A new method that utilizes matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and exploits the hydrogen/deuterium (H/D) exchange properties of proteins was developed for measuring the thermodynamic properties of protein-ligand complexes in solution. Dissociation constants (Kd values) determined by the method for five model protein-ligand complexes that included those with small molecules, nucleic acids, peptides, and other proteins were generally in good agreement with Kd values measured by conventional methods. Important experimental advantages of the described method over existing methods include: the ability to make measurements in a high-throughput and automated fashion, the ability to make measurements using only picomole quantitities of protein, and the ability to analyze either purified or unpurified protein-ligand complexes.

A new H/D exchange- and MALDI mass spectrometry-based technique, termed SUPREX, was used to characterize the thermodynamic properties of a series of model protein-peptide complexes of the Abelson tyrosine kinase SH3 domain (abl-SH3) and the S-Protein (S-Pro). The SUPREX technique was employed to evaluate the folding free energies (DeltaG(f) values) of each model protein in the absence and in the presence of a series of different peptide ligands. Ultimately, these SUPREX-derived DeltaG(f) values were used to calculate dissociation constants (K(d) values) for each of the nine protein-peptide complexes in this study. As part of this work, we describe a new data collection and analysis method that allows the accurate and precise determination of protein folding m-values in the SUPREX experiment. The m-values that we determined for the abl-SH3 domain and the S-Pro system were in good agreement with those determined by conventional techniques. Our results also indicate that the SUPREX-derived K(d) values for the protein-peptide complexes in this work were in reasonably good agreement with those determined by conventional techniques.

SCY-078 (MK-3118) is a novel, semisynthetic derivative of enfumafungin and represents the first compound of the triterpene class of antifungals. SCY-078 exhibits potent inhibition of β-(1,3)-d-glucan synthesis, an essential cell wall component of many pathogenic fungi, including Candida spp. and Aspergillus spp. SCY-078 is currently in phase 2 clinical development for the treatment of invasive fungal diseases. In vitro disposition studies to assess solubility, intestinal permeability, and metabolic stability were predictive of good oral bioavailability. Preclinical pharmacokinetic studies were consistent with once-daily administration to humans. After intravenous delivery, plasma clearance in rodents and dogs was low, representing <15% and <25% of hepatic blood flow, respectively. The terminal elimination-phase half-life was 5.5 to 8.7 h in rodents, and it was ∼9.3 h in dogs. The volume of distribution at steady-state was high (4.7 to 5.3 liters/kg), a finding suggestive of extensive tissue distribution. Exposure of SCY-078 in kidney tissue, a target organ for invasive fungal disease such as candidiasis, exceeded plasma by 20- to 25-fold for the area under the concentration-time curve from 0 h to infinity (AUC0–∞) and Cmax. SCY-078 achieved efficacy endpoints following oral delivery across multiple murine models of disseminated candidiasis. The pharmacokinetic/pharmacodynamic indices Cmax/MIC and AUC/MIC correlated with outcome. Target therapeutic exposure, expressed as the plasma AUC0–24, was comparable across models, with an upper value of 11.2 μg·h/ml (15.4 μM·h); the corresponding mean value for free drug AUC/MIC was ∼0.75. Overall, these results demonstrate that SCY-078 has the oral and intravenous (i.v.) pharmacokinetic properties and potency in murine infection models of disseminated candidiasis to support further investigation as a novel i.v. and oral treatment for invasive fungal diseases.

SUPREX (stability of unpurified proteins from rates of H/D exchange) is a H/D exchange- and matrix-assisted laser desorption/ionization (MALDI)-based technique for characterizing the equilibrium unfolding/refolding properties of proteins and protein-ligand complexes. Here, we describe the application of SUPREX to the thermodynamic analysis of synergistic anion binding to iron-loaded ferric-binding protein (Fe(3+)FbpA-X, X = synergistic anion). The in vivo function of FbpA is to transport unchelated Fe(3+) across the periplasmic space of certain Gram-negative bacteria, a process that requires simultaneous binding of a synergistic anion. Our results indicate that Fe(3+)FbpA-X is not a so-called "ideal" protein system for SUPREX analyses because it does not exhibit two-state folding properties and it does not exhibit EX2 H/D exchange behavior. However, despite these nonideal properties of the Fe(3+)FbpA-X protein-folding/unfolding reaction, we demonstrate that the SUPREX technique is still amenable to the quantitative thermodynamic analysis of synergistic anion binding to Fe(3+)FbpA. As part of this work, the SUPREX technique was used to evaluate the DeltaDeltaG(f) values of four synergistic anion-containing complexes of Fe(3+)FbpA (i.e., Fe(3+)FbpA-PO(4), Fe(3+)FbpA-citrate, Fe(3+)FbpA-AsO(4), and Fe(3+)FbpA-SO(4)). The DeltaDeltaG(f) value obtained for Fe(3+)FbpA-citrate relative to Fe(3+)FbpA-PO(4) (1.45 +/- 0.44 kcal/mol), is in good agreement with that reported previously (1.98 kcal/mol). The value obtained for Fe(3+)FbpA-AsO(4) (0.58 +/- 0.45 kcal/mol) was also consistent with that reported previously (0.68 kcal/mol), but the measurement error is very close to the magnitude of the value. This work (i) demonstrates the utility of the SUPREX method for studying anion binding by FbpA, (ii) provides the first evaluation of a DeltaDeltaG(f) value for Fe(3+)FbpA-SO(4), -1.43 +/- 0.17 kcal/mol, and (iii) helps substantiate our hypothesis that the synergistic anion plays a role in controlling the lability of iron bound to FbpA in the transport process.

Sustained and high concentrations were encountered with 7-day approved administration of 1% azithromycin formulation (AzaSite, Inspire Pharmaceuticals, Inc., Durham, NC) within all ocular surface tissues, particularly the lids. Many ocular surface disorders involving the tear film, eyelids, and adnexal structures are associated with chronic, low-grade bacterial infection and may potentially lead to decreased vision secondary to corneal scarring. Various topical antibiotic and steroid combinations with or without oral tetracyclines are commonly used with variable clinical response and known potential side effects. The clinical relevance of this study is unknown; however, the long-lasting antibacterial and additional anti-inflammatory properties of topical azithromycin might offer an effective alternative treatment option and should be explored further in clinical studies.

Recently, we reported on a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI)-based technique, termed SUPREX, that can be used to measure the thermodynamic stability of a protein (Ghaemmaghami, S.; Fitzgerald, M. C.; Oas, T. G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8296-8301). In the work described here, we report on our efforts to optimize the sensitivity of SUPREX analyses. We describe a new sample handling protocol for SUPREX that involves the use of batch chromatography methods with reversed-phase chromatographic media for the microconcentration and desalting of SUPREX samples. Using ribonuclease A as a model protein system, we demonstrate that our new protocol permits the SUPREX analysis of as little as 10 pmol of protein. This amount of protein is 100-fold less than the amount of material required in our initial SUPREX protocol, and it is 1-2 orders of magnitude less than the amount of material required in conventional spectroscopy-based methods for measuring the thermodynamic stability of a protein.

We report on the development of a series of pyrimidine carboxylic acids that are potent and selective inhibitors of kynurenine monooxygenase and competitive for kynurenine. We describe the SAR for this novel series and report on their inhibition of KMO activity in biochemical and cellular assays and their selectivity against other kynurenine pathway enzymes. We describe the optimization process that led to the identification of a program lead compound with a suitable ADME/PK profile for therapeutic development. We demonstrate that systemic inhibition of KMO in vivo with this lead compound provides pharmacodynamic evidence for modulation of kynurenine pathway metabolites both in the periphery and in the central nervous system.

We recently reported on a new H/D exchange-and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based technique, termed SUPREX, that removes several important limitations associated with measuring the thermodynamic stability of proteins. In contrast to conventional spectroscopy-based techniques for characterizing the equilibrium unfolding behavior of proteins, SUPREX is amenable to the thermodynamic analysis of both purified and unpurified proteins using mg to ng quantities of material. Here we report on the application of SUPREX to the analysis of multimeric protein systems. Included in this work are the SUPREX results we obtained in studies on six model multimeric proteins including the GCN4p1 dimer, the coil-V a L d trimer, the 4-oxalocrotonate tautomerase (4-OT) hexamer, the Trp repressor (TrpR) dimer, the Arc repressor (ArcR) dimer, and an ArcR mutant (the (DOA20)ArcR) dimer which contained two destabilizing mutations including an Asp to Ala mutation at position 20 and an amide to ester bond mutation between amino acid (aa) residues 19 and 20. As part of the work described here, we present a new method for the analysis of SUPREX data that is generally applicable to both monomeric and multimeric protein systems. Our results on the model proteins in this study indicate that this new method can be used to determine folding free energies for proteins with the accuracy and precision of conventional spectroscopybased methods.Keywords: H/D exchange; MALDI; thermodynamic stability; protein folding Conventional, spectroscopy-based methods for measuring the thermodynamic stability of proteins have the disadvantage that they require relatively large amounts of pure protein. This limits the thermodynamic analysis of proteins to those that can be purified in large quantities. We recently reported a new H/D exchange-and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based technique, termed SUPREX, that can be used to quantitate the thermodynamic stability of proteins (Ghaemmaghami et al. 2000). In contrast to conventional methods, the SUPREX technique can be used to quantitate the stability of mg to ng quantities of both purified and unpurified proteins (Powell and Fitzgerald 2001). In SUPREX, protein samples are subjected to H/D exchange by dilution into a series of deuterated exchange buffers containing different concentrations of a chemical denaturant such as guanidinium chloride (GdmCl). After a specified exchange time, the deuterium content of each protein sample is determined using MALDI mass spectrometry. Ultimately, the change in mass relative to the fully protonated sample is plotted as a function of [GdmCl] to generate a SUPREX curve.SUPREX curves can be used to extract accurate thermodynamic parameters for a protein's folding reaction provided that the protein's equilibrium unfolding behavior is