Jaime L. Stark

Application of NMR and molecular docking in structure-based drug discovery

Drug discovery is a complex and costly endeavor, where few drugs that reach the clinical testing phase make it to market. High-throughput screening (HTS) is the primary method used by the pharmaceutical industry to identify initial lead compounds. Unfortunately, HTS has a high failure rate and is not particularly efficient at identifying viable drug leads. These shortcomings have encouraged the development of alternative methods to drive the drug discovery process. Specifically, nuclear magnetic resonance (NMR) spectroscopy and molecular docking are routinely being employed as important components of drug discovery research. Molecular docking provides an extremely rapid way to evaluate likely binders from a large chemical library with minimal cost. NMR ligand-affinity screens can directly detect a protein-ligand interaction, can measure a corresponding dissociation constant, and can reliably identify the ligand binding site and generate a co-structure. Furthermore, NMR ligand affinity screens and molecular docking are perfectly complementary techniques, where the combination of the two has the potential to improve the efficiency and success rate of drug discovery. This review will highlight the use of NMR ligand affinity screens and molecular docking in drug discovery and describe recent examples where the two techniques were combined to identify new and effective therapeutic drugs.

Structure and Function of Human DnaJ Homologue Subfamily A Member 1 (DNAJA1) and Its Relationship to Pancreatic Cancer

Pancreatic cancer has a dismal 5 year survival rate of 5.5% that has not been improved over the past 25 years despite an enormous amount of effort. Thus, there is an urgent need to identify truly novel yet druggable protein targets for drug discovery. The human protein DnaJ homologue subfamily A member 1 (DNAJA1) was previously shown to be downregulated 5-fold in pancreatic cancer cells and has been targeted as a biomarker for pancreatic cancer, but little is known about the specific biological function for DNAJA1 or the other members of the DnaJ family encoded in the human genome. Our results suggest the overexpression of DNAJA1 suppresses the stress response capabilities of the oncogenic transcription factor, c-Jun, and results in the diminution of cell survival. DNAJA1 likely activates a DnaK protein by forming a complex that suppresses the JNK pathway, the hyperphosphorylation of c-Jun, and the anti-apoptosis state found in pancreatic cancer cells. A high-quality nuclear magnetic resonance solution structure of the J-domain of DNAJA1 combined with a bioinformatics analysis and a ligand affinity screen identifies a potential DnaK binding site, which is also predicted to overlap with an inhibitory binding site, suggesting DNAJA1 activity is highly regulated.

PROFESS: a PROtein Function, Evolution, Structure and Sequence database

The proliferation of biological databases and the easy access enabled by the Internet is having a beneficial impact on biological sciences and transforming the way research is conducted. There are ∼1100 molecular biology databases dispersed throughout the Internet. To assist in the functional, structural and evolutionary analysis of the abundant number of novel proteins continually identified from whole-genome sequencing, we introduce the PROFESS (PROtein Function, Evolution, Structure and Sequence) database. Our database is designed to be versatile and expandable and will not confine analysis to a pre-existing set of data relationships. A fundamental component of this approach is the development of an intuitive query system that incorporates a variety of similarity functions capable of generating data relationships not conceived during the creation of the database. The utility of PROFESS is demonstrated by the analysis of the structural drift of homologous proteins and the identification of potential pancreatic cancer therapeutic targets based on the observation of protein–protein interaction networks. Database URL: http://cse.unl.edu/∼profess/

Solution structure and function of YndB, an AHSA1 protein from Bacillus subtilis

The solution structure of the Bacillus subtilis protein YndB has been solved using NMR to investigate proposed biological functions. The YndB structure exhibits the helix‐grip fold, which consists of a β‐sheet with two small and one long α‐helix, forming a hydrophobic cavity that preferentially binds lipid‐like molecules. Sequence and structure comparisons with proteins from eukaryotes, prokaryotes, and archaea suggest that YndB is very similar to the eukaryote protein Aha1, which binds to the middle domain of Hsp90 and induces ATPase activity. On the basis of these similarities, YndB has been classified as a member of the activator of Hsp90 ATPase homolog 1‐like protein (AHSA1) family with a function that appears to be related to stress response. An in silico screen of a compound library of ∼18,500 lipids was used to identify classes of lipids that preferentially bind YndB. The in silico screen identified, in order of affinity, the chalcone/hydroxychalcone, flavanone, and flavone/flavonol classes of lipids, which was further verified by 2D 1H‐15N HSQC NMR titration experiments with trans‐chalcone, flavanone, flavone, and flavonol. All of these compounds are typically found in plants as precursors to various flavonoid antibiotics and signaling molecules. The sum of the data suggests an involvement of YndB with the stress response of B. subtilis to chalcone‐like flavonoids released by plants due to a pathogen infection. The observed binding of chalcone‐like molecules by YndB is likely related to thesymbiotic relationship between B. subtilis and plants. Proteins 2010.

NMRmix: A Tool for the Optimization of Compound Mixtures in 1D (1)H NMR Ligand Affinity Screens.

NMR ligand affinity screening is a powerful technique that is routinely used in drug discovery or functional genomics to directly detect protein–ligand binding events. Binding events can be identified by monitoring differences in the 1D 1H NMR spectrum of a compound with and without protein. Although a single NMR spectrum can be collected within a short period (2—10 min per sample), one-by-one screening of a protein against a library of hundreds or thousands of compounds requires a large amount of spectrometer time and a large quantity of protein. Therefore, compounds are usually evaluated in mixtures ranging in size from 3 to 20 compounds to improve the efficiency of these screens in both time and material. Ideally, the NMR signals from individual compounds in the mixture should not overlap so that spectral changes can be associated with a particular compound. We have developed a software tool, NMRmix, to assist in creating ideal mixtures from a large panel of compounds with known chemical shifts. Input to NMRmix consists of an 1H NMR peak list for each compound, a user-defined overlap threshold, and additional user-defined parameters if default settings are not used. NMRmix utilizes a simulated annealing algorithm to optimize the composition of the mixtures to minimize spectral peak overlaps so that each compound in the mixture is represented by a maximum number of nonoverlapping chemical shifts. A built-in graphical user interface simplifies data import and visual evaluation of the results.

Correlation between Protein Function and Ligand Binding Profiles

We report that proteins with the same function bind the same set of small molecules from a standardized chemical library. This observation led to a quantifiable and rapidly adaptable method for protein functional analysis using experimentally derived ligand binding profiles. Ligand binding is measured using a high-throughput NMR ligand affinity screen with a structurally diverse chemical library. The method was demonstrated using a set of 19 proteins with a range of functions. A statistically significant similarity in ligand binding profiles was only observed between the two functionally identical albumins and between the five functionally similar amylases. This new approach is independent of sequence, structure, or evolutionary information and, therefore, extends our ability to analyze and functionally annotate novel genes.

Identification of low-molecular-weight compounds inhibiting growth of corynebacteria: potential lead compounds for antibiotics.

The bacterial genus Corynebacteria contains several pathogenic species that cause diseases such as diphtheria in humans and “cheesy gland” in goats and sheep. Thus, identifying new therapeutic targets to treat Corynebacteria infections is both medically and economically important. CG2496, a functionally uncharacterized protein from Corynebacterium glutamicum, was evaluated using an NMR ligand‐affinity screen. A total of 11 compounds from a library of 460 biologically active compounds were shown to selectively bind CG2496 in a highly conserved region of the protein. The best binder was identified to be methiothepin (KD=54±19 µM), an FDA‐approved serotonin receptor antagonist. Methiothepin was also shown to inhibit the growth of C. glutamicum, but not bacteria that lack CG2496 homologs. Our results suggest that CG2496 is a novel therapeutic target and methiothepin is a potential lead compound or structural scaffold for developing new antibiotics specifically targeting Corynebacteria.

Conserved lipid and small molecule modulation of COQ8 reveals regulation of the ancient UbiB family

Human COQ8A (ADCK3) and Saccharomyces cerevisiae Coq8p (collectively COQ8) are UbiB family proteins essential for mitochondrial coenzyme Q (CoQ) biosynthesis. However, the biochemical activity of COQ8 and its direct role in CoQ production remain unclear, in part due to lack of known endogenous regulators of COQ8 function and of effective small molecules for probing its activity in vivo. Here we demonstrate that COQ8 possesses evolutionarily conserved ATPase activity that is activated by binding to membranes containing cardiolipin and by phenolic compounds that resemble CoQ pathway intermediates. We further create an analog-sensitive version of Coq8p and reveal that acute chemical inhibition of its endogenous activity in yeast is sufficient to cause respiratory deficiency concomitant with CoQ depletion. Collectively, this work defines lipid and small molecule modulators of an ancient family of atypical kinase-like proteins and establishes a chemical genetic system for further exploring the mechanistic role of COQ8 in CoQ biosynthesis.