Tower 1978

1978 yearbook of Westbrook College in Portland, Maine.https://dune.une.edu/wchc_yearbooks/1007/thumbnail.jp

Inhibition of CDC25B Phosphatase Through Disruption of Protein–Protein Interaction

CDC25 phosphatases are key cell cycle regulators and represent very attractive but challenging targets for anticancer drug discovery. Here, we explored whether fragment-based screening represents a valid approach to identify inhibitors of CDC25B. This resulted in identification of 2-fluoro-4-hydroxybenzonitrile, which directly binds to the catalytic domain of CDC25B. Interestingly, NMR data and the crystal structure demonstrate that this compound binds to the pocket distant from the active site and adjacent to the protein–protein interaction interface with CDK2/Cyclin A substrate. Furthermore, we developed a more potent analogue that disrupts CDC25B interaction with CDK2/Cyclin A and inhibits dephosphorylation of CDK2. Based on these studies, we provide a proof of concept that targeting CDC25 phosphatases by inhibiting their protein–protein interactions with CDK2/Cyclin A substrate represents a novel, viable opportunity to target this important class of enzymes

Two Loops Undergoing Concerted Dynamics Regulate the Activity of the ASH1L Histone Methyltransferase

ASH1L (absent, small, or homeotic-like 1) is a histone methyltransferase (HMTase) involved in gene activation that is overexpressed in multiple forms of cancer. Previous studies of ASH1L’s catalytic SET domain identified an autoinhibitory loop that blocks access of histone substrate to the enzyme active site. Here, we used both nuclear magnetic resonance and X-ray crystallography to identify conformational dynamics in the ASH1L autoinhibitory loop. Using site-directed mutagenesis, we found that point mutations in the autoinhibitory loop that perturb the structure of the SET domain result in decreased enzyme activity, indicating that the autoinhibitory loop is not a simple gate to the active site but is rather a key feature critical to ASH1L function. We also identified a second loop in the SET-I subdomain of ASH1L that experiences conformational dynamics, and we trapped two different conformations of this loop using crystallographic studies. Mutation of the SET-I loop led to a large decrease in ASH1L enzymatic activity in addition to a significant conformational change in the SET-I loop, demonstrating the importance of the structure and dynamics of the SET-I loop to ASH1L function. Furthermore, we found that three C-terminal chromatin-interacting domains greatly enhance ASH1L enzymatic activity and that ASH1L requires native nucleosome substrate for robust activity. Our study illuminates the role of concerted conformational dynamics in ASH1L function and identifies structural features important for ASH1L enzymatic activity

Profiling the Dynamic Interfaces of Fluorinated Transcription Complexes for Ligand Discovery and Characterization

The conformationally dynamic binding surfaces of transcription complexes present a particular challenge for ligand discovery and characterization. In the case of the KIX domain of the master coactivator CBP/p300, few small molecules have been reported that target its two allosterically regulated binding sites despite the important roles that KIX plays in processes ranging from memory formation to hematopoiesis. Taking advantage of the enrichment of aromatic amino acids at protein interfaces, here we show that the incorporation of six ¹⁹F-labeled aromatic side chains within the KIX domain enables recapitulation of the differential binding footprints of three natural activator peptides (MLL, c-Myb, and pKID) in complex with KIX and effectively reports on allosteric changes upon binding using 1D NMR spectroscopy. Additionally, the examination of both the previously described KIX protein–protein interaction inhibitor Napthol-ASE-phosphate and newly discovered ligand 1-10 rapidly revealed both the binding sites and the affinities of these small molecules. Significantly, the utility of using fluorinated transcription factors for ligand discovery was demonstrated through a fragment screen leading to a new low molecular weight fragment ligand for CBP/p300, 1G7. Aromatic amino acids are enriched at protein–biomolecule interfaces; therefore, this quantitative and facile approach will be broadly useful for studying dynamic transcription complexes and screening campaigns complementing existing biophysical methods for studying these dynamic interfaces

Rational Design of Orthogonal Multipolar Interactions with Fluorine in Protein–Ligand Complexes

Multipolar interactions involving fluorine and the protein backbone have been frequently observed in protein–ligand complexes. Such fluorine–backbone interactions may substantially contribute to the high affinity of small molecule inhibitors. Here we found that introduction of trifluoromethyl groups into two different sites in the thienopyrimidine class of menin–MLL inhibitors considerably improved their inhibitory activity. In both cases, trifluoromethyl groups are engaged in short interactions with the backbone of menin. In order to understand the effect of fluorine, we synthesized a series of analogues by systematically changing the number of fluorine atoms, and we determined high-resolution crystal structures of the complexes with menin. We found that introduction of fluorine at favorable geometry for interactions with backbone carbonyls may improve the activity of menin–MLL inhibitors as much as 5- to 10-fold. In order to facilitate the design of multipolar fluorine–backbone interactions in protein–ligand complexes, we developed a computational algorithm named FMAP, which calculates fluorophilic sites in proximity to the protein backbone. We demonstrated that FMAP could be used to rationalize improvement in the activity of known protein inhibitors upon introduction of fluorine. Furthermore, FMAP may also represent a valuable tool for designing new fluorine substitutions and support ligand optimization in drug discovery projects. Analysis of the menin–MLL inhibitor complexes revealed that the backbone in secondary structures is particularly accessible to the interactions with fluorine. Considering that secondary structure elements are frequently exposed at protein interfaces, we postulate that multipolar fluorine–backbone interactions may represent a particularly attractive approach to improve inhibitors of protein–protein interactions

Rational Design of Orthogonal Multipolar Interactions with Fluorine in Protein–Ligand Complexes

Multipolar interactions involving fluorine and the protein backbone have been frequently observed in protein–ligand complexes. Such fluorine–backbone interactions may substantially contribute to the high affinity of small molecule inhibitors. Here we found that introduction of trifluoromethyl groups into two different sites in the thienopyrimidine class of menin–MLL inhibitors considerably improved their inhibitory activity. In both cases, trifluoromethyl groups are engaged in short interactions with the backbone of menin. In order to understand the effect of fluorine, we synthesized a series of analogues by systematically changing the number of fluorine atoms, and we determined high-resolution crystal structures of the complexes with menin. We found that introduction of fluorine at favorable geometry for interactions with backbone carbonyls may improve the activity of menin–MLL inhibitors as much as 5- to 10-fold. In order to facilitate the design of multipolar fluorine–backbone interactions in protein–ligand complexes, we developed a computational algorithm named FMAP, which calculates fluorophilic sites in proximity to the protein backbone. We demonstrated that FMAP could be used to rationalize improvement in the activity of known protein inhibitors upon introduction of fluorine. Furthermore, FMAP may also represent a valuable tool for designing new fluorine substitutions and support ligand optimization in drug discovery projects. Analysis of the menin–MLL inhibitor complexes revealed that the backbone in secondary structures is particularly accessible to the interactions with fluorine. Considering that secondary structure elements are frequently exposed at protein interfaces, we postulate that multipolar fluorine–backbone interactions may represent a particularly attractive approach to improve inhibitors of protein–protein interactions

An Evolution-Based Approach to <i>De Novo</i> Protein Design and Case Study on <i>Mycobacterium tuberculosis</i>

<div><p>Computational protein design is a reverse procedure of protein folding and structure prediction, where constructing structures from evolutionarily related proteins has been demonstrated to be the most reliable method for protein 3-dimensional structure prediction. Following this spirit, we developed a novel method to design new protein sequences based on evolutionarily related protein families. For a given target structure, a set of proteins having similar fold are identified from the PDB library by structural alignments. A structural profile is then constructed from the protein templates and used to guide the conformational search of amino acid sequence space, where physicochemical packing is accommodated by single-sequence based solvation, torsion angle, and secondary structure predictions. The method was tested on a computational folding experiment based on a large set of 87 protein structures covering different fold classes, which showed that the evolution-based design significantly enhances the foldability and biological functionality of the designed sequences compared to the traditional physics-based force field methods. Without using homologous proteins, the designed sequences can be folded with an average root-mean-square-deviation of 2.1 Å to the target. As a case study, the method is extended to redesign all 243 structurally resolved proteins in the pathogenic bacteria <i>Mycobacterium tuberculosis</i>, which is the second leading cause of death from infectious disease. On a smaller scale, five sequences were randomly selected from the design pool and subjected to experimental validation. The results showed that all the designed proteins are soluble with distinct secondary structure and three have well ordered tertiary structure, as demonstrated by circular dichroism and NMR spectroscopy. Together, these results demonstrate a new avenue in computational protein design that uses knowledge of evolutionary conservation from protein structural families to engineer new protein molecules of improved fold stability and biological functionality.</p></div

Evaluation of designed sequences.

<p>Data is averaged over 87 test proteins. The details on each protein can be found at <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003298#pcbi.1003298.s007" target="_blank">Table S2</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003298#pcbi.1003298.s008" target="_blank">S3</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003298#pcbi.1003298.s009" target="_blank">S4</a>.</p>a<p>TM-score between the first I-TASSER model and the target scaffold.</p>b<p>RMSD between the first I-TASSER model and the target scaffold.</p>c<p>SS: Secondary structure.</p>d<p>SA: Solvent accessibility.</p>e<p>PBM: Physics-based method using FoldX.</p>f<p>EvBM: Evolution-based method using only evolutionary terms in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003298#pcbi.1003298.e004" target="_blank">Eq. (1)</a>.</p>g<p>EBM: Evolutionary based method using both evolutionary and physics-based terms in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003298#pcbi.1003298.e006" target="_blank">Eq. (3)</a>.</p

Average RMSD between the scaffold structures and I-TASSER models on the proteins designed by PBM and EBM.

<p>The dataset is divided by the TM-score cutoff of the template proteins that were used for constructing the sequence profiles.</p

Average fractional difference in amino acid composition between the target and designed sequences.

<p>(A) EBM; (B) PBM.</p