James C. Burnett

Very empirical treatment of solvation and entropy: a force field derived from Log Po/w

A non-covalent interaction force field model derived from the partition coefficient of 1-octanol/water solubility is described. This model, HINT for Hydropathic INTeractions, is shown to include, in very empirical and approximate terms, all components of biomolecular associations, including hydrogen bonding, Coulombic interactions, hydrophobic interactions, entropy and solvation/desolvation. Particular emphasis is placed on: (1) demonstrating the relationship between the total empirical HINT score and free energy of association, ΔGinteraction; (2) showing that the HINT hydrophobic-polar interaction sub-score represents the energy cost of desolvation upon binding for interacting biomolecules; and (3) a new methodology for treating constrained water molecules as discrete independent small ligands. An example calculation is reported for dihydrofolate reductase (DHFR) bound with methotrexate (MTX). In that case the observed very tight binding, ΔGinteraction≤−13.6xa0kcal mol−1, is largely due to ten hydrogen bonds between the ligand and enzyme with estimated strength ranging between −0.4 and −2.3xa0kcalxa0mol−1. Four water molecules bridging between DHFR and MTX contribute an additional −1.7xa0kcalxa0mol−1 stability to the complex. The HINT estimate of the cost of desolvation is +13.9xa0kcalxa0mol−1.

High–resolution crystal structure of deoxy hemoglobin complexed with a potent allosteric effector

The crystal structure of human deoxy hemoglobin (Hb) complexed with a potent allosteric effector (2‐[4‐[[(3,5‐dimethylanilino)carbonyl]methyl]phenoxy]‐2‐methylpropionic acid) = RSR‐13) is reported at 1.85 Å resolution. Analysis of the hemoglobin:effector complex indicates that two of these molecules bind to the central water cavity of deoxy Hb in a symmetrical fashion, and that each constrains the protein by engaging in hydrogen bonding and hydrophobic interactions with three of its four subunits. Interestingly, we also find that water‐mediated interactions between the bound effectors and the protein make significant contributions to the overall binding. Physiologically, the interaction of RSR‐13 with Hb results in increased oxygen delivery to peripheral tissues. Thus, this compound has potential therapeutic application in the treatment of hypoxia, ischemia, and trauma‐related blood loss. Currently, RSR‐13 is in phase III clinical trials as a radiosensitizing agent in the treatment of brain tumors. A detailed structural analysis of this compound complexed with deoxy Hb has important implications for the rational design of future analogs.

Computationally accessible method for estimating free energy changes resulting from site-specific mutations of biomolecules: Systematic model building and structural/hydropathic analysis of deoxy and oxy hemoglobins

A practical computational method for the molecular modeling of free‐energy changes associated with protein mutations is reported. The de novo generation, optimization, and thermodynamic analysis of a wide variety of deoxy and oxy hemoglobin mutants are described in detail. Hemoglobin is shown to be an ideal candidate protein for study because both the native deoxy and oxy states have been crystallographically determined, and a large and diverse population of its mutants has been thermodynamically characterized. Noncovalent interactions for all computationally generated hemoglobin mutants are quantitatively examined with the molecular modeling program HINT (Hydropathic INTeractions). HINT scores all biomolecular noncovalent interactions, including hydrogen bonding, acid–base, hydrophobic–hydrophobic, acid–acid, base–base, and hydrophobic–polar, to generate dimer–dimer interface “scores” that are translated into free‐energy estimates. Analysis of 23 hemoglobin mutants, in both deoxy and oxy states, indicates that the effects of mutant residues on structurally bound waters (and visa versa) are important for generating accurate free‐energy estimates. For several mutants, the addition/elimination of structural waters is key to understanding the thermodynamic consequences of residue mutation. Good agreement is found between calculated and experimental data for deoxy hemoglobin mutants (r = 0.79, slope = 0.78, standard error = 1.4 kcal mol−1, n = 23). Less accurate estimates were initially obtained for oxy hemoglobin mutants (r = 0.48, slope = 0.47, standard error = 1.4 kcal mol−1, n = 23). However, the elimination of three outliers from this data set results in a better correlation of r = 0.87 (slope = 0.72, standard error = 0.75, n = 20). These three mutations may significantly perturb the hemoglobin quaternary structure beyond the scope of our structural optimization procedure. The method described is also useful in the examination of residue ionization states in protein structures. Specifically, we find an acidic residue within the native deoxy hemoglobin dimer–dimer interface that may be protonated at physiological pH. The final analysis is a model design of novel hemoglobin mutants that modify cooperative free energy (ΔGc)—the energy barrier between the allosteric transition from deoxy to oxy hemoglobin. Proteins 2001;42:355–377.

Structure of human carbonmonoxyhemoglobin at 2.16 Å: a snapshot of the allosteric transition

A 2.16 A resolution structure of high-salt human carbonmonoxyhemoglobin crystallized at pH 6.4 is reported. The quaternary structure is similar to that of classic R-state hemoglobin; however, subtle but significant tertiary structural changes are observed at the alpha(1)beta(2) and symmetrically equivalent alpha(2)beta(1) interfaces--these are the key subunit interfaces that govern the allosteric transition between the R and T states. Specifically, the movement and weakening of two important hydrogen bonds that are diagnostic for R-state structures, beta(2)His97-alpha(1)Thr38 and beta(2)Arg40-alpha(1)Thr41, have been observed. In addition, a phosphate molecule bound between the two beta-subunits (at the entrance to the central water cavity) has been identified and electron density indicates that this molecule occupies two alternate positions that are related by the dyad axis. Both positions superimpose on the 2,3-diphosphoglycerate binding site. One phosphate conformer interacts with beta(2)Asn139, beta(1)His143 and beta(1)His146, while the second interacts with symmetry-related counterparts (beta(1)Asn139, beta(2)His143 and beta(2)His146).

Recent Advances in Botulinum Neurotoxin Inhibitor Development

Botulinum neurotoxins (BoNTs) are endopeptidases that target motor neurons and block acetylcholine neurotransmitter release. This action results in the muscle paralysis that defines the disease botulism. To date, there are no FDA-approved therapeutics to treat BoNT-mediated paralysis after intoxication of the motor neuron. Importantly, the rationale for pursuing treatments to counter these toxins is driven by their potential misuse. Current drug discovery efforts have mainly focused on small molecules, peptides, and peptidomimetics that can directly and competitively inhibit BoNT light chain proteolytic activity. Although this is a rational approach, direct inhibition of the Zn(2+) metalloprotease activity has been elusive as demonstrated by the dearth of candidates undergoing clinical evaluation. Therefore, broadening the scope of viable targets beyond that of active site protease inhibitors represents an additional strategy that could move the field closer to the clinic. Here we review the rationale, and discuss the outcomes of earlier approaches and highlight potential new targets for BoNT inhibition. These include BoNT uptake and processing inhibitors, enzymatic inhibitors, and modulators of neuronal processes associated with toxin clearance, neurotransmitter potentiation, and other pathways geared towards neuronal recovery and repair.

Post-Intoxication Inhibition of Botulinum Neurotoxin Serotype A within Neurons by Small-Molecule, Non-Peptidic Inhibitors

Botulinum neurotoxins (BoNTs) comprise seven distinct serotypes that inhibit the release of neurotransmitter across neuromuscular junctions, resulting in potentially fatal flaccid paralysis. BoNT serotype A (BoNT/A), which targets synaptosomal-associated protein of 25kDa (SNAP-25), is particularly long-lived within neurons and requires a longer time for recovery of neuromuscular function. There are currently no treatments available to counteract BoNT/A after it has entered the neuronal cytosol. In this study, we examined the ability of small molecule non-peptidic inhibitors (SMNPIs) to prevent SNAP-25 cleavage post-intoxication of neurons. The progressive cleavage of SNAP-25 observed over 5 h following 1 h BoNT/A intoxication was prevented by addition of SMNPIs. In contrast, anti-BoNT/A neutralizing antibodies that strongly inhibited SNAP-25 cleavage when added during intoxication were completely ineffective when added post-intoxication. Although Bafilomycin A1, which blocks entry of BoNT/A into the cytosol by preventing endosomal acidification, inhibited SNAP-25 cleavage post-intoxication, the degree of inhibition was significantly reduced versus addition both during and after intoxication. Post-intoxication application of SMNPIs, on the other hand, was nearly as effective as application both during and after intoxication. Taken together, the results indicate that competitive SMNPIs of BoNT/A light chain can be effective within neurons post-intoxication.

Analysis of botulinum neurotoxin serotype A metalloprotease inhibitors: analogs of a chemotype for therapeutic development in the context of a three-zone pharmacophore

Botulinum neurotoxins (BoNTs), and in particular serotype A, are the most poisonous of known biological substances, and are responsible for the flaccid paralysis of the disease state botulism. Because of the extreme toxicity of these enzymes, BoNTs are considered highest priority biothreat agents. To counter BoNT serotype A (BoNT/A) poisoning, the discovery and development of small molecule, drug-like inhibitors as post-intoxication therapeutic agents has been/is being pursued. Specifically, we are focusing on inhibitors of the BoNT/A light chain (LC) (ie, a metalloprotease) subunit, since such compounds can enter neurons and provide post-intoxication protection of the enzyme target substrate. To aid/facilitate this drug development effort, a pharmacophore for inhibition of the BoNT/A LC subunit was previously developed, and is continually being refined via the incorporation of novel and diverse inhibitor chemotypes. Here, we describe several analogs of a promising therapeutic chemotype in the context of the pharmacophore for BoNT/A LC inhibition. Specifically, we describe: 1) the pharmacophoric fits of the analogs and how these fits rationalize the in vitro inhibitory potencies of the analogs and 2) pharmacophore refinement via the inclusion of new components from the most potent of the presented analogs.

X-ray crystallographic analyses of symmetrical allosteric effectors of hemoglobin: compounds designed to link primary and secondary binding sites

The rational design and X-ray crystallographic analyses of two symmetrical allosteric effectors of hemoglobin (Hb) are reported. Compound design was directed by the previously solved co-crystal structure of one of the most potent allosteric effectors of Hb, 2-[4-[(3,5-dichlorophenylcarbamoyl)-methyl]-phenoxy]-2-methylpropionic acid (RSR4), which revealed two distinct binding sites for this compound in the Hb central water cavity. The primary binding site has been observed for all compounds of this structural class, which stabilize deoxy Hb by engaging in inter-dimer contacts with three of the four protein subunits. Interactions at the secondary binding site of RSR4 occur primarily between the beta(1) and beta(2) subunits and serve to further constrain the deoxy state. Based on these observations, it was hypothesized that compounds with the ability to simultaneously span and link both of these sites would possess increased potency, but at a lower molar concentration than RSR4. Two symmetrical compounds were designed and synthesized based on this hypothesis. The symmetrical effector approach was taken to minimize the number of compound orientations needed to successfully bind at either of the distinct allosteric sites. X-ray crystallographic analyses of these two effectors in complex with Hb revealed that they successfully spanned the RSR4 primary and secondary binding sites. However, the designed compounds interacted with the secondary binding site in such a way that intra-dimer, as opposed to inter-dimer, interactions were generated. In agreement with these observations, in vitro evaluation of the symmetrical effectors in Hb solution indicated that neither compound possessed the potency of RSR4. A detailed analysis of symmetrical effector-Hb contacts and comparisons with the binding contacts of RSR4 are discussed.