Comparative kinetics of cofactor association and dissociation for the human and trypanosomal S-adenosylhomocysteine hydrolases. 3. Role of lysyl and tyrosyl residues of the C-terminal extension.

On the basis of the available X-ray structures of S-adenosylhomocysteine hydrolases (SAHHs), free energy simulations employing the MM-GBSA approach were applied to predict residues important to the differential cofactor binding properties of human and trypanosomal SAHHs (Hs-SAHH and Tc-SAHH), within 5 Šof the cofactor NAD(+)/NADH binding site. Among the 38 residues in this region, only four are different between the two enzymes. Surprisingly, the four nonidentical residues make no major contribution to differential cofactor binding between Hs-SAHH and Tc-SAHH. On the other hand, four pairs of identical residues are shown by free energy simulations to differentiate cofactor binding between Hs-SAHH and Tc-SAHH. Experimental mutagenesis was performed to test these predictions for a lysine residue and a tyrosine residue of the C-terminal extension that penetrates a partner subunit to form part of the cofactor binding site. The K431A mutant of Tc-SAHH (TcK431A) loses its cofactor binding affinity but retains the wild type's tetrameric structure, while the corresponding mutant of Hs-SAHH (HsK426A) loses both cofactor affinity and tetrameric structure [Ault-Riche, D. B., et al. (1994) J. Biol. Chem. 269, 31472-31478]. The tyrosine mutants HsY430A and TcY435A alter the NAD(+) association and dissociation kinetics, with HsY430A increasing the cofactor equilibrium dissociation constant from approximately 10 nM (Hs-SAHH) to ∼800 nM and TcY435A increasing the cofactor equilibrium dissociation constant from approximately 100 nM (Tc-SAHH) to ∼1 mM. Both changes result from larger increases in the off rate combined with smaller decreases in the on rate. These investigations demonstrate that computational free energy decomposition may be used to guide experimental studies by suggesting sensitive sites for mutagenesis. Our finding that identical residues in two orthologous proteins may give significantly different binding free energy contributions strongly suggests that comparative studies of homologous proteins should investigate not only different residues but also identical residues in these proteins.

Molecular dynamics simulations of domain motions of substrate-free S-adenosyl- L-homocysteine hydrolase in solution.

S-Adenosyl-L-homocysteine hydrolase (SAHH) is an enzyme regulating intracellular methylation reactions. The homotetrameric SAHH exists in an open conformation in absence of substrate, while enzyme:inhibitor complexes crystallize in the closed conformation, in which the ligands are engulfed by the protein due to an 18 degrees domain reorientation within each of the four subunits. We present a microscopic description of the structure and dynamics of the substrate-free, NAD(+)-bound SAHH in solution, based on a 15-ns molecular dynamics simulation in explicit solvent. In the trajectory, the four cofactor-binding domains formed a relatively rigid core with structure very similar to the crystal conformation. The four substrate-binding domains, located at the protein exterior, also retained internal structures similar to the crystal, while undergoing large amplitude rigid-body reorientations. The trajectory domain motions exhibited two interesting properties. First, within each subunit the domains fluctuated between open and closed conformations, while at the tetramer level 80% of the domain motions were perpendicular to the direction of the open-to-closed structural transition. Second, the domain reorientations in solution could be represented as a sum of two components, faster, with 20-50 ps correlation time and 3-4 degrees amplitude, and slower, with 8-23 ns correlation time and amplitude of 14-22 degrees . The faster motion is similar to the 1.5 cm(-1) frequency hinge-bending vibrations found in our recent normal mode analysis (Wang et al., Biochemistry 2005;44:7228-7239). The slower motion agrees with fluorescence anisotropy decay measurements, which detected a 10-20 ns domain reorientation of ca. 26 degrees amplitude in the substrate-free enzyme (Wang et al., Biochemistry 2006;45:7778-7786). Our simulations are thus in excellent agreement with experimental data. The simulations allow us to assign the observed nanosecond fluorescence anisotropy signal to fluctuations in domain orientations, and indicate that the microscopic mechanism of the motion involves rotational diffusion within a cone of 10-20 degrees . Overall, our simulation results complement the existing experimental data and provide important new insights into SAHH domain motions in solution, which play a crucial role in the catalytic mechanism of SAHH.

Synthesis of 5'-functionalized nucleosides: S-Adenosylhomocysteine analogues with the carbon-5' and sulfur atoms replaced by a vinyl or halovinyl unit.

Adenosine and uridine analogues functionalized with alkenyl or fluoroalkenyl chain at C5' were prepared employing cross-metathesis, Negishi couplings, and Wittig reactions. Metathesis of the protected 5'-deoxy-5'-methyleneadenosine or uridine analogues with six-carbon amino acids (homoallylglycines) in the presence of Grubbs catalysts gave nucleoside analogues with the C5'-C6' double bond. Alternatively, the Pd-catalyzed cross-coupling between the protected 5'-deoxy-5'-(iodomethylene) nucleosides and suitable alkylzinc bromides also provided analogues with alkenyl unit. Stereoselective Pd-catalyzed monoalkylation of 5'-(bromofluoromethylene)-5'-deoxyadenosine with alkylzinc bromides afforded adenosylhomocysteine analogues with a 6'-(fluoro)vinyl motif. The vinylic adenine nucleosides produced time-dependent inactivation of the S-adenosyl-l-homocysteine hydrolases.

The effect of cosolutes on the isomerization of aspartic acid residues and conformational stability in a monoclonal antibody.

The aspartate residue (Asp 32) located in the complementarity-determining region (CDR) of a recombinant humanized monoclonal antibody (MAb I) is highly susceptible to the isomerization reaction. The modification of Asp 32 residue due to the isomerization reaction results in a significant reduction in the binding affinity of MAb I to IgE. The binding of a MAb I therapeutic to IgE is important for its desired pharmacological effect. In earlier investigations, we demonstrated that the conformational flexibility and residue exposure are factors that are responsible for the observed reactivity of Asp 32 in MAb I. This report explores the role of cosolutes such as glycerol and sucrose in the modulation of Asp 32 reactivity in MAb I. These cosolutes are routinely incorporated in injectable pharmaceutical formulations. The reactivity of the Asp residue in MAb I in these different cosolute-based formulations was compared to its reactivity in a peptide model VDYDG comprising residues 29-33 of MAb I. The formulations of MAb I and VDYDG containing varying concentrations of glycerol and sucrose were incubated at 50 degrees C for a period of 5-7 days. The isomerization of the Asp residue in VDYDG and MAb I was monitored using rp-HPLC and hydrophobic interaction chromatography (HIC), respectively. Structural analysis of MAb I using differential scanning calorimetry (DSC) demonstrated that the structural stability of MAb I was increased in formulations containing glycerol and sucrose. However, the stability of Asp 32 in MAb I was significantly decreased in these formulations. This research suggests that a formulation approach that relies purely on enhancing the structural stability of proteins through addition of these cosolutes could result in problems associated with the chemical stability of these biomolecules.

Effects of amino acid chirality and the chemical linker on the cell permeation characteristics of cyclic prodrugs of opioid peptides.

Previously, our laboratory showed that the oxymethyl-modified coumarinic acid (OMCA) cyclic prodrug of the opioid peptide DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH) exhibited low permeation across both the intestinal mucosa and the blood-brain barrier (BBB). This low cell permeation arose from its strong substrate activity for efflux transporters in these biological barriers. In an attempt to determine whether the chirality of the amino acid asymmetric centers could influence the solution structure of the cyclic prodrugs and thus their substrate activities for efflux transporters, we synthesized cyclic prodrugs of the opioid peptides H-Tyr-Ala-Gly-Phe-D-Leu-OH ([Ala2,D-Leu5]-Enk), H-Tyr-D-Ala-Gly-Phe-Leu-OH ([D-Ala2,Leu5]-Enk), and H-Tyr-Ala-Gly-Phe-Leu-OH ([Ala2,Leu5]-Enk). In an attempt to determine whether the chemical linker (OMCA) bestowed efflux substrate activity on the cyclic prodrugs, we synthesized capped linear derivatives (acetylated on the N-terminal and amidated on the C-terminal end) of [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk. The solution conformations of the cyclic prodrugs were determined by molecular dynamics simulations using two-dimensional NMR data. The physicochemical properties (molecular surface area, polar surface area, and cLogP) were estimated computationally using Sybyl. Cell permeation characteristics were assessed using Caco-2 cells in the presence and absence of known inhibitors of efflux transporters. Despite apparent differences in their solution conformations and their physicochemical properties, the cyclic prodrugs of DADLE, [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk all exhibited strong substrate activity for efflux transporters in Caco-2 cells. In contrast, the capped linear derivatives of [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk exhibited very poor substrate activity for efflux transporters in Caco-2 cells. Therefore, the substrate activities of the cyclic prodrugs for efflux transporters in Caco-2 cells and in the intestinal mucosa and the BBB in vivo are most likely due to the chemical linker used to prepare these molecules and/or its effect on solution structures of the prodrugs.

Enzymes involved in the bioconversion of ester-based prodrugs.

Enzymes are essential for the activation of many prodrugs. In this review, the most important enzymes (e.g., paraoxonase, carboxylesterase, acetylcholinesterase, cholinesterase) involved in the bioconversion of ester-based prodrugs will be discussed in terms of their biology and biochemistry. Most of these enzymes fall into the category of hydrolytic enzymes. However, nonhydrolytic enzymes, including cytochrome P450s, can also catalyze the bioconversion of ester prodrugs and thus will be discussed here. Other factors influencing the ability of these enzymes to catalyze the bioconversion of ester-based prodrugs, particularly species and interindividual differences and stereochemical and structural features of the prodrugs, will be discussed.

Formulation considerations for proteins susceptible to asparagine deamidation and aspartate isomerization.

The asparagine (Asn) deamidation and aspartate (Asp) isomerization reactions are nonenzymatic intra-molecular reactions occurring in peptides and proteins that are a source of major stability concern in the formulation of these biomolecules. The mechanisms for the deamidation and isomerization reactions are similar since they both proceed through an intra-molecular cyclic imide (Asu) intermediate. The formation of the Asu intermediate, which involves the attack by nitrogen of the peptide backbone on the carbonyl carbon of the Asn or the Asp side chain, is the rate-limiting step in both the deamidation and the isomerization reactions at physiological pH. In this article, the influence of factors such as formulation conditions, protein primary sequence, and protein structure on the reactivity of Asn and Asp residues in proteins are reviewed. The importance of formulation conditions such as pH and solvent dielectric in influencing deamidation and isomerization reaction rates is addressed. Formulation strategies that could improve the stability of proteins to deamidation and isomerization reactions are described. The review is intended to provide information to formulation scientists, based on protein sequence and structure, to predict potential degradative sites on a protein molecule and to enable formulation scientists to set appropriate formulation conditions to minimize reactivity of Asn and Asp residues in protein therapeutics.

Are L-adenosine and its derivatives substrates for S-adenosyl-L-homocysteine hydrolase?

Moffatt oxidation of 2',3'-O-isopropylidene-L-adenosine and treatment of the resulting crude 5'-aldehyde with hydroxylamine followed by deprotection gave L-adenosine 5'-carboxaldehyde oximes, whose enantiomers are known to be potent inhibitors of S-adenosyl-L-homocysteine (AdoHcy) hydrolase. The L-adenosine and its 5'-aldehyde oxime derivatives were found to be inactive as inhibitors of AdoHcy hydrolase. Docking calculations showed that binding of L-adenosine to AdoHcy hydrolase is weaker (higher energy) and less specific (larger number of clusters) compared to D-Ado.

Stability of oxymethyl-modified coumarinic acid cyclic prodrugs of diastereomeric opioid peptides in biological media from various animal species including human.

In vitro stability studies of oxymethyl-modified coumarinic acid (OMCA) cyclic prodrugs of the diastereomeric opioid peptides DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), [Ala2,D-Leu5]-Enk (H-Tyr-Ala-Gly-Phe-D-Leu-OH), [D-Ala2,Leu5]-Enk (H-Tyr-D-Ala-Gly-Phe-Leu-OH), and [Ala2,Leu5]-Enk (H-Tyr-Ala-Gly-Phe-Leu-OH) were conducted to evaluate how the chirality of specific amino acid residues (Ala2 and Leu5) in the peptide portion affects their bioconversion by esterases. The stability studies were conducted at 37 degrees C in plasma and tissue homogenates (liver and brain) from five animal species (rat, mouse, canine, guinea pig, and hamster) and human in an attempt to identify an animal species that had a "prodrug bioconversion profile" comparable to that of humans. Initially, the total esterase activity in these biological media was measured using p-nitrophenyl butyrate (PNPB) as a substrate. By repeating this activity assay in the presence of paraoxon, a potent esterase B inhibitor, it was possible to estimate the relative amounts of esterases B and esterases A/C in a biological sample. Stability studies of the cyclic prodrugs were carried out under identical conditions, that is, in the presence and absence of paraoxon. Significant differences in the rates of hydrolysis of the cyclic prodrugs were observed, particularly between cyclic prodrugs with differences in the chirality of the amino acid on the C-terminus of the peptide portion, for example, L-amino acids at the C-terminus hydrolyzed more rapidly than D-amino acids. This stereoselective hydrolysis was independent of the animal species but tended to be more pronounced in brain and liver homogenates compared to plasma. Increased esterase specific activity, as measured by PNPB, in the biological media did not necessarily correlate with increased bioconversion rates of the cyclic prodrugs. The enzymatic stability profiles of the cyclic prodrugs in biological media from canine and guinea pig most closely resembled the profiles from human biological media. Therefore, canine and guinea pig appear to be the most relevant animal models for conducting pharmacokinetic studies on these cyclic prodrugs of opioid peptides.

Significant differences in the disposition of cyclic prodrugs of opioid peptides in rats and guinea pigs following IV administration.

The stabilities of DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), the capped derivative Ac-DADLE-NH2, and the oxymethyl-coumarinic acid (OMCA)-based cyclic prodrug of DADLE and [D-Ala2,Leu5]-Enk (H-Tyr-D-Ala-Gly-Phe-Leu-OH) were determined at 37 degrees C in rat and guinea pig liver microsomes in the presence and absence of paraoxon, an esterase B inhibitor, and ketoconazole, a CYP3A4 inhibitor. These studies showed that the order of stability in microsomes was: DADLE >> Ac-DADLE-NH2 > OMCA-DADLE = OMCA-[D-Ala2,Leu5]-Enk. While paraoxon produced no significant effect on the stability of the studied compounds in liver microsomes, ketoconazole inhibited the metabolism, suggesting that the capped peptide and the cyclic prodrugs are substrates for cytochrome P450 enzymes. For pharmacokinetic studies, the cyclic prodrugs of DADLE and [D-Ala2,Leu5]-Enk were administered i.v. to rats and guinea pigs. Various biological fluids and tissue (brain, bile, and blood) were collected and analyzed for the free peptide and the prodrugs by high performance liquid chromatography with tandem mass spectrometric detection (LC-MS-MS). These studies showed that the conversion of the cyclic prodrugs to the respective linear peptides (i.e., DADLE and [D-Ala2,Leu5]-Enk) was rapid in rat and guinea pig. In terms of drug elimination, only trace amounts of OMCA-DADLE and OMCA-[D-Ala2,Leu5]-Enk were recovered in guinea pig bile (3.3% and 0.82%, respectively), while significant amounts were recovered in rat bile (38.1% and 51.7%, respectively). Brain uptake of the cyclic prodrugs in guinea pigs compared to previously determined brain uptake of OMCA-DADLE in rats was also significantly different. For OMCA-DADLE, the brain levels of the cyclic prodrug and DADLE in guinea pigs were approximately 80 and 8.5 times greater, respectively, than the levels observed in rat brain. The brain-to-plasma prodrug concentration ratios in guinea pigs (>or= 0.6) were significantly higher than the ratio observed in rats (0.01). These species differences are most likely due to the different substrate specificities of the efflux transporters that facilitate liver clearance of these prodrugs and limit their permeation into the brain.

Effects of sucrose and mannitol on asparagine deamidation rates of model peptides in solution and in the solid state.

Asparagine (Asn) degradation kinetics in two model peptides, Gly-Gln-Asn-Gly-Gly (GQNGG) and Val-Tyr-Pro-Asn-Gly-Ala (VYPNGA), were studied at 50 degrees C in pH 7 buffer solutions in the presence and absence of 5% (w/v) sucrose or mannitol and at 50 degrees C and 30% relative humidity in solid samples lyophilized from these solutions. Solid formulations were characterized using Karl Fischer coulometric titration, thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), Fourier-transform infrared spectrometry (FTIR), and solid-state nuclear magnetic resonance (NMR) spectroscopy. GQNGG and VYPNGA showed similar pseudo first-order deamidation rates in solution in the absence of sucrose and mannitol. Adding 5% sucrose or mannitol decreased the rates by no more than 17%. The model peptides degraded 2- to 80-fold more slowly in the solid formulations of sucrose and mannitol than in 5% solutions of these carbohydrates. Ratios of deamidation rates of the model peptides depended upon the solid matrix. In the mannitol solid, the ratio of deamidation rates of GQNGG and VYPNGA (GQNGG:VYPNGA) was approximately 8, while in the sucrose solid, the model peptides deamidated at similar rates (GQNGG:VYPNGA congruent with 1). DSC showed the mannitol formulations to be largely amorphous immediately after lyophilization with some ordered, crystalline-like structure; the extent of ordered structure increased during storage as shown by FTIR and ssNMR. In contrast, the sucrose formulation was largely amorphous after lyophilization and remained so during storage. Together, the results showed that 5% sucrose or mannitol in solution does not significantly change the rates of Asn deamidation of the model peptides, while sucrose stabilizes the model peptides against deamidation more than mannitol in the solid state.

Effects of acidic N + 1 residues on asparagine deamidation rates in solution and in the solid state.

The deamidation kinetics of four model peptides (AcGQNGG, AcGQNDG, AcGQNEG, and AcGQNQG) were studied in solution (70 degrees C, pH 5-10) and in lyophilized solids [70 degrees C, 50% relative humidity, "effective pH" ('pH') 5-10] containing polyvinyl pyrrolidone. AcGQNGG, AcGQNEG, and AcGQNQG degraded exclusively through Asn deamidation, whereas AcGQNDG also displayed Asp isomerization, and Asp-Gly peptide bond cleavage. The pH/'pH'-rate profiles were consistent with a shift in the rate-determining step of Asn deamidation from carbonyl addition to expulsion of ammonia with increasing pH. In solution, AcGQNGG deamidated up to 38-fold faster than the other peptides, indicating the importance of steric effects of the N + 1 residue. AcGQNGG and AcGQNQG had up to 60 times slower rates of deamidation in the solid state than in solution. In contrast, the deamidation rates of AcGQNEG and AcGQNDG in the solid state were similar to those in solution. N + 1 Glu or Asp residue may enhance local hydration, so that the deamidation of Asn in the solid formulations actually proceeds in a solution-like environment.

Deamidation of model beta-turn cyclic peptides in the solid state.

To investigate the importance of secondary structure on peptide deamidation in the solid state, two cyclic beta-turn peptides and their linear analogs were used as models of Asn residues in structured and unstructured domains, and incorporated into poly(vinyl pyrrolidone) (PVP)-based lyophilized solids. The secondary structure of the model peptides was determined in solution and the solid state using a combination of nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD), and Fourier transform infrared (FTIR) spectroscopy. The model beta-turn cyclic peptides were found to be type II beta-turns while the linear analogs were determined to be predominantly unstructured. Quantitatively, the cyclic peptides consisted of approximately 80% beta-turn while the linear analogs contained only 30%-35% beta-turn. To characterize the solid environment, T(g), and moisture content of the solid-state formulations were determined. Accelerated stability studies were conducted in the solid state at 37 degrees C using formulations lyophilized from solutions at pH 8.8 (0.1 M borate buffer). The effect of matrix mobility on solid-state deamidation was investigated by altering the moisture content through variation of relative humidity or the addition of a plasticizer. Cyclic peptides degraded 1.2-8 times slower than the linear analogs under all of the conditions studied. The observed rate constants, however, for all of the peptides decreased dramatically (four orders of magnitude) in the glassy solids. This suggests the greater importance of matrix mobility in solid-state degradation. Molecular dynamics (MD) simulations were also performed to explore the low energy, preferred state of the peptides, and determine the structure around the beta-turn.

Phe-Gly dipeptidomimetics designed for the di-/tripeptide transporters PEPT1 and PEPT2: synthesis and biological investigations.

A series of five Phe-Gly dipeptidomimetics containing different amide bond replacements have been synthesized in a facile way from the readily available unsaturated ketoester 1, and their affinities for the di-/tripeptide transporters hPEPT1 (Caco-2 cells) and rPEPT2 (SKPT cells) were tested. The compounds contained the amide bond isosteres ketomethylene (2a), (R)- and (S)-hydroxyethylidene (3a and 4a), and (R)- and (S)-hydroxyethylene (5a and 6a) to provide information on the conformational and stereochemical requirements for hPEPT1 and rPEPT2 affinity. The affinity studies showed that for rPEPT2 there is no significant difference in affinity between the ketomethylene isostere 2a and the natural substrate Phe-Gly (K(i) values of 18.8 and 14.6 microM, respectively). Also the affinities for hPEPT1 are in the same range (K(i) values of 0.40 and 0.20 mM, respectively). This corroborates earlier findings that the amide bond as such is not essential for binding to PEPTX, but the results also reveal possible differences in the binding of ketomethylene isosteres to hPEPT1 and rPEPT2. The trans-hydroxyethylidene and hydroxyethylene isosteres proved to be poor substrates for PEPTX. These results provide new information about the importance of flexibility and of the stereochemistry at the C(4)-position for this class of compounds. Furthermore, the intracellular uptake of 2a-4a in Caco-2 cells was investigated, showing a 3-fold reduction of the uptake of 2a in the presence of the competetive inhibitor Gly-Pro, indicating contribution from an active transport component. No active uptake of 3a and 4a was observed. Transepithelial transport studies also indicated active transport of 2a across Caco-2 monolayers.