Azurin as a Protein Scaffold for a Low-coordinate Nonheme Iron Site with a Small-molecule Binding Pocket

AbstractOver one billion people are currently infected with a parasitic nematode. Symptoms can include anemia, malnutrition, developmental delay, and in severe cases, death. Resistance is emerging to the anthelmintics currently used to treat nematode infection, prompting the need to develop new anthelmintics. Towards this end, we identified a set of kinases that may be targeted in a nematode-selective manner. We first screened 2040 inhibitors of vertebrate kinases for those that impair the model nematode Caenorhabditis elegans. By determining whether the terminal phenotype induced by each kinase inhibitor matched that of the predicted target mutant in C. elegans, we identified 17 druggable nematode kinase targets. Of these, we found that nematode EGFR, MEK1, and PLK1 kinases have diverged from vertebrates within their drug-binding pocket. For each of these targets, we identified small molecule scaffolds that may be further modified to develop nematode-selective inhibitors. Nematode EGFR, MEK1, and PLK1 therefore represent key targets for the development of new anthelmintic medicines.

Download Full-text

Ligands with poly-fluorophenyl moieties promote a local structural rearrangement in the Spinach2 and Broccoli aptamers that increases ligand affinities

10.1101/2021.10.05.463302 ◽

2021 ◽

Author(s):

Sharif Anisuzzaman ◽

Ivan M Geraskin ◽

Muslum Ilgu ◽

Lee Bendickson ◽

George A Kraus ◽

...

Keyword(s):

Nucleic Acids ◽

Ligand Binding ◽

Small Molecule ◽

Binding Pocket ◽

Structural Rearrangement ◽

Structural Reorganization ◽

Ligand Binding Pocket ◽

Rna Remodeling ◽

Small Molecule Ligands ◽

Target Molecules

The interaction of nucleic acids with their molecular targets often involves structural reorganization that may traverse a complex folding landscape. With the more recent recognition that many RNAs, both coding and noncoding, may regulate cellular activities by interacting with target molecules, it becomes increasingly important to understand the means by which nucleic acids interact with their targets and how drugs might be developed that can influence critical folding transitions. We have extensively investigated the interaction of the Spinach2 and Broccoli aptamers with a library of small molecule ligands modified by various extensions from the imido nitrogen of DFHBI (3,5-difluoro-4-hydroxybenzylidene imidazolinone) that reach out from the Spinach2 ligand binding pocket. Studies of the interaction of these compounds with the aptamers revealed that poly-fluorophenyl-modified ligands initiate a slow change in aptamer affinity that takes an extended time (half-life of ~40 min) to achieve. The change in affinity appears to involve an initial disruption of the entrance to the ligand binding pocket followed by a gradual lockdown for which the most likely driving force is an interaction of the gateway adenine with a nearby 2'OH group. These results suggest that poly-fluorophenyl modifications might increase the ability of small molecule drugs to disrupt local structure and promote RNA remodeling.

Download Full-text

Solution NMR readily reveals distinct structural folds and interactions in doubly 13C- and 19F-labeled RNAs

Science Advances ◽

10.1126/sciadv.abc6572 ◽

2020 ◽

Vol 6 (41) ◽

pp. eabc6572

Author(s):

Owen B. Becette ◽

Guanghui Zong ◽

Bin Chen ◽

Kehinde M. Taiwo ◽

David A. Case ◽

...

Keyword(s):

Small Molecule ◽

High Sensitivity ◽

Binding Pocket ◽

In Vitro Transcription ◽

Rapid Identification ◽

Spin Pair ◽

Encapsidation Signal ◽

Spin Pairs ◽

Critical Components

RNAs form critical components of biological processes implicated in human diseases, making them attractive for small-molecule therapeutics. Expanding the sites accessible to nuclear magnetic resonance (NMR) spectroscopy will provide atomic-level insights into RNA interactions. Here, we present an efficient strategy to introduce 19F-13C spin pairs into RNA by using a 5-fluorouridine-5′-triphosphate and T7 RNA polymerase–based in vitro transcription. Incorporating the 19F-13C label in two model RNAs produces linewidths that are twice as sharp as the commonly used 1H-13C spin pair. Furthermore, the high sensitivity of the 19F nucleus allows for clear delineation of helical and nonhelical regions as well as GU wobble and Watson-Crick base pairs. Last, the 19F-13C label enables rapid identification of a small-molecule binding pocket within human hepatitis B virus encapsidation signal epsilon (hHBV ε) RNA. We anticipate that the methods described herein will expand the size limitations of RNA NMR and aid with RNA-drug discovery efforts.

Download Full-text

An allosteric site on MKP5 reveals a strategy for small-molecule inhibition

Science Signaling ◽

10.1126/scisignal.aba3043 ◽

2020 ◽

Vol 13 (646) ◽

pp. eaba3043 ◽

Cited By ~ 1

Author(s):

Zachary T. K. Gannam ◽

Kisuk Min ◽

Shanelle R. Shillingford ◽

Lei Zhang ◽

James Herrington ◽

...

Keyword(s):

Small Molecule ◽

Binding Pocket ◽

Muscle Disease ◽

Mitogen Activated Protein Kinase ◽

Allosteric Site ◽

Dystrophic Muscle ◽

Smad2 Phosphorylation ◽

Mitogen Activated Protein ◽

P38α Mapk ◽

Tgf Β1

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) have been considered “undruggable,” but their position as regulators of the MAPKs makes them promising therapeutic targets. MKP5 has been suggested as a potential target for the treatment of dystrophic muscle disease. Here, we identified an inhibitor of MKP5 using a p38α MAPK–derived, phosphopeptide-based small-molecule screen. We solved the structure of MKP5 in complex with this inhibitor, which revealed a previously undescribed allosteric binding pocket. Binding of the inhibitor to this pocket collapsed the MKP5 active site and was predicted to limit MAPK binding. Treatment with the inhibitor recapitulated the phenotype of MKP5 deficiency, resulting in activation of p38 MAPK and JNK. We demonstrated that MKP5 was required for TGF-β1 signaling in muscle and that the inhibitor blocked TGF-β1–mediated Smad2 phosphorylation. TGF-β1 pathway antagonism has been proposed for the treatment of dystrophic muscle disease. Thus, allosteric inhibition of MKP5 represents a therapeutic strategy against dystrophic muscle disease.

Download Full-text

Fragment-based drug design of nature-inspired compounds

Physical Sciences Reviews ◽

10.1515/psr-2018-0110 ◽

2019 ◽

Vol 4 (9) ◽

Cited By ~ 1

Author(s):

Abdulkarim Najjar ◽

Abdurrahman Olğaç ◽

Fidele Ntie-Kang ◽

Wolfgang Sippl

Keyword(s):

Drug Design ◽

Small Molecules ◽

Small Molecule ◽

Binding Pocket ◽

Biological Macromolecules ◽

Drug Candidates ◽

Fragment Screening ◽

Small Molecule Drugs ◽

Lead Generation ◽

Hit Identification

Abstract Natural product (NP)-derived drugs can be extracts, biological macromolecules, or purified small molecule substances. Small molecule drugs can be originally purified from NPs, can represent semisynthetic molecules, natural fragments containing small molecules, or are fully synthetic molecules that mimic natural compounds. New semisynthetic NP-like drugs are entering the pharmaceutical market almost every year and reveal growing interests in the application of fragment-based approaches for NPs. Thus, several NP databases were constructed to be implemented in the fragment-based drug design (FBDD) workflows. FBDD has been established previously as an approach for hit identification and lead generation. Several biophysical and computational methods are used for fragment screening to identify potential hits. Once the fragments within the binding pocket of the protein are identified, they can be grown, linked, or merged to design more active compounds. This work discusses applications of NPs and NP scaffolds to FBDD. Moreover, it briefly reviews NP databases containing fragments and reports on case studies where the approach has been successfully applied for the design of antimalarial and anticancer drug candidates.

Download Full-text

Biochemical and Genetic Characterizations of a Novel Human Immunodeficiency Virus Type 1 Inhibitor That Blocks gp120-CD4 Interactions

Journal of Virology ◽

10.1128/jvi.77.19.10528-10536.2003 ◽

2003 ◽

Vol 77 (19) ◽

pp. 10528-10536 ◽

Cited By ~ 112

Author(s):

Qi Guo ◽

Hsu-Tso Ho ◽

Ira Dicker ◽

Li Fan ◽

Nannan Zhou ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Small Molecule ◽

Viral Entry ◽

Binding Pocket ◽

Gp120 Protein ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT BMS-378806 is a recently discovered small-molecule human immunodeficiency virus type 1 (HIV-1) attachment inhibitor with good antiviral activity and pharmacokinetic properties. Here, we demonstrate that the compound targets viral entry by inhibiting the binding of the HIV-1 envelope gp120 protein to cellular CD4 receptors via a specific and competitive mechanism. BMS-378806 binds directly to gp120 at a stoichiometry of approximately 1:1, with a binding affinity similar to that of soluble CD4. The potential BMS-378806 target site was localized to a specific region within the CD4 binding pocket of gp120 by using HIV-1 gp120 variants carrying either compound-selected resistant substitutions or gp120-CD4 contact site mutations. Mapping of resistance substitutions to the HIV-1 envelope, and the lack of compound activity against a CD4-independent viral infection confirm the gp120-CD4 interactions as the target in infected cells. BMS-378806 therefore serves as a prototype for this new class of antiretroviral agents and validates gp120 as a viable target for small-molecule inhibitors.

Download Full-text

Characterization of the Raf Kinase Inhibitory Protein (RKIP) Binding Pocket: NMR-Based Screening Identifies Small-Molecule Ligands

PLoS ONE ◽

10.1371/journal.pone.0010479 ◽

2010 ◽

Vol 5 (5) ◽

pp. e10479 ◽

Cited By ~ 21

Author(s):

Anne N. Shemon ◽

Gary L. Heil ◽

Alexey E. Granovsky ◽

Mathew M. Clark ◽

Dan McElheny ◽

...

Keyword(s):

Small Molecule ◽

Binding Pocket ◽

Inhibitory Protein ◽

Raf Kinase ◽

Raf Kinase Inhibitory Protein ◽

Small Molecule Ligands

Download Full-text

Graph Neural Networks Bootstrapped for Synthetic Selection and Validation of Small Molecule Immunomodulators

10.33774/chemrxiv-2021-r4xnx-v2 ◽

2021 ◽

Author(s):

Prageeth R. Wijewardhane ◽

Krupal P. Jethava ◽

Jonathan A Fine ◽

Gaurav Chopra

Keyword(s):

Neural Network ◽

Machine Learning ◽

Small Molecule ◽

Model Performance ◽

Cost Effective ◽

Bioactive Compound ◽

Binding Pocket ◽

Chemical Diversity ◽

Training Data ◽

Kappa Score

The Programmed Cell Death Protein 1/Programmed Death-Ligand 1 (PD-1/PD-L1) interaction is an immune checkpoint utilized by cancer cells to enhance immune suppression. There is a huge need to develop small molecule drugs that are fast acting, cost effective, and readily bioavailable compared to antibodies. Unfortunately, synthesizing and validating large libraries of small- molecules to inhibit PD-1/PD-L1 interaction in a blind manner is both time-consuming and expensive. To improve this drug discovery pipeline, we have developed a machine learning methodology trained on patent data to identify, synthesize, and validate PD-1/PD-L1 small molecule inhibitors. Our model incorporates two features: docking scores to represent the energy of binding (E) as a global feature and sub-graph features through a graph neural network (GNN) of molecular topology to represent local features. This interaction energy-based Graph Neural Network (EGNN) model outperforms traditional machine learning methods and a simple GNN with a F1 score of 0.9524 and Cohen’s kappa score of 0.8861 for the hold out test set, suggesting that the topology of the small molecule, the structural interaction in the binding pocket, and chemical diversity of the training data are all important considerations for enhancing model performance. A Bootstrapped EGNN model was used to select compounds for synthesis and experimental validation with predicted high and low potency to inhibit PD-1/PD-L1 interaction. The potent inhibitor, (4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2- methylbenzyl)oxy)-2,6-dimethoxybenzyl)-D-serine, is a hybrid of two known bioactive scaffolds, with an IC50 of 339.9 nM that is comparatively better than the known bioactive compound. We conclude that our bootstrapped EGNN model will be useful to identify target-specific high potency molecules designed by scaffold hopping, a well-known medicinal chemistry technique.

Download Full-text

Compensatory Substitutions in the HIV-1 Capsid Reduce the Fitness Cost Associated with Resistance to a Capsid-Targeting Small-Molecule Inhibitor

Journal of Virology ◽

10.1128/jvi.01411-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 208-219 ◽

Cited By ~ 18

Author(s):

Jiong Shi ◽

Jing Zhou ◽

Upul D. Halambage ◽

Vaibhav B. Shah ◽

Mallori J. Burse ◽

...

Keyword(s):

Amino Acid ◽

Small Molecule ◽

Therapeutic Target ◽

Binding Pocket ◽

Host Protein ◽

Viral Capsid ◽

Inhibitory Protein ◽

Hiv 1 ◽

Ca Protein

ABSTRACTThe HIV-1 capsid plays multiple roles in infection and is an emerging therapeutic target. The small-molecule HIV-1 inhibitor PF-3450074 (PF74) blocks HIV-1 at an early postentry stage by binding the viral capsid and interfering with its function. Selection for resistance resulted in accumulation of five amino acid changes in the viral CA protein, which collectively reduced binding of the compound to HIV-1 particles. In the present study, we dissected the individual and combinatorial contributions of each of the five substitutions Q67H, K70R, H87P, T107N, and L111I to PF74 resistance, PF74 binding, and HIV-1 infectivity. Q67H, K70R, and T107N each conferred low-level resistance to PF74 and collectively conferred strong resistance. The substitutions K70R and L111I impaired HIV-1 infectivity, which was partially restored by the other substitutions at positions 67 and 107. PF74 binding to HIV-1 particles was reduced by the Q67H, K70R, and T107N substitutions, consistent with the location of these positions in the inhibitor-binding pocket. Replication of the 5Mut virus was markedly impaired in cultured macrophages, reminiscent of the previously reported N74D CA mutant. 5Mut substitutions also reduced the binding of the host protein CPSF6 to assembled CA complexesin vitroand permitted infection of cells expressing the inhibitory protein CPSF6-358. Our results demonstrate that strong resistance to PF74 requires accumulation of multiple substitutions in CA to inhibit PF74 binding and compensate for fitness impairments associated with some of the sequence changes.IMPORTANCEThe HIV-1 capsid is an emerging drug target, and several small-molecule compounds have been reported to inhibit HIV-1 infection by targeting the capsid. Here we show that resistance to the capsid-targeting inhibitor PF74 requires multiple amino acid substitutions in the binding pocket of the CA protein. Three changes in CA were necessary to inhibit binding of PF74 while maintaining viral infectivity. Replication of the PF74-resistant HIV-1 mutant was impaired in macrophages, likely owing to altered interactions with host cell factors. Our results suggest that HIV-1 resistance to capsid-targeting inhibitors will be limited by functional constraints on the viral capsid protein. Therefore, this work enhances the attractiveness of the HIV-1 capsid as a therapeutic target.

Download Full-text