Synthetic, Biochemical, X-ray Crystallographic, Computational and High-Throughput Screening Approaches Toward Anthrax Toxin Lethal Factor Inhibition

University of Minnesota Ph.D. dissertation.October 2015. Major: Medicinal Chemistry. Advisor: Elizabeth Amin. 1 computer file (PDF); xvi, 227 pages.The lethal factor (LF) enzyme secreted by Bacillus anthracis is chiefly responsible for anthrax-related cytotoxicity. In this dissertation, I present the computational design, synthesis, biochemical testing, structural biology, and virtual and high-throughput screening approaches to identify binding requirements for LF inhibition. To this end, we designed ~50 novel compounds to probe design principles and structural requirements for LF. Specifically, in Chapters 2 and 3, computational, synthetic, biochemical and structural biology methods to explore the underinvestigated LF S2′ binding subsite are described. We discovered that LF domain 3 is very flexible and results in a relatively unconstrained S2′ binding site region. Additionally, we found that the S1′ subsite can undergo a novel conformational change resulting in a previously unreported tunnel region, which we term S1′*, that we expect can further be explored to design potent and selective LF inhibitors. Using this novel LF configuration, we virtually screened ~11 million drug-like compounds for activity against LF and have identified a novel compound that inhibits LF with an IC50 of 126 μM. In the course of this work, we found that reliable representation of zinc and other transition metal centers in macromolecules is nontrivial, due to the complexity of the coordination environment and charge distribution at the catalytic center. In Chapter 7, I will present work on applying and optimizing quantum mechanical methods developed by the Truhlar group to accurately calculate bond dissociation energies at low computational cost for various representative Zn2+ and Cd2+ model systems. By analyzing errors, we developed a prescription for an optimal system fragmentation strategy for our models. With this scheme, we find that the EE-3B-CE method is able to reproduce 53 conventionally calculated bond energies with an average absolute error of only 0.59 kcal/mol. Therefore, one could use the EE 3B CE approximation to obtain accurate results for large systems and/or identify better parameters for Zn centers for use in virtual screening. Finally, we present the results of a large-scale in vitro HTS campaign of ~250,000 small-molecules against LF. After extensive validation, involving secondary assays and hit synthesis we were able to prioritize a key lead for further prosecution

The Power of Language and Speech in Movement: Speech Effects and its Classifications

This article examines the structural, semantic, and functional features of the speech effect phenomenon. It also elucidates the expression of speech effect through language and means of speech, its reflection in socially oriented and mass media, and its impact on the addressee

Electrostatically Embedded Many-Body Expansion for Neutral and Charged Metalloenzyme Model Systems

The electrostatically embedded many-body (EE-MB) method has proven accurate for calculating cohesive and conformational energies in clusters, and it has recently been extended to obtain bond dissociation energies for metal–ligand bonds in positively charged inorganic coordination complexes. In the present paper, we present four key guidelines that maximize the accuracy and efficiency of EE-MB calculations for metal centers. Then, following these guidelines, we show that the EE-MB method can also perform well for bond dissociation energies in a variety of neutral and negatively charged inorganic coordination systems representing metalloenzyme active sites, including a model of the catalytic site of the zinc-bearing anthrax toxin lethal factor, a popular target for drug development. In particular, we find that the electrostatically embedded three-body (EE-3B) method is able to reproduce conventionally calculated bond-breaking energies in a series of pentacoordinate and hexacoordinate zinc-containing systems with an average absolute error (averaged over 25 cases) of only 0.98 kcal/mol

Analysis of the Errors in the Electrostatically Embedded Many-Body Expansion of the Energy and the Correlation Energy for Zn and Cd Coordination Complexes with Five and Six Ligands and Use of the Analysis to Develop a Generally Successful Fragmentation Strategy

In the present paper, we apply the electrostatically embedded many-body expansion of the correlation energy (EE-MB-CE) to the calculation of zinc–ligand and cadmium–ligand bond dissociation energies, and we analyze the errors due to various fragmentation schemes in a variety of neutral, positively charged, and negatively charged Zn²⁺ and Cd²⁺ coordination complexes. As a result of the analysis, we are able to present a new, simple, and unambiguous fragmentation strategy. Following this strategy, we show that both methods perform well for zinc–ligand and cadmium–ligand bond dissociation energies for all systems studied in the paper, including a model of the catalytic site of the zinc-bearing anthrax toxin lethal factor (LF), which has garnered substantial attention as a target for drug development. To draw general conclusions, we consider ten pentacoordinate and hexacoordinate zinc and cadmium containing coordination complexes, each with 10 or 15 different fragmentation schemes. By analyzing errors, we developed a prescription for the optimal fragmentation strategy. With this scheme, and using MP2 correlation energies as a test, we find that the electrostatically embedded three-body expansion of the correlation energy (EE-3B-CE) method is able to reproduce all 53 conventionally calculated bond energies with an average absolute error of only 0.59 kcal/mol. The paper also presents EE-MB-CE calculations using the CCSD­(T) level of theory on an LF model system. With CCSD­(T), EE-3B-CE has an average error of 0.30 kcal/mol

Catch and Anchor Approach To Combat Both Toxicity and Longevity of Botulinum Toxin A

Botulinum neurotoxins have remarkable persistence (∼weeks to months in cells), outlasting the small-molecule inhibitors designed to target them. To address this disconnect, inhibitors bearing two pharmacophores-a zinc binding group and a Cys-reactive warhead-were designed to leverage both affinity and reactivity. A series of first-generation bifunctional inhibitors was achieved through structure-based inhibitor design. Through X-ray crystallography, engagement of both the catalytic Zn2+ and Cys165 was confirmed. A second-generation series improved on affinity by incorporating known reversible inhibitor pharmacophores; the mechanism was confirmed by exhaustive dialysis, mass spectrometry, and in vitro evaluation against the C165S mutant. Finally, a third-generation inhibitor was shown to have good cellular activity and low toxicity. In addition to our findings, an alternative method of modeling time-dependent inhibition that simplifies assay setup and allows comparison of inhibition models is discussed

Probing the S2′ Subsite of the Anthrax Toxin Lethal Factor Using Novel N‑Alkylated Hydroxamates

The lethal factor (LF) enzyme secreted by <i>Bacillus anthracis</i> is a zinc hydrolase that is chiefly responsible for anthrax-related cell death. Although many studies of the design of small molecule LF inhibitors have been conducted, no LF inhibitor is yet available as a therapeutic agent. Inhibitors with considerable chemical diversity have been developed and investigated; however, the LF S2′ subsite has not yet been systematically explored as a potential target for lead optimization. Here we present synthesis, experimental evaluation, modeling, and structural biology for a novel series of sulfonamide hydroxamate LF inhibitor analogues specifically designed to extend into, and probe chemical preferences of, this S2′ subsite. We discovered that this region accommodates a wide variety of chemical functionalities and that a broad selection of ligand structural modifications directed to this area can be incorporated without significant deleterious alterations in biological activity. We also identified key residues in this subsite that can potentially be targeted to improve inhibitor binding