Free Energy Perturbation Approach for Accurate Crystalline Aqueous Solubility Predictions.

Early assessment of crystalline thermodynamic solubility continues to be elusive for drug discovery and development despite its critical importance, especially for the ever-increasing fraction of poorly soluble drug candidates. Here we present a detailed evaluation of a physics-based free energy perturbation (FEP+) approach for computing the thermodynamic aqueous solubility. The predictive power of this approach is assessed across diverse chemical spaces, spanning pharmaceutically relevant literature compounds and more complex AbbVie compounds. Our approach achieves predictive (RMSE = 0.86) and differentiating power (R2 = 0.69) and therefore provides notably improved correlations to experimental solubility compared to state-of-the-art machine learning approaches that utilize quantum mechanics-based descriptors. The importance of explicit considerations of crystalline packing in predicting solubility by the FEP+ approach is also highlighted in this study. Finally, we show how computed energetics, including hydration and sublimation free energies, can provide further insights into molecule design to feed the medicinal chemistry DMTA cycle.

Machine-Learning-Based Single-Molecule Quantification of Circulating MicroRNA Mixtures.

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression and are emerging as powerful indicators of diseases. MiRs are secreted in blood plasma and thus may report on systemic aberrations at an early stage via liquid biopsy analysis. We present a method for multiplexed single-molecule detection and quantification of a selected panel of miRs. The proposed assay does not depend on sequencing, requires less than 1 mL of blood, and provides fast results by direct analysis of native, unamplified miRs. This is enabled by a novel combination of compact spectral imaging and a machine learning-based detection scheme that allows simultaneous multiplexed classification of multiple miR targets per sample. The proposed end-to-end pipeline is extremely time efficient and cost-effective. We benchmark our method with synthetic mixtures of three target miRs, showcasing the ability to quantify and distinguish subtle ratio changes between miR targets.

Discovery of a Potent and Selective Tyrosine Kinase 2 Inhibitor: TAK-279.

TYK2 is a key mediator of IL12, IL23, and type I interferon signaling, and these cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genome-wide association studies and clinical results, TYK2 inhibition through small molecules is an attractive therapeutic strategy to treat these diseases. Herein, we report the discovery of a series of highly selective pseudokinase (Janus homology 2, JH2) domain inhibitors of TYK2 enzymatic activity. A computationally enabled design strategy, including the use of FEP+, was instrumental in identifying a pyrazolo-pyrimidine core. We highlight the utility of computational physics-based predictions used to optimize this series of molecules to identify the development candidate 30, a potent, exquisitely selective cellular TYK2 inhibitor that is currently in Phase 2 clinical trials for the treatment of psoriasis and psoriatic arthritis.

Modulation of biliverdin dynamics and spectral properties by Sandercyanin.

Biliverdin IX-alpha (BV), a tetrapyrrole, is found ubiquitously in most living organisms. It functions as a metabolite, pigment, and signaling compound. While BV is known to bind to diverse protein families such as heme-metabolizing enzymes and phytochromes, not many BV-bound lipocalins (ubiquitous, small lipid-binding proteins) have been studied. The molecular basis of binding and conformational selectivity of BV in lipocalins remains unexplained. Sandercyanin (SFP)-BV complex is a blue lipocalin protein present in the mucus of the Canadian walleye (Stizostedion vitreum). In this study, we present the structures and binding modes of BV to SFP. Using a combination of designed site-directed mutations, X-ray crystallography, UV/VIS, and resonance Raman spectroscopy, we have identified multiple conformations of BV that are stabilized in the binding pocket of SFP. In complex with the protein, these conformers generate varied spectroscopic signatures both in their absorption and fluorescence spectra. We show that despite no covalent anchor, structural heterogeneity of the chromophore is primarily driven by the D-ring pyrrole of BV. Our work shows how conformational promiscuity of BV is correlated to the rearrangement of amino acids in the protein matrix leading to modulation of spectral properties.

Potent and selective TYK2-JH1 inhibitors highly efficacious in rodent model of psoriasis.

TYK2 is a member of the JAK family of kinases and a key mediator of IL-12, IL-23, and type I interferon signaling. These cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genetic association studies, TYK2 inhibition is an attractive therapeutic strategy for these diseases. Herein, we report the discovery of a series of highly selective catalytic site TYK2 inhibitors designed using FEP+ and structurally enabled design starting from a virtual screen hit. We highlight the structure-based optimization to identify a lead candidate 30, a potent cellular TYK2 inhibitor with excellent selectivity, pharmacokinetic properties, and in vivo efficacy in a mouse psoriasis model.

Divalent Ion-Induced Switch in DNA Cleavage of KpnI Endonuclease Probed through Surface-Enhanced Raman Spectroscopy.

We demonstrate the remarkable ability of surface-enhanced Raman spectroscopy (SERS) to track the allosteric changes in restriction endonuclease KpnI (R.KpnI) caused by metal ions. R.KpnI binds and promiscuously cleaves DNA upon activation by Mg2+ ions. However, the divalent ion Ca2+ induces high fidelity cleavage, which can be overcome by higher concentrations of Mg2+ ions. In the absence of any 3D crystal structure, for the first time, we have elucidated the structural underpinnings of such a differential effect of divalent ions on the endonuclease activity. A combined SERS and molecular dynamics (MD) approach showed that Ca2+ ion activates an enzymatic switch in the active site, which is responsible for the high fidelity activity of the enzyme. Thus, SERS in combination with MD simulations provides a powerful tool for probing the link between the structure and activity of enzyme molecules that play vital roles in DNA transactions.

Novel Physics-Based Ensemble Modeling Approach That Utilizes 3D Molecular Conformation and Packing to Access Aqueous Thermodynamic Solubility: A Case Study of Orally Available Bromodomain and Extraterminal Domain Inhibitor Lead Optimization Series.

Drug design with patient centricity for ease of administration and pill burden requires robust understanding of the impact of chemical modifications on relevant physicochemical properties early in lead optimization. To this end, we have developed a physics-based ensemble approach to predict aqueous thermodynamic crystalline solubility, with a 2D chemical structure as the input. Predictions for the bromodomain and extraterminal domain (BET) inhibitor series show very close match (0.5 log unit) with measured thermodynamic solubility for cases with low crystal anisotropy and good match (1 log unit) for high anisotropy structures. The importance of thermodynamic solubility is clearly demonstrated by up to a 4 log unit drop in solubility compared to kinetic (amorphous) solubility in some cases and implications thereof, for instance on human dose. We have also demonstrated that incorporating predicted crystal structures in thermodynamic solubility prediction is necessary to differentiate (up to 4 log unit) between solubility of molecules within the series. Finally, our physics-based ensemble approach provides valuable structural insights into the origins of 3-D conformational landscapes, crystal polymorphism, and anisotropy that can be leveraged for both drug design and development.

Reliable and Accurate Solution to the Induced Fit Docking Problem for Protein-Ligand Binding.

We present a reliable and accurate solution to the induced fit docking problem for protein-ligand binding by combining ligand-based pharmacophore docking, rigid receptor docking, and protein structure prediction with explicit solvent molecular dynamics simulations. This novel methodology in detailed retrospective and prospective testing succeeded to determine protein-ligand binding modes with a root-mean-square deviation within 2.5 Å in over 90% of cross-docking cases. We further demonstrate these predicted ligand-receptor structures were sufficiently accurate to prospectively enable predictive structure-based drug discovery for challenging targets, substantially expanding the domain of applicability for such methods.

Topological phase transition induced by band structure modulation in a Chern insulator.

We study a systematic evolution of the topological properties of a Chern insulator upon smooth variation of a hopping parameter (t1) of the electrons among a pair of nearest neighbour sites on a honeycomb lattice, while keeping the other two hopping terms (t) fixed. In the absence of a Haldane flux, the tuning oft1results in gradual shifting of the Dirac cones which eventually merge into one at theMpoint in the Brillouin zone (BZ) att1= 2twith a gapless semi-Dirac dispersion at low energies. In the presence of a Haldane flux, the system becomes a Chern insulator fort1< 2t, but turns gapless att1= 2twith the semi-Dirac dispersion being transformed to an anisotropic Dirac one. The spectrum eventually gaps out and transforms into a trivial insulator fort1> 2t. The Chern number phase diagram obtained via integrating the Berry curvature over the BZ shows a gradual shrinking of the 'topological' lobes, and vanishes just beyondt1= 2t, where a small but a finite Berry curvature still exists. Thus, there is a phase transition from a topological phase to a trivial phase across the semi-Dirac point (t1= 2t). The vanishing of the anomalous Hall conductivity plateau and the merger of the chiral edge states with the bulk bands near theMpoint provide robust support of the observed phase transition.

Role of Explicit Solvation in the Simulation of Resonance Raman Spectra within Short-Time Dynamics Approximation.

In short-time dynamics approximation, relative resonance Raman (RR) intensity of a vibrational mode primarily depends on the magnitude of square of the excited-state gradient along the corresponding normal coordinate, ground-state normal mode eigenvector, and harmonic vibrational wavenumbers. In this study, through simulation of RR spectra of guanosine-5'-monophosphate (GMP) in two ππ* singlet excited states, we analyze how the explicitly hydrogen-bonded local solvation structure of the chromophore dictates intensities of the RR active modes in an unprecedented manner. We show that the accuracy of the structural model of solvated chromophore plays a decisive role in determining an optimal theoretical method for prediction of the Franck-Condon region of the ππ* excited states. 9-Methylguanine (9-meG) in complex with six water molecules (9-meG·6H2O) is found out to be the most accurate one for describing GMP in two different bright electronic states. We find that explicit hydrogen-bonded water molecules strongly influence computed RR intensities of GMP by modulating both the ground-state normal mode vectors and the excited-state energy gradients. We find that simultaneous inclusion of six explicit waters to describe the solute-solvent interaction near all hydration sites is essential for reliable prediction of the features of RR spectra in Lb and Bb electronic states of GMP.

Noncovalent inhibitors reveal BTK gatekeeper and auto-inhibitory residues that control its transforming activity.

Inhibition of Bruton tyrosine kinase (BTK) is a breakthrough therapy for certain B cell lymphomas and B cell chronic lymphatic leukemia. Covalent BTK inhibitors (e.g., ibrutinib) bind to cysteine C481, and mutations of this residue confer clinical resistance. This has led to the development of noncovalent BTK inhibitors that do not require binding to cysteine C481. These new compounds are now entering clinical trials. In a systematic BTK mutagenesis screen, we identify residues that are critical for the activity of noncovalent inhibitors. These include a gatekeeper residue (T474) and mutations in the kinase domain. Strikingly, co-occurrence of gatekeeper and kinase domain lesions (L512M, E513G, F517L, L547P) in cis results in a 10- to 15-fold gain of BTK kinase activity and de novo transforming potential in vitro and in vivo. Computational BTK structure analyses reveal how these lesions disrupt an intramolecular mechanism that attenuates BTK activation. Our findings anticipate clinical resistance mechanisms to a new class of noncovalent BTK inhibitors and reveal intramolecular mechanisms that constrain BTK's transforming potential.

OPLS3e: Extending Force Field Coverage for Drug-Like Small Molecules.

Building upon the OPLS3 force field we report on an enhanced model, OPLS3e, that further extends its coverage of medicinally relevant chemical space by addressing limitations in chemotype transferability. OPLS3e accomplishes this by incorporating new parameter types that recognize moieties with greater chemical specificity and integrating an on-the-fly parametrization approach to the assignment of partial charges. As a consequence, OPLS3e leads to greater accuracy against performance benchmarks that assess small molecule conformational propensities, solvation, and protein-ligand binding.

Kinetics of Melanin Polymerization during Enzymatic and Nonenzymatic Oxidation.

Melanin is an abundant biopigment in the animal kingdom, but its structure remains poorly understood. This is a substantial impediment to understanding the mechanistic origin of its observed functions. Proposed models of melanin structure include aggregates of both linear and macrocyclic units and noncovalently held monomers. Both models are broadly in agreement with current experimental data. To constrain the structural and kinetic models of melanin, experimental data of high resolution with chemical specificity accompanied by atomistic modeling are required. We have addressed this by obtaining electronic absorption, infrared, and ultraviolet resonance Raman (RR) spectra of melanin at several wavelengths of excitation that are sensitive to small changes in structure. From these experiments, we observed kinetics of the formation of different species en route to melanin polymerization. Exclusive chemical signatures of monomer 3,4-dihydroxyphenylalanine (dopa), intermediate dopachrome (DC), and early-time polymer are established through their vibrational bands at 1292, 1670, and 1616 cm-1 respectively. Direct evidence of reduced heterogeneity of melanin oligomers in tyrosinase-induced formation is provided from experimental measurements of vibrational bandwidths. Models made with density functional theory show that the linear homopolymeric structures of 5,6-dihydroxyindole can account for experimentally observed wavenumbers and broad bandwidth in Raman spectra of dopa-melanin. We capture resonance Raman (RR) signature of DC, the intermediate stabilized by the enzyme tyrosinase, for the first time in an enzyme-assisted melanization reaction using 488 nm excitation wavelength and propose that this wavelength can be used to probe reaction intermediates of melanin formation in solution.

Ultrafast structural dynamics of photoexcited adenine.

We report deep UV initiated excited state dynamics of the canonical nucleobase adenine (Ade) through Resonance Raman (RR) intensity analysis. RR spectra of Ade at excitation wavelengths throughout the Bb absorption band in the 210-230 nm wavelength range are measured and subsequently converted to scattering cross-sections. The time-dependent wave packet (TDWP) formalism has been employed for self-consistent simulations of the resulting wavelength dependent Raman excitation profiles (REP) and absorption spectrum of Ade. These simulations yield instantaneous nuclear dynamics of Ade within tens of femtoseconds (fs) of photoabsorption as structural distortions, linewidth broadening and solvation parameters. The instantaneous geometrical distortions of the purine ring following photoexcitation into the Bb state are analyzed vis-à-vis the low energy La state (∼260 nm) of Ade. We find that while photoabsorption by the La state causes major distortions of the imidazole ring, pyrimidine ring suffers maximal changes following Bb excitation. Seven in-plane stretching vibrations out of fifteen resonantly enhanced modes of Ade are found to contribute 76% of the total internal reorganization energy (981 cm-1) in the Bb excited state. In addition, the inertial response of the solvation shell to photoexcitation is found to be of 1190 cm-1 in magnitude, and with a relaxation time of 26.5 fs. A parallel comparison is drawn between the UV-C initiated photodynamics of Ade (6-aminopurine) with that of two substituted purines, viz., 6-chloroguanine (6-ClG or 2-amino-6-chloropurine) and guanine (2-amino-6-oxo-purie) which were reported earlier.

Ultrafast Nuclear Dynamics of Photoexcited Guanosine-5'-Monophosphate in Three Singlet States.

We report measurement of resonance Raman (RR) spectra of guanosine-5'-monophosphate (GMP), a DNA nucleotide at excitation wavelengths throughout its ππ* absorption band (Bb) in the 210-230 nm range. From these data, we constructed wavelength-dependent Raman intensity excitation profiles (REPs) for all observed modes. These profiles and the absorption spectrum were then modeled using self-consistent simulations based on the time-dependent wave packet propagation formalism. We inferred the initial structural dynamics of GMP immediately after photoexcitation in terms of dimensionless displacements. The simulations also provide linewidth-broadening parameters that in turn report on the time scale of dynamics. We compared deduced structural changes in the purine ring upon photoabsorption into the Bb state with those deduced for the two lowest lying ππ* (La and Lb at 280 and 248 nm, respectively) excited states of GMP. We find that excitation to the Lb state lengthens C6-N1 and C2═N3 bonds, which lie along the formation coordinate of various oxidative adducts but Bb excitation does not. We also find that photoabsorption by the Bb state weakens the C8-N9 bond and thus might assist imidazole ring opening via cleavage of the same bond. Electronic excitation to different ππ* states of the guanine chromophore results in contrasting structural changes; although absorption by the La and Lb states induces expansion of pyrimidine and contraction of imidazole rings, excitation results in overall shrinkage of both the rings. Computed absolute changes in internal coordinates imply that photoexcitation to any of the three singlet states of GMP does not lead directly to the formation of a cation radical of guanine.

Accelerating drug discovery through tight integration of expert molecular design and predictive scoring.

Modeling protein-ligand interactions has been a central goal of computational chemistry for many years. We here review recent progress toward this goal, and highlight the role free energy calculation methods and computational solvent analysis techniques are now having in drug discovery. We further describe recent use of these methodologies to advance two separate drug discovery programs targeting acetyl-CoA carboxylase and tyrosine kinase 2. These examples suggest that tight integration of sophisticated chemistry teams with state-of-the-art computational methods can dramatically improve the efficiency of small molecule drug discovery.

Sub-50 fs excited state dynamics of 6-chloroguanine upon deep ultraviolet excitation.

The photophysical properties of natural nucleobases and their respective nucleotides are ascribed to the sub-picosecond lifetime of their first singlet states in the UV-B region (260-350 nm). Electronic transitions of the ππ* type, which are stronger than those in the UV-B region, lie at the red edge of the UV-C range (100-260 nm) in all isolated nucleobases. The lowest energetic excited states in the UV-B region of nucleobases have been investigated using a plethora of experimental and theoretical methods in gas and solution phases. The sub-picosecond lifetime of these molecules is not a general attribute of all nucleobases but specific to the five primary nucleobases and a few xanthine and methylated derivatives. To determine the overall UV photostability, we aim to understand the effect of more energetic photons lying in the UV-C region on nucleobases. To determine the UV-C initiated photophysics of a nucleobase system, we chose a halogen substituted purine, 6-chloroguanine (6-ClG), that we had investigated previously using resonance Raman spectroscopy. We have performed quantitative measurements of the resonance Raman cross-section across the Bb absorption band (210-230 nm) and constructed the Raman excitation profiles. We modeled the excitation profiles using Lee and Heller's time-dependent theory of resonance Raman intensities to extract the initial excited state dynamics of 6-ClG within 30-50 fs after photoexcitation. We found that imidazole and pyrimidine rings of 6-ClG undergo expansion and contraction, respectively, following photoexcitation to the Bb state. The amount of distortions of the excited state structure from that of the ground state structure is reflected by the total internal reorganization energy that is determined at 112 cm(-1). The contribution of the inertial component of the solvent response towards the total reorganization energy was obtained at 1220 cm(-1). In addition, our simulation also yields an instantaneous response of the first solvation shell within an ultrafast timescale of less than 30 fs following photoexcitation.

Not just an oil slick: how the energetics of protein-membrane interactions impacts the function and organization of transmembrane proteins.

The membrane environment, its composition, dynamics, and remodeling, have been shown to participate in the function and organization of a wide variety of transmembrane (TM) proteins, making it necessary to study the molecular mechanisms of such proteins in the context of their membrane settings. We review some recent conceptual advances enabling such studies, and corresponding computational models and tools designed to facilitate the concerted experimental and computational investigation of protein-membrane interactions. To connect productively with the high resolution achieved by cognate experimental approaches, the computational methods must offer quantitative data at an atomistically detailed level. We show how such a quantitative method illuminated the mechanistic importance of a structural characteristic of multihelical TM proteins, that is, the likely presence of adjacent polar and hydrophobic residues at the protein-membrane interface. Such adjacency can preclude the complete alleviation of the well-known hydrophobic mismatch between TM proteins and the surrounding membrane, giving rise to an energy cost of residual hydrophobic mismatch. The energy cost and biophysical formulation of hydrophobic mismatch and residual hydrophobic mismatch are reviewed in the context of their mechanistic role in the function of prototypical members of multihelical TM protein families: 1), LeuT, a bacterial homolog of mammalian neurotransmitter sodium symporters; and 2), rhodopsin and the ß1- and ß2-adrenergic receptors from the G-protein coupled receptor family. The type of computational analysis provided by these examples is poised to translate the rapidly growing structural data for the many TM protein families that are of great importance to cell function into ever more incisive insights into mechanisms driven by protein-ligand and protein-protein interactions in the membrane environment.

Protein and lipid interactions driving molecular mechanisms of in meso crystallization.

The recent advances in the in meso crystallization technique for the structural characterization of G-protein coupled receptor (GPCR) proteins have established the usefulness of the lipidic-cubic phases (LCPs) in the field of crystallography of membrane proteins. It is surprising that despite the success of the approach, the molecular mechanisms of the in meso method are still not well understood. Therefore, the approach must rely on extensive screening for a suitable protein construct, for host and additive lipids, and for the appropriate precipitants and temperature. To shed light on the in meso crystallization mechanisms, we used extensive coarse-grained molecular dynamics simulations to study, in molecular detail, LCPs under different conditions (compositions and temperatures relevant to crystallogenesis) and their interactions with different types of GPCR constructs. The results presented show how the modulation of the lattice constant of the LCP (triggered by the addition of precipitant during the in meso assay), or of the host lipid type, can destabilize monomeric proteins in the bilayer of the LCP and thus drive their aggregation into the stacked lamellae, where the residual hydrophobic mismatch between the protein and the membrane can drive the formation of lateral contacts leading to nucleation and crystal growth. Moreover, we demonstrate how particular protein designs (such as transmembrane proteins engineered to contain large polar regions) can promote protein stacking interactions in the third, out-of-plane, dimension. The insights provided by the new aspects of the specific molecular mechanisms responsible for protein-protein interactions inside the cubic phase presented here should be helpful in guiding the rational design of future in meso trials with successful outcomes.

How the dynamic properties and functional mechanisms of GPCRs are modulated by their coupling to the membrane environment.

Experimental observations of the dependence of function and organization of G protein-coupled receptors (GPCRs) on their lipid environment have stimulated new quantitative studies of the coupling between the proteins and the membrane. It is important to develop such a quantitative understanding at the molecular level because the effects of the coupling are seen to be physiologically and clinically significant. Here we review findings that offer insight into how membrane-GPCR coupling is connected to the structural characteristics of the GPCR, from sequence to 3D structural detail, and how this coupling is involved in the actions of ligands on the receptor. The application of a recently developed computational approach designed for quantitative evaluation of membrane remodeling and the energetics of membrane-protein interactions brings to light the importance of the radial asymmetry of the membrane-facing surface of GPCRs in their interaction with the surrounding membrane. As the radial asymmetry creates adjacencies of hydrophobic and polar residues at specific sites of the GPCR, the ability of membrane remodeling to achieve complete hydrophobic matching is limited, and the residual mismatch carries a significant energy cost. The adjacencies are shown to be affected by ligand-induced conformational changes. Thus, functionally important organization of GPCRs in the cell membrane can depend both on ligand-determined properties and on the lipid composition of various membrane regions with different remodeling capacities. That this functionally important reorganization can be driven by oligomerization patterns that reduce the energy cost of the residual mismatch, suggests a new perspective on GPCR dimerization and ligand-GPCR interactions. The relation between the modulatory effects on GPCRs from the binding of specific cell-membrane components, e.g., cholesterol, and those produced by the non-local energetics of hydrophobic mismatch are discussed in this context.