Development of an improved inhibitor of Lats kinases to promote regeneration of mammalian organs.

The Hippo signaling pathway acts as a brake on regeneration in many tissues. This cascade of kinases culminates in the phosphorylation of the transcriptional cofactors Yap and Taz, whose concentration in the nucleus consequently remains low. Various types of cellular signals can reduce phosphorylation, however, resulting in the accumulation of Yap and Taz in the nucleus and subsequently in mitosis. We earlier identified a small molecule, TRULI, that blocks the final kinases in the pathway, Lats1 and Lats2, and thus elicits proliferation of several cell types that are ordinarily postmitotic and aids regeneration in mammals. In the present study, we present the results of chemical modification of the original compound and demonstrate that a derivative, TDI-011536, is an effective blocker of Lats kinases in vitro at nanomolar concentrations. The compound fosters extensive proliferation in retinal organoids derived from human induced pluripotent stem cells. Intraperitoneal administration of the substance to mice suppresses Yap phosphorylation for several hours and induces transcriptional activation of Yap target genes in the heart, liver, and skin. Moreover, the compound initiates the proliferation of cardiomyocytes in adult mice following cardiac cryolesions. After further chemical refinement, related compounds might prove useful in protective and regenerative therapies.

Using a Function-First "Scout Fragment"-Based Approach to Develop Allosteric Covalent Inhibitors of Conformationally Dynamic Helicase Mechanoenzymes.

Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as translation, ribosome assembly, and genome maintenance. Helicases with essential functions in certain cancer cells have been identified, and helicases expressed by many viruses are required for their pathogenicity. Therefore, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop chemical inhibitors for helicases, enzymes with high conformational dynamics. We envisioned that electrophilic "scout fragments", which have been used in chemical proteomic studies, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest an approach to discover covalent inhibitor starting points and druggable allosteric sites in conformationally dynamic mechanoenzymes.

Enhancing Hit Discovery in Virtual Screening through Absolute Protein-Ligand Binding Free-Energy Calculations.

In the hit identification stage of drug discovery, a diverse chemical space needs to be explored to identify initial hits. Contrary to empirical scoring functions, absolute protein-ligand binding free-energy perturbation (ABFEP) provides a theoretically more rigorous and accurate description of protein-ligand binding thermodynamics and could, in principle, greatly improve the hit rates in virtual screening. In this work, we describe an implementation of an accurate and reliable ABFEP method in FEP+. We validated the ABFEP method on eight congeneric compound series binding to eight protein receptors including both neutral and charged ligands. For ligands with net charges, the alchemical ion approach is adopted to avoid artifacts in electrostatic potential energy calculations. The calculated binding free energies correlate with experimental results with a weighted average of R2 = 0.55 for the entire dataset. We also observe an overall root-mean-square error (RMSE) of 1.1 kcal/mol after shifting the zero-point of the simulation data to match the average experimental values. Through ABFEP calculations using apo versus holo protein structures, we demonstrated that the protein conformational and protonation state changes between the apo and holo proteins are the main physical factors contributing to the protein reorganization free energy manifested by the overestimation of raw ABFEP calculated binding free energies using the holo structures of the proteins. Furthermore, we performed ABFEP calculations in three virtual screening applications for hit enrichment. ABFEP greatly improves the hit rates as compared to docking scores or other methods like metadynamics. The good performance of ABFEP in rank ordering compounds demonstrated in this work confirms it as a useful tool to improve the hit rates in virtual screening, thus facilitating hit discovery.

Scaffold Hopping and Optimization of Small Molecule Soluble Adenyl Cyclase Inhibitors Led by Free Energy Perturbation.

Free energy perturbation is a computational technique that can be used to predict how small changes to an inhibitor structure will affect the binding free energy to its target. In this paper, we describe the utility of free energy perturbation with FEP+ in the hit-to-lead stage of a drug discovery project targeting soluble adenyl cyclase. The project was structurally enabled by X-ray crystallography throughout. We employed free energy perturbation to first scaffold hop to a preferable chemotype and then optimize the binding affinity to sub-nanomolar levels while retaining druglike properties. The results illustrate that effective use of free energy perturbation can enable a drug discovery campaign to progress rapidly from hit to lead, facilitating proof-of-concept studies that enable target validation.

International Union of Basic and Clinical Pharmacology. CVII. Structure and Pharmacology of the Apelin Receptor with a Recommendation that Elabela/Toddler Is a Second Endogenous Peptide Ligand.

The predicted protein encoded by the APJ gene discovered in 1993 was originally classified as a class A G protein-coupled orphan receptor but was subsequently paired with a novel peptide ligand, apelin-36 in 1998. Substantial research identified a family of shorter peptides activating the apelin receptor, including apelin-17, apelin-13, and [Pyr1]apelin-13, with the latter peptide predominating in human plasma and cardiovascular system. A range of pharmacological tools have been developed, including radiolabeled ligands, analogs with improved plasma stability, peptides, and small molecules including biased agonists and antagonists, leading to the recommendation that the APJ gene be renamed APLNR and encode the apelin receptor protein. Recently, a second endogenous ligand has been identified and called Elabela/Toddler, a 54-amino acid peptide originally identified in the genomes of fish and humans but misclassified as noncoding. This precursor is also able to be cleaved to shorter sequences (32, 21, and 11 amino acids), and all are able to activate the apelin receptor and are blocked by apelin receptor antagonists. This review summarizes the pharmacology of these ligands and the apelin receptor, highlights the emerging physiologic and pathophysiological roles in a number of diseases, and recommends that Elabela/Toddler is a second endogenous peptide ligand of the apelin receptor protein.

Optimization of lead compounds into on-demand, nonhormonal contraceptives: leveraging a public-private drug discovery institute collaboration†.

Efforts to develop new male or female nonhormonal, orally available contraceptives assume that to be effective and safe, targets must be (1) essential for fertility; (2) amenable to targeting by small-molecule inhibitors; and (3) restricted to the germline. In this perspective, we question the third assumption and propose that despite its wide expression, soluble adenylyl cyclase (sAC: ADCY10), which is essential for male fertility, is a valid target. We hypothesize that an acute-acting sAC inhibitor may provide orally available, on-demand, nonhormonal contraception for men without adverse, mechanism-based effects. To test this concept, we describe a collaboration between academia and the unique capabilities of a public-private drug discovery institute.

Identification of Optimal Ligand Growth Vectors Using an Alchemical Free-Energy Method.

In this work, a novel method to rationally design inhibitors with improved steric contacts and enhanced binding free energies is presented. This new method uses alchemical single step perturbation calculations to rapidly optimize the van der Waals interactions of a small molecule in a protein-ligand complex in order to maximize its binding affinity. The results of the optimizer are used to predict beneficial growth vectors on the ligand, and good agreement is found between the predictions from the optimizer and a more rigorous free energy calculation, with a Spearman's rank order correlation of 0.59. The advantage of the method presented here is the significant speed up of over 10-fold compared to traditional free energy calculations and sublinear scaling with the number of growth vectors assessed. Where experimental data were available, mutations from hydrogen to a methyl group at sites highlighted by the optimizer were calculated with MBAR, and the mean unsigned error between experimental and calculated values of the binding free energy was 0.83 kcal/mol.

Inhibition of 3-phosphoglycerate dehydrogenase (PHGDH) by indole amides abrogates de novo serine synthesis in cancer cells.

Cancer cells reprogram their metabolism to support growth and to mitigate cellular stressors. The serine synthesis pathway has been identified as a metabolic pathway frequently altered in cancers and there has been considerable interest in developing pharmacological agents to target this pathway. Here, we report a series of indole amides that inhibit human 3-phosphoglycerate dehydrogenase (PHGDH), the enzyme that catalyzes the first committed step of the serine synthesis pathway. Using X-ray crystallography, we show that the indole amides bind the NAD+ pocket of PHGDH. Through structure-based optimization we were able to develop compounds with low nanomolar affinities for PHGDH in an enzymatic IC50 assay. In cellular assays, the most potent compounds inhibited de novo serine synthesis with low micromolar to sub-micromolar activities and these compounds successfully abrogated the proliferation of cancer cells in serine free media. The indole amide series reported here represent an important improvement over previously published PHGDH inhibitors as they are markedly more potent and their mechanism of action is better defined.

Computational Fluorine Scanning Using Free-Energy Perturbation.

We present perturbative fluorine scanning, a computational fluorine scanning approach using free-energy perturbation. This method can be applied to molecular dynamics simulations of a single compound and make predictions for the best binders out of numerous fluorinated analogues. We tested the method on nine test systems: renin, DPP4, menin, P38, factor Xa, CDK2, AKT, JAK2, and androgen receptor. The predictions were in excellent agreement with more rigorous alchemical free-energy calculations and in good agreement with experimental data for most of the test systems. However, the agreement with experiment was very poor in some of the test systems, and this highlights the need for improved force fields in addition to accurate treatment of tautomeric and protonation states. The method is of particular interest due to the wide use of fluorine in medicinal chemistry to improve binding affinity and ADME properties. The promising results on this test case suggest that perturbative fluorine scanning will be a useful addition to the available arsenal of free-energy methods.

Assimilating Radial Distribution Functions To Build Water Models with Improved Structural Properties.

The structural properties of three- and four-site water models are improved by extending the ForceBalance parametrization code to include a new methodology allowing for the targeting of any radial distribution function (RDF) during the parametrization of a force field. The mean squared difference (MSD) between the experimental and simulated RDFs contributes to an objective function, allowing for the systematic optimization of force field parameters to reach closer overall agreement with experiment. RDF fitting is applied to develop modified versions of the TIP3P and TIP4P/2005 water models in which the Lennard-Jones potential is replaced by a Buckingham potential. The optimized TIP3P-Buckingham and TIP4P-Buckingham potentials feature 93 and 98% lower MSDs in the OO RDF compared to the TIP3P and TIP4P/2005 models respectively, with marked decreases in the height of the first peak. Additionally, these Buckingham models predict the entropy of water more accurately, reducing the error in the entropy of TIP3P from 11 to 3% and the error in the entropy of TIP4P/2005 from 11 to 2%. These new Buckingham models have improved predictive power for many nonfitted properties particularly in the case of TIP3P. Our work directly demonstrates how the Buckingham potential can improve the description of water's structural properties beyond the Lennard-Jones potential. Moreover, adding a Buckingham potential is a favorable alternative to adding interaction sites in terms of computational speed on modern GPU hardware.

On the accuracy of one- and two-particle solvation entropies.

Evaluating solvation entropies directly and combining with direct energy calculations is one way of calculating free energies of solvation and is used by Inhomogeneous Fluid Solvation Theory (IFST). The configurational entropy of a fluid is a function of the interatomic correlations and can thus be expressed in terms of correlation functions. The entropies in this work are directly calculated from a truncated series of integrals over these correlation functions. Many studies truncate all terms higher than the solvent-solute correlations. This study includes an additional solvent-solvent correlation term and assesses the associated free energy when IFST is applied to a fixed Lennard-Jones particle solvated in neon. The strength of the central potential is varied to imitate larger solutes. Average free energy estimates with both levels of IFST are able to reproduce the estimate made using the Free energy Perturbation (FEP) to within 0.16 kcal/mol. We find that the signal from the solvent-solvent correlations is very weak. Our conclusion is that for monatomic fluids simulated by pairwise classical potentials the correction term is relatively small in magnitude. This study shows it is possible to reproduce the free energy from a path based method like FEP, by only considering the endpoints of the path. This method can be directly applied to more complex solutes which break the spherical symmetry of this study.

Studying the role of cooperative hydration in stabilizing folded protein states.

Understanding and modelling protein folding remains a key scientific and engineering challenge. Two key questions in protein folding are (1) why many proteins adopt a folded state and (2) how these proteins transition from the random coil ensemble to a folded state. In this paper we employ molecular dynamics simulations to address the first of these questions. Computational methods are well-placed to address this issue due to their ability to analyze systems at atomic-level resolution. Traditionally, the stability of folded proteins has been ascribed to the balance of two types of intermolecular interactions: hydrogen-bonding interactions and hydrophobic contacts. In this study, we explore a third type of intermolecular interaction: cooperative hydration of protein surface residues. To achieve this, we consider multiple independent simulations of the villin headpiece domain to quantify the contributions of different interactions to the energy of the native and fully extended states. In addition, we consider whether these findings are robust with respect to the protein forcefield, the water model, and the presence of salt. In all cases, we identify many cooperatively hydrated interactions that are transient but energetically favor the native state. Whilst further work on additional protein structures, forcefields, and water models is necessary, these results suggest a role for cooperative hydration in protein folding that should be explored further. Rational design of cooperative hydration on the protein surface could be a viable strategy for increasing protein stability.

Quantifying the entropy of binding for water molecules in protein cavities by computing correlations.

Protein structural analysis demonstrates that water molecules are commonly found in the internal cavities of proteins. Analysis of experimental data on the entropies of inorganic crystals suggests that the entropic cost of transferring such a water molecule to a protein cavity will not typically be greater than 7.0 cal/mol/K per water molecule, corresponding to a contribution of approximately +2.0 kcal/mol to the free energy. In this study, we employ the statistical mechanical method of inhomogeneous fluid solvation theory to quantify the enthalpic and entropic contributions of individual water molecules in 19 protein cavities across five different proteins. We utilize information theory to develop a rigorous estimate of the total two-particle entropy, yielding a complete framework to calculate hydration free energies. We show that predictions from inhomogeneous fluid solvation theory are in excellent agreement with predictions from free energy perturbation (FEP) and that these predictions are consistent with experimental estimates. However, the results suggest that water molecules in protein cavities containing charged residues may be subject to entropy changes that contribute more than +2.0 kcal/mol to the free energy. In all cases, these unfavorable entropy changes are predicted to be dominated by highly favorable enthalpy changes. These findings are relevant to the study of bridging water molecules at protein-protein interfaces as well as in complexes with cognate ligands and small-molecule inhibitors.

Comparing distance metrics for rotation using the k-nearest neighbors algorithm for entropy estimation.

Distance metrics facilitate a number of methods for statistical analysis. For statistical mechanical applications, it is useful to be able to compute the distance between two different orientations of a molecule. However, a number of distance metrics for rotation have been employed, and in this study, we consider different distance metrics and their utility in entropy estimation using the k-nearest neighbors (KNN) algorithm. This approach shows a number of advantages over entropy estimation using a histogram method, and the different approaches are assessed using uniform randomly generated data, biased randomly generated data, and data from a molecular dynamics (MD) simulation of bulk water. The results identify quaternion metrics as superior to a metric based on the Euler angles. However, it is demonstrated that samples from MD simulation must be independent for effective use of the KNN algorithm and this finding impacts any application to time series data.

Next-generation neuropeptide Y receptor small-molecule agonists inhibit mosquito-biting behavior.

BACKGROUND: Female Aedes aegypti mosquitoes can spread disease-causing pathogens when they bite humans to obtain blood nutrients required for egg production. Following a complete blood meal, host-seeking is suppressed until eggs are laid. Neuropeptide Y-like receptor 7 (NPYLR7) plays a role in endogenous host-seeking suppression and previous work identified small-molecule NPYLR7 agonists that inhibit host-seeking and blood-feeding when fed to mosquitoes at high micromolar doses. METHODS: Using structure-activity relationship analysis and structure-guided design we synthesized 128 compounds with similarity to known NPYLR7 agonists. RESULTS: Although in vitro potency (EC50) was not strictly predictive of in vivo effect, we identified three compounds that reduced blood-feeding from a live host when fed to mosquitoes at a dose of 1 µM-a 100-fold improvement over the original reference compound. CONCLUSIONS: Exogenous activation of NPYLR7 represents an innovative vector control strategy to block mosquito biting behavior and prevent mosquito-human host interactions that lead to pathogen transmission.

Next Generation Neuropeptide Y Receptor Small Molecule Agonists Inhibit Mosquito Biting Behavior.

Female Aedes aegypti mosquitoes can spread disease-causing pathogens when they bite humans to obtain blood nutrients required for egg production. Following a complete blood meal, host-seeking is suppressed until eggs are laid. Neuropeptide Y-like Receptor 7 (NPYLR7) plays a role in endogenous host-seeking suppression and previous work identified small molecule NPYLR7 agonists that suppress host-seeking and blood feeding when fed to mosquitoes at high micromolar doses. Using structure activity relationship analysis and structure-guided design we synthesized 128 compounds with similarity to known NPYLR7 agonists. Although in vitro potency (EC50) was not strictly predictive of in vivo effect, we identified 3 compounds that suppressed blood feeding from a live host when fed to mosquitoes at a 1 µM dose, a 100-fold improvement over the original reference compound. Exogenous activation of NPYLR7 represents an innovative vector control strategy to block mosquito biting behavior and prevent mosquito/human host interactions that lead to pathogen transmission.

Lead Optimization of Small Molecule ENL YEATS Inhibitors to Enable In Vivo Studies: Discovery of TDI-11055.

Eleven-nineteen leukemia (ENL) is an epigenetic reader protein that drives oncogenic transcriptional programs in acute myeloid leukemia (AML). AML is one of the deadliest hematopoietic malignancies, with an overall 5-year survival rate of 27%. The epigenetic reader activity of ENL is mediated by its YEATS domain that binds to acetyl and crotonyl marks on histone tails and colocalizes with promoters of actively transcribed genes that are essential for leukemia. Prior to the discovery of TDI-11055, existing inhibitors of ENL YEATS showed in vitro potency, but had not shown efficacy in in vivo animal models. During the course of the medicinal chemistry campaign described here, we identified ENL YEATS inhibitor TDI-11055 that has an improved pharmacokinetic profile and is appropriate for in vivo evaluation of the ENL YEATS inhibition mechanism in AML.

Combining solvent thermodynamic profiles with functionality maps of the Hsp90 binding site to predict the displacement of water molecules.

Intermolecular interactions in the aqueous phase must compete with the interactions between the two binding partners and their solvating water molecules. In biological systems, water molecules in protein binding sites cluster at well-defined hydration sites and can form strong hydrogen-bonding interactions with backbone and side-chain atoms. Displacement of such water molecules is only favorable when the ligand can form strong compensating hydrogen bonds. Conversely, water molecules in hydrophobic regions of protein binding sites make only weak interactions, and the requirements for favorable displacement are less stringent. The propensity of water molecules for displacement can be identified using inhomogeneous fluid solvation theory (IFST), a statistical mechanical method that decomposes the solvation free energy of a solute into the contributions from different spatial regions and identifies potential binding hotspots. In this study, we employed IFST to study the displacement of water molecules from the ATP binding site of Hsp90, using a test set of 103 ligands. The predicted contribution of a hydration site to the hydration free energy was found to correlate well with the observed displacement. Additionally, we investigated if this correlation could be improved by using the energetic scores of favorable probe groups binding at the location of hydration sites, derived from a multiple copy simultaneous search (MCSS) method. The probe binding scores were not highly predictive of the observed displacement and did not improve the predictivity when used in combination with IFST-based hydration free energies. The results show that IFST alone can be used to reliably predict the observed displacement of water molecules in Hsp90. However, MCSS can augment IFST calculations by suggesting which functional groups should be used to replace highly displaceable water molecules. Such an approach could be very useful in improving the hit-to-lead process for new drug targets.

On-demand male contraception via acute inhibition of soluble adenylyl cyclase.

Nearly half of all pregnancies are unintended; thus, existing family planning options are inadequate. For men, the only choices are condoms and vasectomy, and most current efforts to develop new contraceptives for men impact sperm development, meaning that contraception requires months of continuous pretreatment. Here, we provide proof-of-concept for an innovative strategy for on-demand contraception, where a man would take a birth control pill shortly before sex, only as needed. Soluble adenylyl cyclase (sAC) is essential for sperm motility and maturation. We show a single dose of a safe, acutely-acting sAC inhibitor with long residence time renders male mice temporarily infertile. Mice exhibit normal mating behavior, and full fertility returns the next day. These studies define sAC inhibitors as leads for on-demand contraceptives for men, and they provide in vivo proof-of-concept for previously untested paradigms in contraception; on-demand contraception after just a single dose and pharmacological contraception for men.