Thomas L Poulos

Thomas L Poulos
University of California, Irvine | UCI · Center for Cognitive Neuroscience

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221
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Publications

Publications (221)
Article
A series of potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors (hnNOS) based on the 2-aminopyridine scaffold with a shortened amino sidechain is reported. A rapid and simple protocol was developed to access these inhibitors in excellent yields. Neuronal nitric oxide synthase (nNOS) is a novel therapeutic target...
Article
Full-text available
A characteristic feature of cytochromes P450* is that the complex formed between the ferrous heme iron and carbon monoxide generates an intense absorption band at 450 nm. This unique feature of P450s is due to the proximal thiolate Cys ligand coordinated to the heme iron. Various harsh treatments shift this band to 420 nm, thereby giving P420 which...
Article
Full-text available
AmphL is a cytochrome P450 enzyme that catalyzes the C8 oxidation of 8-deoxyamphotericin B to the polyene macrolide antibiotic, amphotericin B. To understand this substrate selectivity, we solved the crystal structure of AmphL to a resolution of 2.0Å in complex with amphotericin B and performed molecular dynamics simulations. A detailed comparison...
Article
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Three well-characterized heme peroxidases (cytochrome c peroxidase = CCP, ascorbate peroxidase = APX, and Leishmania major peroxidase = LMP) all have a Trp residue tucked under the heme stacked against the proximal His heme ligand. The reaction of peroxidases with H 2 O 2 to give Compound I results in the oxidation of this Trp to a cationic radical...
Article
Cytochrome P450s are amongst nature’s most powerful catalysts. Their ability to activate molecular dioxygen to form high-valent ferryl intermediates (Compounds I and II) enables a wide array of chemistries ranging from simple epoxidations to more complicated C-H bond oxidation. Oxygen activation is achieved by reduction of the ferrous dioxygen comp...
Article
The bacterial cytochrome P450cam catalyzes the oxidation of camphor to 5-exo-hydroxycamphor as the first step in the oxidative assimilation of camphor as a carbon/energy source. CYP101D1 is another bacterial P450 that catalyzes the same reaction. A third P450 (P450tcu) has recently been discovered that has ≈86% sequence identity to P450cam as well...
Article
Inhibition of neuronal nitric oxide synthase (nNOS), an enzyme implicated in neurodegenerative disorders, is an attractive strategy for treating or preventing these diseases. We previously developed several classes of 2-aminoquinoline-based nNOS inhibitors, but these compounds had drawbacks including off-target promiscuity, low activity against hum...
Article
Genes encoding cytochrome P450 (CYP; P450) enzymes occur widely in the Archaea, Bacteria, and Eukarya, where they play important roles in metabolism of endogenous regulatory molecules and exogenous chemicals. We now report that genes for multiple and unique P450s occur commonly in giant viruses in the Mimiviridae, Pandoraviridae, and other families...
Article
Effective delivery of therapeutic drugs into the human brain is one of the most challenging tasks in central nervous system drug development because of the blood-brain barrier (BBB). To overcome the BBB, both passive permeability and efflux transporter liability of a compound must be addressed. Herein, we report our optimization related to BBB pene...
Article
It has become increasingly clear that cytochromes P450 can cycle back and forth between two extreme conformational states termed the closed and open states. In the well-studied cytochrome P450cam, the binding of its redox partner, putidaredoxin (Pdx), shifts P450cam toward the open state. Shifting to the open state is thought to be important in the...
Article
The over production of nitric oxide in the brain by neuronal nitric oxide synthase (nNOS) is associated with a number of neurodegenerative diseases. Although inhibiting nNOS is an important therapeutic goal, it is important not to inhibit endothelial NOS (eNOS) owing to the critical role played by eNOS in maintaining vascular tone. While it has bee...
Article
Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan dioxygenase (hTDO) catalyze the same dioxygenation reaction of Trp to generate N-formyl kynurenine (NFK). They share high structural similarity, especially in the active site. However, hIDO1 possesses a unique inhibitory substrate binding site (Si) that is absent in hTDO. In addition, in hI...
Article
CooAs are dimeric bacterial CO sensing transcription factors that activate a series of enzymes responsible for CO oxidation. The crystal structure of Rhodspirillum rubrum (rrCooA) shows that the N-terminal Pro from monomer A of the dimer coordinates the heme of monomer B which locks rrCooA in the “off” state. Upon CO binding, it is postulated that...
Article
We have compared the thermodynamics of substrate and redox partner binding of P450cam to its close homologue, CYP101D1, using isothermal titration calorimetry (ITC). CYP101D1 binds camphor about 10-fold more weakly than P450cam which is consistent with the inability of camphor to cause a complete low- to high-spin shift in CYP101D1. Even so molecul...
Article
Inhibition of neuronal nitric oxide synthase (nNOS) is a promising therapeutic approach to treat neurodegenerative diseases. Recently, we have achieved considerable progress in improving the potency and isoform selectivity of human nNOS inhibitors bearing a 2-aminopyridine scaffold. However, these inhibitors still suffered from too low cell membran...
Article
Full-text available
The ultrafast kinetics of CO rebinding to carbon monoxide oxidation activator protein (ChCooA) are measured over a wide temperature range and compared with the kinetics of CO binding in other heme systems such as myoglobin (Mb) and hemoglobin (Hb). The Arrhenius prefactor for CO binding to ChCooA and protoheme (~10¹¹ s⁻¹) is similar to what is foun...
Article
The mechanisms by which cancer cell-intrinsic CYP monooxygenases promote tumor progression are largely unknown. CYP3A4 was unexpectedly associated with breast cancer mitochondria and synthesized arachidonic acid (AA)-derived epoxyeicosatrienoic acids (EETs), which promoted the electron transport chain/respiration and inhibited AMPKα. CYP3A4 knockdo...
Article
Leishmania major peroxidase (LmP) is structurally and functionally similar to the well-studied yeast cytochrome c peroxidase (CCP). A recent Brownian dynamics study showed that L. major cytochrome c (LmCytc) associates with LmP by forming an initial complex with the N-terminal helix A of LmP, followed by a movement toward the electron transfer (ET)...
Article
Previous crystal structures of cytochrome P450cam complexed with its redox partner, putidaredoxin (Pdx), shows that P450cam adopts the open conformation. It has been hypothesized that the Pdx induced shift toward the open state frees the essential Asp251 from salt bridges with Arg186 and Lys178 so that Asp251 can participate in a proton relay netwo...
Article
Neuronal nitric oxide synthase (nNOS) is a target for development of antineurodegenerative agents. Most nNOS inhibitors mimic l-arginine and have poor bioavailability. 2-Aminoquinolines showed promise as bioavailable nNOS inhibitors but suffered from low human nNOS inhibition, low selectivity versus human eNOS, and significant binding to other CNS...
Article
Leishmania major pseudoperoxidase (LmPP) is a recently discovered heme protein expressed by the human pathogen. Previous in vivo and in vitro studies suggest that LmPP is a crucial element of the pathogen's defense mechanism against the reactive nitrogen species peroxynitrite produced during the host immune response. To shed light on the potential...
Article
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,...
Article
Human cytochrome P450 3A4 (CYP3A4) is a major hepatic and intestinal enzyme that oxidizes more than 60% of administered therapeutics. Knowledge of how CYP3A4 adjusts and reshapes the active site to regioselectively oxidize chemically diverse compounds is critical for better understanding structure-function relations in this important enzyme, improv...
Article
Full-text available
The camphor monooxygenase, cytochrome P450cam, exhibits a strict requirement for its own redox partner, putidaredoxin (Pdx), a two iron-sulfur ferredoxin. The closest homolog to P450cam is CYP101D1 which is structurally very similar, uses a similar redox partner, and exhibits nearly identical enzymatic properties in the monooxygenation of camphor t...
Article
Full-text available
Once it was discovered that the enzyme nitric oxide synthase (NOS) is responsible for the biosynthesis of NO, NOS became a drug target. Particularly important is the over production of NO by neuronal NOS (nNOS) in various neurodegenerative disorders. After the various NOS isoforms were identified, inhibitor development proceeded rapidly. It soon be...
Article
Nitric oxide (NO) is produced in Gram-positive pathogens Bacillus anthracis and Staphylococcus aureus by the bacterial isoform of nitric oxide synthase (NOS). Inhibition of bacterial nitric oxide synthase (bNOS) has been identified as a promising antibacterial strategy for targeting methicillin-resistant Staphylocoocus aureus1. One class of NOS inh...
Article
The heme iron of cytochromes P450 must be reduced to bind and activate molecular oxygen for substrate oxidation. Reducing equivalents are derived from a redox partner, which requires the formation of a protein-protein complex. A subject of increasing discussion is the role that redox partner binding plays, if any, in favoring significant structural...
Article
Cytochrome P450 3A4 (CYP3A4) promotes ER+HER2- breast cancer cell proliferation and survival, in part, by biosynthesis of epoxyeicosatrienoic acids (EETs). EETs are known to regulate mitochondrial function in non-transformed cells, but the roles of CYP3A4 and EETs in regulation of breast cancer bioenergetics are unknown. Hexyl-benzyl-biguanide (HBB...
Article
The incidence of cutaneous melanoma (CM) has increased markedly over the past four decades although there have been some dramatic advances recently in the treatment of advanced melanoma. The development of novel therapeutic interventions blocking melanoma progression would be of high impact. Recently, our group has identified that neuronal NO synth...
Article
Development of potent and isoform selective nitric oxide synthase (NOS) inhibitors is challenging owing to the structural similarity in the heme active sites. One amino acid difference between NOS isoforms, Asp597 in rat nNOS versus Asn368 in bovine eNOS, has been identified as the structural basis for why some dipeptide amide inhibitors bind more...
Article
Neuronal nitric oxide synthase (nNOS) is an important therapeutic target for the treatment of various neurodegenerative disorders. A major challenge in the design of nNOS inhibitors focuses on potency in humans and selectivity over other NOS isoforms. Here we report potent and selective human nNOS inhibitors based on the 2-aminopyridine scaffold wi...
Article
Full-text available
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 f...
Article
Leishmania major, the parasitic causative agent of leishmaniasis, produces a heme peroxidase (LmP), which catalyzes the peroxide dependent oxidation of mitochondrial cytochrome c (LmCytc) for protection from reactive oxygen species produced by both the host and L. major mitochondrial respiration. The association of LmP and LmCytc, which is known fr...
Article
Nitric oxide synthase (NOS) is a multidomain enzyme that catalyzes the production of nitric oxide (NO) by oxidizing L-Arg to NO and L-citrulline. NO production requires multiple interdomain electron transfer steps between the FMN and heme domain. Specifically, NADPH derived electrons are transferred to the heme containing oxygenase domain via the F...
Article
Human cytochrome P450 3A4 (CYP3A4) is a key xenobiotic-metabolizing enzyme that oxidizes and clears the majority of drugs. CYP3A4 inhibition may lead to drug-drug interactions, toxicity and other adverse effects but, in some cases, could be beneficial and enhance therapeutic efficiency of co-administered pharmaceuticals that are metabolized by CYP3...
Article
While cytochrome P450 enzymes (CYPs) are implicated in tumor angiogenesis through biosynthesis of epoxyeicosatrienoic acids (EETs), little is known about breast cancer cell-intrinsic CYPs that exhibit epoxygenase activity, such as CYP3A4. In an orthotopic breast cancer model, silencing of epithelial CYP3A4 suppressed angiogenesis-related escape of...
Article
Methods for melanoma treatment and prevention with selective nitric oxide synthase inhibitor compounds and related pharmaceutical compositions, alone or in conjunction with one or more other melanoma therapies.
Article
We have analyzed a recently obtained crystal structure of human neuronal nitric oxide synthase (nNOS), then designed and synthesized several 2-aminopyridine derivatives containing a truncated side chain to avoid the hydrophobic pocket that differentiates human and rat nNOS in an attempt to explore alternative binding poses along the substrate acces...
Article
Bacterial infections associated with methicillin-resistant Staphylococcus aureus (MRSA) are a major economic burden to hospitals, and confer high rates of morbidity and mortality among those infected. Exploitation of novel therapeutic targets is thus necessary to combat this dangerous pathogen. Here, we report on the identification and characteriza...
Article
We co-crystallized human cytochrome P450 3A4 (CYP3A4) with progesterone (PRG) under two different conditions but the resulting complexes contained only one PRG molecule bound to the previously identified peripheral site. A novel feature in one of our structures is a citrate ion, originating from the crystallization solution. The citrate-binding sit...
Article
Nitric oxide generated by bacterial nitric oxide synthase (NOS) increases the susceptibility of the Gram-positive pathogen Staphylococcus aureus and Bacillus anthracis to oxidative stress including antibiotic-induced oxidative stress. Previous work has shown that NOS inhibitors improve the effectiveness of antimicrobials. Development of potent and...
Article
Oxidation of L-arginine (L-Arg) to nitric oxide (NO) by NO synthase (NOS) takes place at the heme active site. It is of current interest to study structures of the heme species that activates O2 and transforms the substrate. The NOS ferrous-NO complex is a close mimic of the obligatory ferric (hydro)peroxo intermediate in NOS catalysis. In this wor...
Article
Full-text available
Cytochrome P450 3A4 (CYP3A4) is the major and most important drug-metabolizing enzyme in humans that oxidizes and clears over a half of all administered pharmaceuticals. This is possible because CYP3A4 is promiscuous with respect to substrate binding and has the ability to catalyze diverse oxidative chemistries in addition to traditional hydroxylat...
Article
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, FeIV=O;Trp.+. Cytochrome c (Cytc) reduces the Trp radical to give Compound II, FeIV=O;T...
Article
Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and the second messenger nitric oxide. Three mechanistic pathways are proposed for the inactivation of neuronal NOS (nNOS) by (S)-2-amino-5-(2-(methylthio)acetimidamido)pentanoic acid (1): sulfide oxidation, oxidative dethiolation, and oxidative demethylation. Four p...
Article
Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide. N5-(1-Iminoethyl)-L-ornithine (L-NIO), an amidine-containing molecule, is a natural product known to be an inactivator of inducible NOS (iNOS). Because of the presence of the amidine methyl group in place of the guanidine amino group of substrate L-...
Article
Progesterone receptor membrane component 1 (PGRMC1) is a 25 KDa protein with an N-terminal transmembrane domain and a putative C-terminal cytochrome b5 domain. Heme-binding activity of PGRMC1 has been shown in various homologues of PGRMC1. Albeit the general definition of PGRMC1 as a progesterone receptor, progesterone-binding activity has not been...
Article
Cytochrome P450cam catalyzes the stereo and regio-specific hydroxylation of camphor to 5-exo-hydroxylcamphor. The two electrons for the oxidation of camphor are provided by putidaredoxin (Pdx), a Fe2S2 containing protein. Two recent crystal structures of the P450cam-Pdx complex, one solved with the aid of covalent cross-linking and one without, hav...
Article
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-imidaz...
Article
Full-text available
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,...
Article
Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Metformin is a widely used biguanide diabetes drug that is associated with decreased breast cancer risk and is currently being studied for treatment and prevention of breast cancer. While metformin and biguanides buformin and phenformin exhibit inhibitory activity against breast...
Article
Full-text available
Production of nitric oxide (NO) by nitric oxide synthase (NOS) requires electrons to reduce the heme iron for substrate oxidation. Both FAD and FMN flavin groups mediate the transfer of NADPH derived electrons to NOS. Unlike mammalian NOS that contain both FAD and FMN binding domains within a single polypeptide chain, bacterial NOS is only composed...
Article
To develop potent and selective nNOS inhibitors, a new series of double-headed molecules with chiral linkers that derive from natural amino acid derivatives have been designed and synthesized. The new structures integrate a thiophenecarboximidamide head with two types of chiral linkers, presenting easy synthesis and good inhibitory properties. Inhi...
Article
Many pyrrolidine-based inhibitors highly selective for neuronal nitric oxide synthase (nNOS) over endothelial NOS (eNOS) exhibit dramatically different binding modes. In some cases the inhibitor binds in a 180o flipped orientation in nNOS relative to eNOS. From the several crystal structures we have solved we know that isoform selectivity correlate...
Article
The binding of calmodulin (CaM) to neuronal nitric oxide synthase (nNOS) enables formation of the output state of nNOS for nitric oxide (NO) production. Essential to NOS function is the geometry and dynamics of CaM docking to the NOS oxygenase domain, but little is known about these details. In the present work, the domain docking in a CaM-bound ox...
Chapter
The unique P450 architecture is designed, in part, to enable the structure to adopt open and closed conformations. These changes are required to provide substrate access to the active site. Some of these motions also appear to be important in proton-coupled electron transfer associated with O2 activation. Solvent protons must have access to the act...
Article
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 about 15 Å from the Zn2+. We have solved the crystal structures...
Article
Inactivation of human drug-metabolizing cytochrome P450 3A4 (CYP3A4) could lead to serious adverse events such as drug-drug interactions and toxicity. However, when properly controlled, CYP3A4 inhibition may be beneficial as it can improve clinical efficacy of co-administered therapeutics that otherwise are quickly metabolized by CYP3A4. Currently,...
Article
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 struc...
Article
The crystal structure of the FMN containing redox partner to P450cin, cindoxin (Cdx), has been solved to 1.3 Å resolution. The overall structure is similar to the FMN domain of human cytochrome P450 reductase. A Brownian dynamics/molecular dynamics docking method was used to produce a model of Cdx with its redox partner, P450cin. This Cdx-P450cin m...
Patent
Full-text available
Methods for melanoma treatment and prevention with selective nitric oxide synthase inhibitor compounds and related pharmaceutical compositions, alone or in conjunction with one or more other melanoma therapies.
Article
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 hope th...
Article
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 struct...
Article
The three important mammalian isozymes of nitric oxide synthase (NOS) are neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Inhibitors of nNOS show promise as treatments for neurodegenerative diseases. Eight easily-synthesized compounds containing either one (20a,b) or two (9a-d; 15a,b) 2-amino-4-methylpyridine groups with a ch...
Article
Although CYP101D1 and P450cam catalyze the same reaction at similar rates and share strikingly similar active site architectures, there are significant functional differences. CYP101D1 thus provides an opportunity to probe what structural and functional features must be shared and what features can differ but maintain the high catalytic efficiency....
Article
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...