Glen E. Kellogg

Target Flexibility: An Emerging Consideration in Drug Discovery and Design†

Department of General and Inorganic Chemistry, UniVersity of Parma, Via G.P. Usberti 17/A 43100, Parma, Italy, National Institute for Biosystems and Biostructures, Rome, Italy, Department of Medicinal Chemistry and Institute for Structural Biology & Drug DiscoVery, Virginia Commonwealth UniVersity, Richmond, Virginia 23298-0540, Department of Pharmaceutics, UniVersity of Parma, Via GP Usberti 27/A, 43100 Parma, Italy, High Performance Systems, CINECA Supercomputing Centre, Casalecchio di Reno, Bologna, Italy, Dulbecco Telethon Institute, Department of Chemistry, UniVersity of Modena and Reggio Emilia, Via Campi 183, 41100 Modena, Italy, Department of Mathematics and Natural Sciences, Pharmaceutical Institute, Christian-Albrechts-UniVersity, Gutenbergstrasse 76, 24118 Kiel, Germany, Departments of Biochemistry & Molecular Biology, Computer Science & Engineering, and Physics & Astronomy, Michigan State UniVersity, East Lansing, Michigan 48824-1319, Department of Molecular Biology, MB-5, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037-1000, Molecular Modeling and Bioinformatics Unit, Institute of Biomedical Research, Scientific Park of Barcelona, Department of Biochemistry and Molecular Biology, UniVersity of Barcelona, Josep Samitier 1-5, Barcelona 08028, Spain, Department of Experimental Medicine, UniVersity of Parma, Via Volturno, 39, 43100, Parma, Italy, Department of Chemical, Food, Pharmaceutical and Pharmacological Sciences, UniVersity of Piemonte Orientale “Amedeo AVogadro”, Via BoVio 6, 28100 NoVara, Italy, Institute of Pharmacy and Food Chemistry, UniVersity of Wurzburg, Am Hubland, D-97074 Wurzburg, Germany

Hydrophobicity: is LogPo/w more than the sum of its parts?

The empirically calculated parameter LogPo/w, the log10 of the coefficient for solvent partitioning between 1-octanol and water, has been used to provide the key data for a unique non-covalent interaction force field called HINT (Hydropathic INTeractions). This experimentally-derived force field encodes entropic as well as enthalpic information and also includes some representation of solvation and desolvation energetics in biomolecular associations. The theoretical basis for the HINT model is discussed. This review includes: 1) discussion of calculational representation of the hydrophobic effect, 2) the rationale for describing the experimental LogP o/w based descriptors used in the HINT force field and model as free energy-like, 3) the relationship between hydrophobic fragment constants and partial group electrostatic charge, and 4) the implications of structurally-conserved water molecules on free energy of molecular association. Several recent applications of HINT in structure-based and ligand-based drug discovery are reviewed. Finally, future directions in the HINT model development are proposed.

E-STATE FIELDS : APPLICATIONS TO 3D QSAR

SummaryThe derivation of a new 3D QSAR field based on the electrotopological state (E-state) formalism is described. A complementary index and its associated field, the HE-state, describing the polarity of hydrogens is also defined. These new fields are constructed from a nonempirical index that incorporates electronegativity, the inductive influence of neighboring atoms, and the topological state into a single atomistic descriptor. The classic CoMFA steroid test data set was examined with models incorporating the E-state and HE-state fields alone and in combination with steric, electrostatic and hydropathic fields. The single best model was the E-state/HE-state combination with q2=0.803 (three components) and r2=0.979. Using the E-state and/or HE-state fields with other fields consistently produced models with improved statistics, where the E-state fields provided a significant, if not dominant, contribution.

A Second Receptor Binding Site on Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Contributes to Activation of the Fusion Mechanism

ABSTRACT The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three discrete activities that each affect the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. The interrelationship between the receptor binding and fusion-triggering functions of HN has not been clear. For human parainfluenza type 3 (HPIV3), one bifunctional site on HN can carry out both receptor binding and neuraminidase activities, and this sites receptor binding can be inhibited by the small receptor analog zanamivir. We now report experimental evidence, complemented by computational data, for a second receptor binding site near the HPIV3 HN dimer interface. This second binding site can mediate receptor binding even in the presence of zanamivir, and it differs from the second receptor binding site of the paramyxovirus Newcastle disease virus in its function and its relationship to the primary binding site. This second binding site of HPIV3 HN is involved in triggering F. We suggest that the two receptor binding sites on HPIV3 HN each contribute in distinct ways to virus-cell interaction; one is the multifunctional site that contains both binding and neuraminidase activities, and the other contains binding activity and also is involved in fusion promotion.

1,2-Dithiole-3-Ones as Potent Inhibitors of the Bacterial 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)

ABSTRACT The enzyme FabH catalyzes the initial step of fatty acid biosynthesis via a type II dissociated fatty acid synthase. The pivotal role of this essential enzyme, combined with its unique structural features and ubiquitous occurrence in bacteria, has made it an attractive new target for the development of antibacterial and antiparasitic compounds. We have searched the National Cancer Institute database for compounds bearing structural similarities to thiolactomycin, a natural product which exhibits a weak activity against FabH. This search has yielded several substituted 1,2-dithiole-3-ones that are potent inhibitors of FabH from both Escherichia coli (ecFabH) and Staphylococcus aureus (saFabH). The most potent inhibitor was 4,5-dichloro-1,2-dithiole-3-one, which had 50% inhibitory concentration (IC50) values of 2 μM (ecFabH) and 0.16 μM (saFabH). The corresponding 3-thione analog exhibited comparable activities. Analogs in which the 4-chloro substituent was replaced with a phenyl group were also potent inhibitors, albeit somewhat less effectively (IC50 values of 5.7 and 0.98 μM for ecFabH and saFabH, respectively). All of the 5-chlorinated inhibitors were most effective when they were preincubated with FabH in the absence of substrates. The resulting enzyme-inhibitor complex did not readily regain activity after excess inhibitor was removed, suggesting that a slow dissociation occurs. In stark contrast, a series of inhibitors in which the 5-chloro substituent was replaced with the isosteric and isoelectronic trifluoromethyl group were poorer inhibitors (IC50 values typically ranging from 25 to >100 μM for both ecFabH and saFabH), did not require a preincubation period for maximal activity, and generated an enzyme-inhibitor complex which readily dissociated. Possible modes of binding of 5-chloro-1,2-dithiole-3-ones and 5-chloro-1,2-dithiole-3-thiones with FabH which account for the role of the 5-chloro substituent were considered.

Molecular Determinants of Antiviral Potency of Paramyxovirus Entry Inhibitors

ABSTRACT Hendra virus (HeV) and Nipah virus (NiV) constitute the Henipavirus genus of paramyxoviruses, both fatal in humans and with the potential for subversion as agents of bioterrorism. Binding of the HeV/NiV attachment protein (G) to its receptor triggers a series of conformational changes in the fusion protein (F), ultimately leading to formation of a postfusion six-helix bundle (6HB) structure and fusion of the viral and cellular membranes. The ectodomain of paramyxovirus F proteins contains two conserved heptad repeat regions, the first (the N-terminal heptad repeat [HRN]) adjacent to the fusion peptide and the second (the C-terminal heptad repeat [HRC]) immediately preceding the transmembrane domain. Peptides derived from the HRN and HRC regions of F are proposed to inhibit fusion by preventing activated F molecules from forming the 6HB structure that is required for fusion. We previously reported that a human parainfluenza virus 3 (HPIV3) F peptide effectively inhibits infection mediated by the HeV glycoproteins in pseudotyped-HeV entry assays more effectively than the comparable HeV-derived peptide, and we now show that this peptide inhibits live-HeV and -NiV infection. HPIV3 F peptides were also effective in inhibiting HeV pseudotype virus entry in a new assay that mimics multicycle replication. This anti-HeV/NiV efficacy can be correlated with the greater potential of the HPIV3 C peptide to interact with the HeV F N peptide coiled-coil trimer, as evaluated by thermal unfolding experiments. Furthermore, replacement of a buried glutamic acid (glutamic acid 459) in the C peptide with valine enhances antiviral potency and stabilizes the 6HB conformation. Our results strongly suggest that conserved interhelical packing interactions in the F protein fusion core are important determinants of C peptide inhibitory activity and offer a strategy for the development of more-potent analogs of F peptide inhibitors.

Design of O-acetylserine sulfhydrylase inhibitors by mimicking Nature

The inhibition of cysteine biosynthesis in prokaryotes and protozoa has been proposed to be relevant for the development of antibiotics. Haemophilus influenzae O-acetylserine sulfhydrylase (OASS), catalyzing l-cysteine formation, is inhibited by the insertion of the C-terminal pentapeptide (MNLNI) of serine acetyltransferase into the active site. Four-hundred MNXXI pentapeptides were generated in silico, docked into OASS active site using GOLD, and scored with HINT. The terminal P5 Ile accounts for about 50% of the binding energy. Glu or Asp at position P4 and, to a lesser extent, at position P3 also significantly contribute to the binding interaction. The predicted affinity of 14 selected pentapeptides correlated well with the experimentally determined dissociation constants. The X-ray structure of three high affinity pentapeptide-OASS complexes were compared with the docked poses. These results, combined with a GRID analysis of the active site, allowed us to define a pharmacophoric scaffold for the design of peptidomimetic inhibitors.

Hydrophobicity - Shake Flasks, Protein Folding and Drug Discovery

Hydrophobic interactions are some of the most important interactions in nature. They are the primary driving force in a number of phenomena. This is mostly an entropic effect and can account for a number of biophysical events such as protein-protein or protein-ligand binding that are of immense importance in drug design. The earliest studies on this phenomenon can be dated back to the end of the 19(th) century when Meyer and Overton independently correlated the hydrophobic nature of gases to their anesthetic potency. Since then, significant progress has been made in this realm of science. This review briefly traces the history of hydrophobicity research along with the theoretical estimation of partition coefficients. Finally, the application of hydrophobicity estimation methods in the field of drug design and protein folding is discussed.

Paramyxovirus Receptor-Binding Molecules: Engagement of One Site on the Hemagglutinin-Neuraminidase Protein Modulates Activity at the Second Site

ABSTRACT The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three different activities: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. These three discrete properties each affect the ability of HN to promote viral fusion and entry. For human parainfluenza type 3, one bifunctional site on HN can carry out both binding and neuraminidase, and the receptor mimic, zanamivir, impairs viral entry by blocking receptor binding. We report here that for Newcastle disease virus, the HN receptor avidity is increased by zanamivir, due to activation of a second site that has higher receptor avidity. Only certain receptor mimics effectively activate the second site (site II) via occupation of site I; yet without activation of this second site, binding is mediated entirely by site I. Computational modeling designed to complement the experimental approaches suggests that the potential for small molecule receptor mimics to activate site II, upon binding to site I, directly correlates with their predicted strengths of interaction with site I. Taken together, the experimental and computational data show that the molecules with the strongest interactions with site I—zanamivir and BCX 2798—lead to the activation of site II. The finding that site II, once activated, shows higher avidity for receptor than site I, suggests paradigms for further elucidating the regulation of HN′s multiple functions in the viral life cycle.

Inhibition of Hendra Virus Fusion

ABSTRACT Hendra virus (HeV) is a recently identified paramyxovirus that is fatal in humans and could be used as an agent of bioterrorism. The HeV receptor-binding protein (G) is required in order for the fusion protein (F) to mediate fusion, and analysis of the triggering/activation of HeV F by G should lead to strategies for interfering with this key step in viral entry. HeV F, once triggered by the receptor-bound G, by analogy with other paramyxovirus F proteins, undergoes multistep conformational changes leading to a six-helix bundle (6HB) structure that accomplishes fusion of the viral and cellular membranes. The ectodomain of paramyxovirus F proteins contains two conserved heptad repeat regions (HRN and HRC) near the fusion peptide and the transmembrane domains, respectively. Peptides derived from the HRN and HRC regions of F are proposed to inhibit fusion by preventing F, after the initial triggering step, from forming the 6HB structure that is required for fusion. HeV peptides have previously been found to be effective at inhibiting HeV fusion. However, we found that a human parainfluenza virus 3 F-peptide is more effective at inhibiting HeV fusion than the comparable HeV-derived peptide.