substrate with a Km of 1.50 +/- 0.04 microM and a Vmax. of 12.00 +/- 0.18 mumol/min per mg. The amino acid sequence of this peptide is not based on any sequence in a known protein substrate of the cyclin-dependent kinase, but rather was designed from structural attributes that appear to be important in the majority of cdc2 substrates. The cyclin-dependent enzyme is remarkably indiscriminate in its ability to recognize and phosphorylate peptides that contain an assortment of structurally diverse residues at the P-2, P-1 and P+2 positions. However, peptides that contain a free N-terminal serine or lack an arginine at the P+4 position are relatively poor substrates. These aspects of the substrate specificity of the cdc2 protein kinase are compared and contrasted with the previously reported substrate specificity of a cdc2-like protein kinase from bovine brain [Beaudette, Lew and Wang (1993) J. Biol. Chem. 268, 20825-20830].
Amino Acid Sequence↗