Balanced Permeability Index: A Multiparameter Index for Improved In Vitro Permeability.

The optimization of passive permeability is a key objective for orally available small molecule drug candidates. For drugs targeting the central nervous system (CNS), minimizing P-gp-mediated efflux is an additional important target for optimization. The physicochemical properties most strongly associated with high passive permeability and lower P-gp efflux are size, polarity, and lipophilicity. In this study, a new metric called the Balanced Permeability Index (BPI) was developed that combines these three properties. The BPI was found to be more effective than any single property in classifying molecules based on their permeability and efflux across a diverse range of chemicals and assays. BPI is easy to understand, allowing researchers to make decisions about which properties to prioritize during the drug development process.

Discovery and Preclinical Pharmacology of NX-2127, an Orally Bioavailable Degrader of Bruton's Tyrosine Kinase with Immunomodulatory Activity for the Treatment of Patients with B Cell Malignancies.

Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, is an essential effector of B-cell receptor (BCR) signaling. Chronic activation of BTK-mediated BCR signaling is a hallmark of many hematological malignancies, which makes it an attractive therapeutic target. Pharmacological inhibition of BTK enzymatic function is now a well-proven strategy for the treatment of patients with these malignancies. We report the discovery and characterization of NX-2127, a BTK degrader with concomitant immunomodulatory activity. By design, NX-2127 mediates the degradation of transcription factors IKZF1 and IKZF3 through molecular glue interactions with the cereblon E3 ubiquitin ligase complex. NX-2127 degrades common BTK resistance mutants, including BTKC481S. NX-2127 is orally bioavailable, exhibits in vivo degradation across species, and demonstrates efficacy in preclinical oncology models. NX-2127 has advanced into first-in-human clinical trials and achieves deep and sustained degradation of BTK following daily oral dosing at 100 mg.

Trends in Neosubstrate Degradation by Cereblon-Based Molecular Glues and the Development of Novel Multiparameter Optimization Scores.

Molecular glues enable the degradation of previously "undruggable" proteins via the recruitment of cereblon (CRBN) to the target. One major challenge in designing CRBN E3 ligase modulating compounds (CELMoDs) is the selectivity profile toward neosubstrates, proteins recruited by CRBN E3 ligase agents for degradation. Common neosubstrates include Aiolos, Ikaros, GSPT1, CK1α, and SALL4. Unlike achieving potency and selectivity for traditional small molecule inhibitors, reducing the degradation of these neosubstrates is complicated by the ternary nature of the complex formed between the protein, CRBN, and CELMoD. The standard guiding principles of medicinal chemistry, such as enforcing hydrogen bond formation, are less predictive of degradation efficiency and selectivity. Disclosed is an analysis of our glutarimide CELMoD library to identify interpretable chemical features correlated to selectivity profiles and general cytotoxicity. Included is a simple multiparameter optimization function using only three parameters to predict whether molecules will have undesired neosubstrate activity.

On Ternary Complex Stability in Protein Degradation: In Silico Molecular Glue Binding Affinity Calculations.

Molecular glues are small molecules that simultaneously bind to two proteins, creating a chemically induced protein-protein interface. CELMoDs (cereblon E3 ligase modulators) are a class of molecular glues that promote recruitment of neosubstrate proteins to the E3 ubiquitin ligase cereblon (CRBN) for poly-Lys48-ubiquitination and proteasomal degradation. Ternary complex structures of clinical CELMoDs CC-885 and CC-90009 bound to CRBN and neosubstrate G1 to S phase transition protein 1 (GSPT1) have been experimentally determined. Although cellular degradation is a downstream event, dependent not only on the affinity of the glue CELMoD in the ternary complex, we test the applicability of established structure-based drug design principles to predict binding affinity of CELMoDs to the protein-protein neointerface and correlation to measured cellular degradation for the neosubstrates GSPT1 and zinc finger Aiolos (IKZF3). For a congeneric series of CELMoDs, which have a similar sequence of binding events and resultant binding modes, we conclude that well-established structure-based methods that measure in silico ternary complex stabilities can predict relative degradation potency by CELMoDs.

Selectivity Challenges in Docking Screens for GPCR Targets and Antitargets.

To investigate large library docking's ability to find molecules with joint activity against on-targets and selectivity versus antitargets, the dopamine D2 and serotonin 5-HT2A receptors were targeted, seeking selectivity against the histamine H1 receptor. In a second campaign, κ-opioid receptor ligands were sought with selectivity versus the µ-opioid receptor. While hit rates ranged from 40% to 63% against the on-targets, they were just as good against the antitargets, even though the molecules were selected for their putative lack of binding to the off-targets. Affinities, too, were often as good or better for the off-targets. Even though it was occasionally possible to find selective molecules, such as a mid-nanomolar D2/5-HT2A ligand with 21-fold selectivity versus the H1 receptor, this was the exception. Whereas false-negatives are tolerable in docking screens against on-targets, they are intolerable against antitargets; addressing this problem may demand new strategies in the field.

Structure-based discovery of selective positive allosteric modulators of antagonists for the M2 muscarinic acetylcholine receptor.

Subtype-selective antagonists for muscarinic acetylcholine receptors (mAChRs) have long been elusive, owing to the highly conserved orthosteric binding site. However, allosteric sites of these receptors are less conserved, motivating the search for allosteric ligands that modulate agonists or antagonists to confer subtype selectivity. Accordingly, a 4.6 million-molecule library was docked against the structure of the prototypical M2 mAChR, seeking molecules that specifically stabilized antagonist binding. This led us to identify a positive allosteric modulator (PAM) that potentiated the antagonist N-methyl scopolamine (NMS). Structure-based optimization led to compound '628, which enhanced binding of NMS, and the drug scopolamine itself, with a cooperativity factor (α) of 5.5 and a KB of 1.1 µM, while sparing the endogenous agonist acetylcholine. NMR spectral changes determined for methionine residues reflected changes in the allosteric network. Moreover, '628 slowed the dissociation rate of NMS from the M2 mAChR by 50-fold, an effect not observed at the other four mAChR subtypes. The specific PAM effect of '628 on NMS antagonism was conserved in functional assays, including agonist stimulation of [35S]GTPγS binding and ERK 1/2 phosphorylation. Importantly, the selective allostery between '628 and NMS was retained in membranes from adult rat hypothalamus and in neonatal rat cardiomyocytes, supporting the physiological relevance of this PAM/antagonist approach. This study supports the feasibility of discovering PAMs that confer subtype selectivity to antagonists; molecules like '628 can convert an armamentarium of potent but nonselective GPCR antagonist drugs into subtype-selective reagents, thus reducing their off-target effects.

GPCR-Bench: A Benchmarking Set and Practitioners' Guide for G Protein-Coupled Receptor Docking.

Virtual screening is routinely used to discover new ligands and in particular new ligand chemotypes for G protein-coupled receptors (GPCRs). To prepare for a virtual screen, we often tailor a docking protocol that will enable us to select the best candidates for further screening. To aid this, we created GPCR-Bench, a publically available docking benchmarking set in the spirit of the DUD and DUD-E reference data sets for validation studies, containing 25 nonredundant high-resolution GPCR costructures with an accompanying set of diverse ligands and computational decoy molecules for each target. Benchmarking sets are often used to compare docking protocols; however, it is important to evaluate docking methods not by "retrospective" hit rates but by the actual likelihood that they will produce novel prospective hits. Therefore, docking protocols must not only rank active molecules highly but also produce good poses that a chemist will select for purchase and screening. Currently, no simple objective machine-scriptable function exists that can do this; instead, docking hit lists must be subjectively examined in a consistent way to compare between docking methods. We present here a case study highlighting considerations we feel are of importance when evaluating a method, intended to be useful as a practitioners' guide.

Decoding the Role of Water Dynamics in Ligand-Protein Unbinding: CRF1R as a Test Case.

The residence time of a ligand-protein complex is a crucial aspect in determining biological effect in vivo. Despite its importance, the prediction of ligand koff still remains challenging for modern computational chemistry. We have developed aMetaD, a fast and generally applicable computational protocol to predict ligand-protein unbinding events using a molecular dynamics (MD) method based on adiabatic-bias MD and metadynamics. This physics-based, fully flexible, and pose-dependent ligand scoring function evaluates the maximum energy (RTscore) required to move the ligand from the bound-state energy basin to the next. Unbinding trajectories are automatically analyzed and translated into atomic solvation factor (SF) values representing the water dynamics during the unbinding event. This novel computational protocol was initially tested on two M3 muscarinic receptor and two adenosine A2A receptor antagonists and then evaluated on a test set of 12 CRF1R ligands. The resulting RTscores were used successfully to classify ligands with different residence times. Additionally, the SF analysis was used to detect key differences in the degree of accessibility to water molecules during the predicted ligand unbinding events. The protocol provides actionable working hypotheses that are applicable in a drug discovery program for the rational optimization of ligand binding kinetics.

Structures of mGluRs shed light on the challenges of drug development of allosteric modulators.

The metabotropic glutamate receptor family includes many potential therapeutic targets for a wide range of neurological disorders however to date no approved drugs have progressed to market. For some receptor subtypes it has been difficult to separate therapeutic benefit from undesirable side effects. For others finding suitable drug like molecules has been challenging. Chemotypes identified from screening have been limited and difficult to optimise away from undesirable groups. Frequently within related series, compounds have switched from agonist to antagonists. Recently the structures of the transmembrane domain of mGlu1 and mGlu5 have been solved revealing the binding site of allosteric modulators which provides an understanding of the difficulties to date and an opportunity for future structure based approaches to drug design.

Millisecond dynamics of RNA polymerase II translocation at atomic resolution.

Transcription is a central step in gene expression, in which the DNA template is processively read by RNA polymerase II (Pol II), synthesizing a complementary messenger RNA transcript. At each cycle, Pol II moves exactly one register along the DNA, a process known as translocation. Although X-ray crystal structures have greatly enhanced our understanding of the transcription process, the underlying molecular mechanisms of translocation remain unclear. Here we use sophisticated simulation techniques to observe Pol II translocation on a millisecond timescale and at atomistic resolution. We observe multiple cycles of forward and backward translocation and identify two previously unidentified intermediate states. We show that the bridge helix (BH) plays a key role accelerating the translocation of both the RNA:DNA hybrid and transition nucleotide by directly interacting with them. The conserved BH residues, Thr831 and Tyr836, mediate these interactions. To date, this study delivers the most detailed picture of the mechanism of Pol II translocation at atomic level.

SAMPL4 & DOCK3.7: lessons for automated docking procedures.

The SAMPL4 challenges were used to test current automated methods for solvation energy, virtual screening, pose and affinity prediction of the molecular docking pipeline DOCK 3.7. Additionally, first-order models of binding affinity were proposed as milestones for any method predicting binding affinity. Several important discoveries about the molecular docking software were made during the challenge: (1) Solvation energies of ligands were five-fold worse than any other method used in SAMPL4, including methods that were similarly fast, (2) HIV Integrase is a challenging target, but automated docking on the correct allosteric site performed well in terms of virtual screening and pose prediction (compared to other methods) but affinity prediction, as expected, was very poor, (3) Molecular docking grid sizes can be very important, serious errors were discovered with default settings that have been adjusted for all future work. Overall, lessons from SAMPL4 suggest many changes to molecular docking tools, not just DOCK 3.7, that could improve the state of the art. Future difficulties and projects will be discussed.

Morphing methods to visualize coarse-grained protein dynamics.

Morphing was initially developed as a cinematic effect, where one image is seamlessly transformed into another image. The technique was widely adopted by biologists to visualize the transition between protein conformational states, generating an interpolated pathway from an initial to a final protein structure. Geometric morphing seeks to create visually suggestive movies that illustrate structural changes between conformations but do not necessarily represent a biologically relevant pathway, while minimum energy path (MEP) interpolations aim at describing the true transition state between the crystal structure minima in the energy landscape.

Muscarinic receptors as model targets and antitargets for structure-based ligand discovery.

G protein-coupled receptors (GPCRs) regulate virtually all aspects of human physiology and represent an important class of therapeutic drug targets. Many GPCR-targeted drugs resemble endogenous agonists, often resulting in poor selectivity among receptor subtypes and restricted pharmacologic profiles. The muscarinic acetylcholine receptor family exemplifies these problems; thousands of ligands are known, but few are receptor subtype-selective and nearly all are cationic in nature. Using structure-based docking against the M2 and M3 muscarinic receptors, we screened 3.1 million molecules for ligands with new physical properties, chemotypes, and receptor subtype selectivities. Of 19 docking-prioritized molecules tested against the M2 subtype, 11 had substantial activity and 8 represented new chemotypes. Intriguingly, two were uncharged ligands with low micromolar to high nanomolar Ki values, an observation with few precedents among aminergic GPCRs. To exploit a single amino-acid substitution among the binding pockets between the M2 and M3 receptors, we selected molecules predicted by docking to bind to the M3 and but not the M2 receptor. Of 16 molecules tested, 8 bound to the M3 receptor. Whereas selectivity remained modest for most of these, one was a partial agonist at the M3 receptor without measurable M2 agonism. Consistent with this activity, this compound stimulated insulin release from a mouse ß-cell line. These results support the ability of structure-based discovery to identify new ligands with unexplored chemotypes and physical properties, leading to new biologic functions, even in an area as heavily explored as muscarinic pharmacology.

Conformation guides molecular efficacy in docking screens of activated ß-2 adrenergic G protein coupled receptor.

A prospective, large library virtual screen against an activated ß2-adrenergic receptor (ß2AR) structure returned potent agonists to the exclusion of inverse-agonists, providing the first complement to the previous virtual screening campaigns against inverse-agonist-bound G protein coupled receptor (GPCR) structures, which predicted only inverse-agonists. In addition, two hits recapitulated the signaling profile of the co-crystal ligand with respect to the G protein and arrestin mediated signaling. This functional fidelity has important implications in drug design, as the ability to predict ligands with predefined signaling properties is highly desirable. However, the agonist-bound state provides an uncertain template for modeling the activated conformation of other GPCRs, as a dopamine D2 receptor (DRD2) activated model templated on the activated ß2AR structure returned few hits of only marginal potency.

Structure-based ligand discovery for the protein-protein interface of chemokine receptor CXCR4.

G-protein-coupled receptors (GPCRs) are key signaling molecules and are intensely studied. Whereas GPCRs recognizing small-molecules have been successfully targeted for drug discovery, protein-recognizing GPCRs, such as the chemokine receptors, claim few drugs or even useful small molecule reagents. This reflects both the difficulties that attend protein-protein interface inhibitor discovery, and the lack of structures for these targets. Imminent structure determination of chemokine receptor CXCR4 motivated docking screens for new ligands against a homology model and subsequently the crystal structure. More than 3 million molecules were docked against the model and then against the crystal structure; 24 and 23 high-scoring compounds from the respective screens were tested experimentally. Docking against the model yielded only one antagonist, which resembled known ligands and lacked specificity, whereas the crystal structure docking yielded four that were dissimilar to previously known scaffolds and apparently specific. Intriguingly, several were potent and relatively small, with IC(50) values as low as 306 nM, ligand efficiencies as high as 0.36, and with efficacy in cellular chemotaxis. The potency and efficiency of these molecules has few precedents among protein-protein interface inhibitors, and supports structure-based efforts to discover leads for chemokine GPCRs.

Optimized torsion-angle normal modes reproduce conformational changes more accurately than cartesian modes.

We present what to our knowledge is a new method of optimized torsion-angle normal-mode analysis, in which the normal modes move along curved paths in Cartesian space. We show that optimized torsion-angle normal modes reproduce protein conformational changes more accurately than Cartesian normal modes. We also show that orthogonalizing the displacement vectors from torsion-angle normal-mode analysis and projecting them as straight lines in Cartesian space does not lead to better performance than Cartesian normal modes. Clearly, protein motion is more naturally described by curved paths in Cartesian space.

RNA polymerase II trigger loop residues stabilize and position the incoming nucleotide triphosphate in transcription.

A structurally conserved element, the trigger loop, has been suggested to play a key role in substrate selection and catalysis of RNA polymerase II (pol II) transcription elongation. Recently resolved X-ray structures showed that the trigger loop forms direct interactions with the beta-phosphate and base of the matched nucleotide triphosphate (NTP) through residues His1085 and Leu1081, respectively. In order to understand the role of these two critical residues in stabilizing active site conformation in the dynamic complex, we performed all-atom molecular dynamics simulations of the wild-type pol II elongation complex and its mutants in explicit solvent. In the wild-type complex, we found that the trigger loop is stabilized in the "closed" conformation, and His1085 forms a stable interaction with the NTP. Simulations of point mutations of His1085 are shown to affect this interaction; simulations of alternative protonation states, which are inaccessible through experiment, indicate that only the protonated form is able to stabilize the His1085-NTP interaction. Another trigger loop residue, Leu1081, stabilizes the incoming nucleotide position through interaction with the nucleotide base. Our simulations of this Leu mutant suggest a three-component mechanism for correctly positioning the incoming NTP in which (i) hydrophobic contact through Leu1081, (ii) base stacking, and (iii) base pairing work together to minimize the motion of the incoming NTP base. These results complement experimental observations and provide insight into the role of the trigger loop on transcription fidelity.

Can morphing methods predict intermediate structures?

Movement is crucial to the biological function of many proteins, yet crystallographic structures of proteins can give us only a static snapshot. The protein dynamics that are important to biological function often happen on a timescale that is unattainable through detailed simulation methods such as molecular dynamics as they often involve crossing high-energy barriers. To address this coarse-grained motion, several methods have been implemented as web servers in which a set of coordinates is usually linearly interpolated from an initial crystallographic structure to a final crystallographic structure. We present a new morphing method that does not extrapolate linearly and can therefore go around high-energy barriers and which can produce different trajectories between the same two starting points. In this work, we evaluate our method and other established coarse-grained methods according to an objective measure: how close a coarse-grained dynamics method comes to a crystallographically determined intermediate structure when calculating a trajectory between the initial and final crystal protein structure. We test this with a set of five proteins with at least three crystallographically determined on-pathway high-resolution intermediate structures from the Protein Data Bank. For simple hinging motions involving a small conformational change, segmentation of the protein into two rigid sections outperforms other more computationally involved methods. However, large-scale conformational change is best addressed using a nonlinear approach and we suggest that there is merit in further developing such methods.

How hydrophobic buckminsterfullerene affects surrounding water structure.

The hydrophobic hydration of fullerenes in water is of significant interest as the most common Buckminsterfullerene (C60) is a mesoscale sphere; C60 also has potential in pharmaceutical and nanomaterial applications. We use an all-atom molecular dynamics simulation lasting hundreds of nanoseconds to determine the behavior of a single molecule of C60 in a periodic box of water, and compare this to methane. A C60 molecule does not induce drying at the surface; however, unlike a hard sphere methane, a hard sphere C60 solute does. This is due to a larger number of attractive Lennard-Jones interactions between the carbon atom centers in C60 and the surrounding waters. In these simulations, water is not uniformly arranged but rather adopts a range of orientations in the first hydration shell despite the spherical symmetry of both solutes. There is a clear effect of solute size on the orientation of the first hydration shell waters. There is a large increase in hydrogen-bonding contacts between waters in the C60 first hydration shell. There is also a disruption of hydrogen bonds between waters in the first and second hydration shells. Water molecules in the first hydration shell preferentially create triangular structures that minimize the net water dipole near the surface near both the methane and C60 surface, reducing the total energy of the system. Additionally, in the first and second hydration shells, the water dipoles are ordered to a distance of 8 A from the solute surface. We conclude that, with a diameter of approximately 1 nm, C60 behaves as a large hydrophobic solute.