P. Nuno Palma

BiGGER: A new (soft) docking algorithm for predicting protein interactions

A new computationally efficient and automated “soft docking” algorithm is described to assist the prediction of the mode of binding between two proteins, using the three‐dimensional structures of the unbound molecules. The method is implemented in a software package called BiGGER (Bimolecular Complex Generation with Global Evaluation and Ranking) and works in two sequential steps: first, the complete 6‐dimensional binding spaces of both molecules is systematically searched. A population of candidate protein‐protein docked geometries is thus generated and selected on the basis of the geometric complementarity and amino acid pairwise affinities between the two molecular surfaces. Most of the conformational changes observed during protein association are treated in an implicit way and test results are equally satisfactory, regardless of starting from the bound or the unbound forms of known structures of the interacting proteins. In contrast to other methods, the entire molecular surfaces are searched during the simulation, using absolutely no additional information regarding the binding sites. In a second step, an interaction scoring function is used to rank the putative docked structures. The function incorporates interaction terms that are thought to be relevant to the stabilization of protein complexes. These include: geometric complementarity of the surfaces, explicit electrostatic interactions, desolvation energy, and pairwise propensities of the amino acid side chains to contact across the molecular interface. The relative functional contribution of each of these interaction terms to the global scoring function has been empirically adjusted through a neural network optimizer using a learning set of 25 protein‐protein complexes of known crystallographic structures. In 22 out of 25 protein‐protein complexes tested, near‐native docked geometries were found with Cα RMS deviations ≤ 4.0 Å from the experimental structures, of which 14 were found within the 20 top ranking solutions. The program works on widely available personal computers and takes 2 to 8 hours of CPU time to run any of the docking tests herein presented. Finally, the value and limitations of the method for the study of macromolecular interactions, not yet revealed by experimental techniques, are discussed. Proteins 2000;39:372–384.

Discovery of a Long-Acting, Peripherally Selective Inhibitor of Catechol-O-methyltransferase

Novel nitrocatechol-substituted heterocycles were designed and evaluated for their ability to inhibit catechol-O-methyltransferase (COMT). Replacement of the pyrazole core of the initial hit 4 with a 1,2,4-oxadiazole ring resulted in a series of compounds endowed with longer duration of COMT inhibition. Incorporation of a pyridine N-oxide residue at position 3 of the 1,2,4-oxadiazole ring led to analogue 37f, which was found to possess activity comparable to entacapone and lower toxicity in comparison to tolcapone. Lead structure 37f was systematically modified in order to improve selectivity and duration of COMT inhibition as well as to minimize toxicity. Oxadiazole 37d (2,5-dichloro-3-(5-(3,4-dihydroxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)-4,6-dimethylpyridine 1-oxide (BIA 9-1067)) was identified as a long-acting, purely peripheral inhibitor, which is currently under clinical evaluation as an adjunct to L-Dopa therapy of Parkinsons disease.

A novel approach for assesing macromolecular complexes combining soft-docking calculations with NMR data

We present a novel and efficient approach for assessing protein–protein complex formation, which combines ab initio docking calculations performed with the protein docking algorithm BiGGER and chemical shift perturbation data collected with heteronuclear single quantum coherence (HSQC) or TROSY nuclear magnetic resonance (NMR) spectroscopy. This method, termed “restrained soft‐docking,” is validated for several known protein complexes. These data demonstrate that restrained soft‐docking extends the size limitations of NMR spectroscopy and provides an alternative method for investigating macromolecular protein complexes that requires less experimental time, effort, and resources. The potential utility of this novel NMR and simulated docking approach in current structural genomic initiatives is discussed.

Modeling protein complexes with BiGGER

This article describes the method and results of our participation in the Critical Assessment of PRediction of Interactions (CAPRI) experiment, using the protein docking program BiGGER (Bimolecular complex Generation with Global Evaluation and Ranking) (Palma et al., Proteins 2000;39:372–384). Of five target complexes (CAPRI targets 2, 4, 5, 6, and 7), only one was successfully predicted (target 6), but BiGGER generated reasonable models for targets 4, 5, and 7, which could have been identified if additional biochemical information had been available. Proteins 2003;52:19–23.

Opicapone: a short lived and very long acting novel catechol-O-methyltransferase inhibitor following multiple dose administration in healthy subjects.

AIMS The aim of this study was to assess the tolerability, pharmacokinetics and inhibitory effect on erythrocyte soluble catechol-O-methyltransferase (S-COMT) activity following repeated doses of opicapone. METHODS This randomized, placebo-controlled, double-blind study enrolled healthy male subjects who received either once daily placebo or opicapone 5, 10, 20 or 30 mg for 8 days. RESULTS Opicapone was well tolerated. Its systemic exposure increased in an approximately dose-proportional manner with an apparent terminal half-life of 1.0 to 1.4 h. Sulphation was the main metabolic pathway. Opicapone metabolites recovered in urine accounted for less than 3% of the amount of opicapone administered suggesting that bile is likely the main route of excretion. Maximum S-COMT inhibition (Emax ) ranged from 69.9% to 98.0% following the last dose of opicapone. The opicapone-induced S-COMT inhibition showed a half-life in excess of 100 h, which was dose-independent and much longer than plasma drug exposure. Such a half-life translates into a putative underlying rate constant that is comparable with the estimated dissociation rate constant of the COMT-opicapone complex. CONCLUSION Despite its short elimination half-life, opicapone markedly and sustainably inhibited erythrocyte S-COMT activity making it suitable for a once daily regimen.

Computation of the binding affinities of catechol-O-methyltransferase inhibitors: multisubstate relative free energy calculations.

Alchemical free energy simulations are amongst the most accurate techniques for the computation of the free energy changes associated with noncovalent protein–ligand interactions. A procedure is presented to estimate the relative binding free energies of several ligands to the same protein target where multiple, low‐energy configurational substates might coexist, as opposed to one unique structure. The contributions of all individual substates were estimated, explicitly, with the free energy perturbation method, and combined in a rigorous fashion to compute the overall relative binding free energies and dissociation constants. It is shown that, unless the most stable bound forms are known a priori, inaccurate results may be obtained if the contributions of multiple substates are ignored. The method was applied to study the complex formed between human catechol‐O‐methyltransferase and BIA 9‐1067, a newly developed tight‐binding inhibitor that is currently under clinical evaluation for the therapy of Parkinsons disease. Our results reveal an exceptionally high‐binding affinity (Kd in subpicomolar range) and provide insightful clues on the interactions and mechanism of inhibition. The inhibitor is, itself, a slowly reacting substrate of the target enzyme and is released from the complex in the form of O‐methylated product. By comparing the experimental catalytic rate (kcat) and the estimated dissociation rate (koff) constants of the enzyme‐inhibitor complex, one can conclude that the observed inhibition potency (Ki) is primarily dependent on the catalytic rate constant of the inhibitors O‐methylation, rather than the rate constant of dissociation of the complex.

Synechocystis ferredoxin/ferredoxin-NADP+-reductase/NADP+ complex: Structural model obtained by NMR-restrained docking

Ferredoxin (Fd) and ferredoxin‐NADP+‐reductase (FNR) are two terminal physiological partners of the photosynthetic electron transport chain. Based on a nuclear magnetic resonance (NMR)‐restrained‐docking approach, two alternative structural models of the Fd–FNR complex in the presence of NADP+ are proposed. The protein docking simulations were performed with the software BiGGER. NMR titration revealed a 1:1 stoichiometry for the complex and allowed the mapping of the interacting residues at the surface of Fd. The NMR chemical shifts were encoded into distance constraints and used with theoretically calculated electronic coupling between the redox cofactors to propose experimentally validated docked complexes.

CHAPTER 4:Catechol‐O‐Methyl‐Transferase Inhibitors: Present Problems and Relevance of the New Ones

Levodopa, in association with a DOPA decarboxylase inhibitor (e.g., carbidopa or benserazide) has for many years been the undisputed gold standard drug for the symptomatic treatment of Parkinson’s disease (PD). However, given its rapid disposition and elimination in the periphery, it was hypothesized that significant enhancements in levodopa bioavailability and clinical efficacy could be achieved through co‐adjuvant therapy with a catechol‐O‐methyl‐transferase (COMT) inhibitor. Early attempts, dating back to the late 1950s, to discover COMT inhibitors were generally hampered by their lack of in vivo efficacy, target selectivity or by considerable toxicity. It was not until the late 1990s that entacapone and tolcapone, representatives of a new class of potent COMT inhibitors (nitrocatechol derivatives), made their way to clinical practice for the treatment of PD. Even though these drugs have since contributed to an increase in the usefulness of levodopa therapy, each of them presents known limitations, namely concerning their clinical efficacy and safety. The unmet medical need for more efficacious and safer COMT inhibitors has motivated intense research in this field over the last decade. Opicapone is the first, third‐generation COMT inhibitor among the nitrocatechol derivatives under clinical development, and demonstrates superior pharmacodynamic and safety profiles in humans, over previous drugs. In this chapter, we review the major advances in this field, summarize the relevant non‐clinical and clinical human pharmacology and discuss new insights into the mechanism of action of opicapone.

Discovery of a Potent, Long‐Acting, and CNS‐Active Inhibitor (BIA 10‐2474) of Fatty Acid Amide Hydrolase

Fatty acid amide hydrolase (FAAH) can be targeted for the treatment of pain associated with various medical conditions. Herein we report the design and synthesis of a novel series of heterocyclic‐N‐carboxamide FAAH inhibitors that have a good alignment of potency, metabolic stability and selectivity for FAAH over monoacylglycerol lipase (MAGL) and carboxylesterases (CEs). Lead optimization efforts carried out with benzotriazolyl‐ and imidazolyl‐N‐carboxamide series led to the discovery of clinical candidate 8 l (3‐(1‐(cyclohexyl(methyl)carbamoyl)‐1H‐imidazol‐4‐yl)pyridine 1‐oxide; BIA 10‐2474) as a potent and long‐acting inhibitor of FAAH. However, during a Phase I clinical trial with compound 8 l, unexpected and unpredictable serious neurological adverse events occurred, affecting five healthy volunteers, including the death of one subject.

Molecular Interactions Between Metalloproteins Involved in Electron Transfer Processes: Tetraheme Cytochrome c 3 and Flavodoxin. Nmr and Molecular Modeling Studies

Flavodoxin (16 kDa) and the tetra-heme cytochrome c 3 (13 kDa) are two low molecular weight proteins, involved in the electron transport system operating in sulfate-reducing bacteria, between organic substrates and terminal electron acceptors: The electron transfer, as observed in vitro between these two proteins, is believed to occur through the formation of a specific complex between the two proteins, being the interaction mainly electrostatic in nature. The nature and properties of the protein-protein complex (stoichiometry, interaction sites, association constants) were probed. Integration and correlation of the experimental results obtained from magnetic resonance studies on protein-protein titrations with the available structural and biochemical data is presented. A structural model for a hypothetical ternary complex, formed between one molecule of flavodoxin and two molecules of cytochrome c3, is proposed using the available X-ray structures of the isolated proteins and, when required, model structures predicted by homology modeling. Open image in new window