Georges Chreifi

Simplified 2-Aminoquinoline-Based Scaffold for Potent and Selective Neuronal Nitric Oxide Synthase Inhibition.

Since high levels of nitric oxide (NO) are implicated in neurodegenerative disorders, inhibition of the neuronal isoform of nitric oxide synthase (nNOS) and reduction of NO levels are therapeutically desirable. Nonetheless, many nNOS inhibitors mimic l-arginine and are poorly bioavailable. 2-Aminoquinoline-based scaffolds were designed with the hope that they could (a) mimic aminopyridines as potent, isoform-selective arginine isosteres and (b) possess chemical properties more conducive to oral bioavailability and CNS penetration. A series of these compounds was synthesized and assayed against purified nNOS enzymes, endothelial NOS (eNOS), and inducible NOS (iNOS). Several compounds built on a 7-substituted 2-aminoquinoline core are potent and isoform-selective; X-ray crystallography indicates that aminoquinolines exert inhibitory effects by mimicking substrate interactions with the conserved active site glutamate residue. The most potent and selective compounds, 7 and 15, were tested in a Caco-2 assay and showed good permeability and low efflux, suggesting high potential for oral bioavailability.

Potent and Selective Double-Headed Thiophene-2-carboximidamide Inhibitors of Neuronal Nitric Oxide Synthase for the Treatment of Melanoma

Selective inhibitors of neuronal nitric oxide synthase (nNOS) are regarded as valuable and powerful agents with therapeutic potential for the treatment of chronic neurodegenerative pathologies and human melanoma. Here, we describe a novel hybrid strategy that combines the pharmacokinetically promising thiophene-2-carboximidamide fragment and structural features of our previously reported potent and selective aminopyridine inhibitors. Two inhibitors, 13 and 14, show low nanomolar inhibitory potency (Ki = 5 nM for nNOS) and good isoform selectivities (nNOS over eNOS [440- and 540-fold, respectively] and over iNOS [260- and 340-fold, respectively]). The crystal structures of these nNOS–inhibitor complexes reveal a new hot spot that explains the selectivity of 14 and why converting the secondary to tertiary amine leads to enhanced selectivity. More importantly, these compounds are the first highly potent and selective nNOS inhibitory agents that exhibit excellent in vitro efficacy in melanoma cell lines.

Crystal structure of the pristine peroxidase ferryl center and its relevance to proton-coupled electron transfer

Significance A major problem in determining the crystal structures of metalloenzymes is that the reducing power of X-rays often changes the oxidation state of the metal center, thereby complicating important mechanistic conclusions on enzyme function. This reduction is especially problematic in studying Fe(IV)=O intermediates, which are powerful oxidants used by many metalloenzymes. This problem can be circumvented using the Stanford Linear Coherent Light Source (LCLS), which generates intense X-ray pulses on the femtosecond time scale and enables structure determinations with no reduction of metal centers. Here, we report the crystal structure of the Fe(IV)=O peroxidase intermediate called compound I using data obtained from the LCLS. We also present kinetic and computational results that, together with crystal structures, provide important mechanistic insights. The reaction of peroxides with peroxidases oxidizes the heme iron from Fe(III) to Fe(IV)=O and a porphyrin or aromatic side chain to a cationic radical. X-ray–generated hydrated electrons rapidly reduce Fe(IV), thereby requiring very short exposures using many crystals, and, even then, some reduction cannot be avoided. The new generation of X-ray free electron lasers capable of generating intense X-rays on the tenths of femtosecond time scale enables structure determination with no reduction or X-ray damage. Here, we report the 1.5-Å crystal structure of cytochrome c peroxidase (CCP) compound I (CmpI) using data obtained with the Stanford Linear Coherent Light Source (LCLS). This structure is consistent with previous structures. Of particular importance is the active site water structure that can mediate the proton transfer reactions required for both CmpI formation and reduction of Fe(IV)=O to Fe(III)-OH. The structures indicate that a water molecule is ideally positioned to shuttle protons between an iron-linked oxygen and the active site catalytic His. We therefore have carried out both computational and kinetic studies to probe the reduction of Fe(IV)=O. Kinetic solvent isotope experiments show that the transfer of a single proton is critical in the peroxidase rate-limiting step, which is very likely the proton-coupled reduction of Fe(IV)=O to Fe(III)-OH. We also find that the pKa of the catalytic His substantially increases in CmpI, indicating that this active site His is the source of the proton required in the reduction of Fe(IV)=O to Fe(IV)-OH.

Novel 2,4-Disubstituted Pyrimidines as Potent, Selective, and Cell-Permeable Inhibitors of Neuronal Nitric Oxide Synthase.

Selective inhibition of neuronal nitric oxide synthase (nNOS) is an important therapeutic approach to target neurodegenerative disorders. However, the majority of the nNOS inhibitors developed are arginine mimetics and, therefore, suffer from poor bioavailability. We designed a novel strategy to combine a more pharmacokinetically favorable 2-imidazolylpyrimidine head with promising structural components from previous inhibitors. In conjunction with extensive structure–activity studies, several highly potent and selective inhibitors of nNOS were discovered. X-ray crystallographic analysis reveals that these type II inhibitors utilize the same hydrophobic pocket to gain strong inhibitory potency (13), as well as high isoform selectivity. Interestingly, select compounds from this series (9) showed good permeability and low efflux in a Caco-2 assay, suggesting potential oral bioavailability, and exhibited minimal off-target binding to 50 central nervous system receptors. Furthermore, even with heme-coordinating groups in the molecule, modifying other pharmacophoric fragments minimized undesirable inhibition of cytochrome P450s from human liver microsomes.

Structures of human constitutive nitric oxide synthases

Mammals produce three isoforms of nitric oxide synthase (NOS): neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). The overproduction of NO by nNOS is associated with a number of neurodegenerative disorders; therefore, a desirable therapeutic goal is the design of drugs that target nNOS but not the other isoforms. Crystallography, coupled with computational approaches and medicinal chemistry, has played a critical role in developing highly selective nNOS inhibitors that exhibit exceptional neuroprotective properties. For historic reasons, crystallography has focused on rat nNOS and bovine eNOS because these were available in high quality; thus, their structures have been used in structure-activity-relationship studies. Although these constitutive NOSs share more than 90% sequence identity across mammalian species for each NOS isoform, inhibitor-binding studies revealed that subtle differences near the heme active site in the same NOS isoform across species still impact enzyme-inhibitor interactions. Therefore, structures of the human constitutive NOSs are indispensible. Here, the first structure of human neuronal NOS at 2.03 Å resolution is reported and a different crystal form of human endothelial NOS is reported at 1.73 Å resolution.

Nitric oxide synthase inhibitors that interact with both heme propionate and tetrahydrobiopterin show high isoform selectivity

Overproduction of NO by nNOS is implicated in the pathogenesis of diverse neuronal disorders. Since NO signaling is involved in diverse physiological functions, selective inhibition of nNOS over other isoforms is essential to minimize side effects. A series of α-amino functionalized aminopyridine derivatives (3–8) were designed to probe the structure–activity relationship between ligand, heme propionate, and H4B. Compound 8R was identified as the most potent and selective molecule of this study, exhibiting a Ki of 24 nM for nNOS, with 273-fold and 2822-fold selectivity against iNOS and eNOS, respectively. Although crystal structures of 8R complexed with nNOS and eNOS revealed a similar binding mode, the selectivity stems from the distinct electrostatic environments in two isoforms that result in much lower inhibitor binding free energy in nNOS than in eNOS. These findings provide a basis for further development of simple, but even more selective and potent, nNOS inhibitors.

Chiral linkers to improve selectivity of double-headed neuronal nitric oxide synthase inhibitors.

To develop potent and selective nNOS inhibitors, new double-headed molecules with chiral linkers that derive from natural amino acids or their derivatives have been designed. The new structures contain two ether bonds, which greatly simplifies the synthesis and accelerates structure optimization. Inhibitor (R)-6b exhibits a potency of 32nM against nNOS and is 475 and 244 more selective for nNOS over eNOS and iNOS, respectively. Crystal structures show that the additional binding between the aminomethyl moiety of 6b and the two heme propionates in nNOS, but not eNOS, is the structural basis for its high selectivity. This work demonstrates the importance of stereochemistry in this class of molecules, which significantly influences the potency and selectivity of the inhibitors. The structure-activity information gathered here provides a guide for future structure optimization.

Communication between the zinc and tetrahydrobiopterin binding sites in nitric oxide synthase.

The nitric oxide synthase (NOS) dimer is stabilized by a Zn2+ ion coordinated to four symmetry-related Cys residues exactly along the dimer 2-fold axis. Each of the two essential tetrahydrobiopterin (H4B) molecules in the dimer interacts directly with the heme, and each H4B molecule is ∼15 Å from the Zn2+. We have determined the crystal structures of the bovine endothelial NOS dimer oxygenase domain bound to three different pterin analogues, which reveal an intimate structural communication between the H4B and Zn2+ sites. The binding of one of these compounds, 6-acetyl-2-amino-7,7-dimethyl-7,8-dihydro-4(3H)-pteridinone (1), to the pterin site and Zn2+ binding are mutually exclusive. Compound 1 both directly and indirectly disrupts hydrogen bonding between key residues in the Zn2+ binding motif, resulting in destabilization of the dimer and a complete disruption of the Zn2+ site. Addition of excess Zn2+ stabilizes the Zn2+ site at the expense of weakened binding of 1. The unique structural features of 1 that disrupt the dimer interface are extra methyl groups that extend into the dimer interface and force a slight opening of the dimer, thus resulting in disruption of the Zn2+ site. These results illustrate a very delicate balance of forces and structure at the dimer interface that must be maintained to properly form the Zn2+, pterin, and substrate binding sites.

Nitrile in the Hole: Discovery of a Small Auxiliary Pocket in Neuronal Nitric Oxide Synthase Leading to the Development of Potent and Selective 2-Aminoquinoline Inhibitors.

Neuronal nitric oxide synthase (nNOS) inhibition is a promising strategy to treat neurodegenerative disorders, but the development of nNOS inhibitors is often hindered by poor pharmacokinetics. We previously developed a class of membrane-permeable 2-aminoquinoline inhibitors and later rearranged the scaffold to decrease off-target binding. However, the resulting compounds had decreased permeability, low human nNOS activity, and low selectivity versus human eNOS. In this study, 5-substituted phenyl ether-linked aminoquinolines and derivatives were synthesized and assayed against purified NOS isoforms. 5-Cyano compounds are especially potent and selective rat and human nNOS inhibitors. Activity and selectivity are mediated by the binding of the cyano group to a new auxiliary pocket in nNOS. Potency was enhanced by methylation of the quinoline and by introduction of simple chiral moieties, resulting in a combination of hydrophobic and auxiliary pocket effects that yielded high (∼500-fold) n/e selectivity. Importantly, the Caco-2 assay also revealed improved membrane permeability over previous compounds.

Enzymatic Mechanism of Leishmania Major Peroxidase and the Critical Role of Specific Ionic Interactions.

Leishmania major peroxidase (LmP) is very similar to the well-known yeast cytochrome c peroxidase (CcP). Both enzymes catalyze the peroxidation of cytochrome c. Like CcP, LmP reacts with H2O2 to form Compound I, which consists of a ferryl heme and a Trp radical, Fe(IV)═O;Trp(•+). Cytochrome c (Cytc) reduces the Trp radical to give Compound II, Fe(IV)═O;Trp, which is followed by an intramolecular electron transfer to give Fe(III)-OH;Trp(•+), and in the last step, Cytc reduces the Trp radical. In this study, we have used steady-state and single-turnover kinetics to improve our understanding of the overall mechanism of LmP catalysis. While the activity of CcP greatly increases with ionic strength, the kcat for LmP remains relatively constant at all ionic strengths tested. Therefore, unlike CcP, where dissociation of oxidized Cytc is limiting at low ionic strengths, association/dissociation reactions are not limiting at any ionic strength in LmP. We conclude that in LmP, the intramolecular electron transfer reaction, Fe(IV)═O;Trp to Fe(III)-OH;Trp(•+), is limiting at all ionic strengths. Unlike CcP, LmP depends on key intermolecular ion pairs to form the electron transfer competent complex. Mutating these sites causes the initial rate of association to decrease by 2 orders of magnitude and a substantial decrease in kcat. The drop in kcat is due to a switch in the rate-limiting step of the mutants from intramolecular electron transfer to the rate of association in forming the LmP-LmCytc complex. These studies show that while LmP and CcP form very similar complexes and exhibit similar activities, they substantially differ in how their activity changes as a function of ionic strength. This difference is primarily due to the heavy reliance of LmP on highly specific intermolecular ion pairs, while CcP relies mainly on nonpolar interactions.