Preclinical characterization of a non-peptidomimetic HIV protease inhibitor with improved metabolic stability.

Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents.

Scaffold-hopping from xanthines to tricyclic guanines: A case study of dipeptidyl peptidase 4 (DPP4) inhibitors.

Molecular modeling of unbound tricyclic guanine scaffolds indicated that they can serve as effective bioisosteric replacements of xanthines. This notion was further confirmed by a combination of X-ray crystallography and SAR studies, indicating that tricyclic guanine DPP4 inhibitors mimic the binding mode of xanthine inhibitors, exemplified by linagliptin. Realization of the bioisosteric relationship between these scaffolds potentially will lead to a wider application of cyclic guanines as xanthine replacements in drug discovery programs for a variety of biological targets. Newly designed DPP4 inhibitors achieved sub-nanomolar potency range and demonstrated oral activity in vivo in mouse glucose tolerance test.

Iminopyrimidinones: a novel pharmacophore for the development of orally active renin inhibitors.

The development of renin inhibitors with favorable oral pharmacokinetic profiles has been a longstanding challenge for the pharmaceutical industry. As part of our work to identify inhibitors of BACE1, we have previously developed iminopyrimidinones as a novel pharmacophore for aspartyl protease inhibition. In this letter we describe how we modified substitution around this pharmacophore to develop a potent, selective and orally active renin inhibitor.

eCounterscreening: using QSAR predictions to prioritize testing for off-target activities and setting the balance between benefit and risk.

During drug development, compounds are tested against counterscreens, a panel of off-target activities that would be undesirable for a drug to have. Testing every compound against every counterscreen is generally too costly in terms of time and money, and we need to find a rational way of prioritizing counterscreen testing. Here we present the eCounterscreening paradigm, wherein predictions from QSAR models for counterscreen activity are used to generate a recommendation as to whether a specific compound in a specific project should be tested against a specific counterscreen. The rules behind the recommendations, which can be summarized in a risk-benefit plot specific for a counterscreen/project combination, are based on a previously assembled database of prospective QSAR predictions. The recommendations require two user-defined cutoffs: the level of activity in a specific counterscreen that is considered undesirable and the level of risk the chemist is willing to accept that an undesired counterscreen activity will go undetected. We demonstrate in a simulated prospective experiment that eCounterscreening can be used to postpone a large fraction of counterscreen testing and still have an acceptably low risk of undetected counterscreen activity.

Discovery of potent iminoheterocycle BACE1 inhibitors.

The synthesis of a series of iminoheterocycles and their structure-activity relationships (SAR) as inhibitors of the aspartyl protease BACE1 will be detailed. An effort to access the S3 subsite directly from the S1 subsite initially yielded compounds with sub-micromolar potency. A subset of compounds from this effort unexpectedly occupied a different binding site and displayed excellent BACE1 affinities. Select compounds from this subset acutely lowered Aß40 levels upon subcutaneous and oral administration to rats.

Combining NMR and X-ray crystallography in fragment-based drug discovery: discovery of highly potent and selective BACE-1 inhibitors.

Fragment-based drug discovery (FBDD) has become increasingly popular over the last decade. We review here how we have used highly structure-driven fragment-based approaches to complement more traditional lead discovery to tackle high priority targets and those struggling for leads. Combining biomolecular nuclear magnetic resonance (NMR), X-ray crystallography, and molecular modeling with structure-assisted chemistry and innovative biology as an integrated approach for FBDD can solve very difficult problems, as illustrated in this chapter. Here, a successful FBDD campaign is described that has allowed the development of a clinical candidate for BACE-1, a challenging CNS drug target. Crucial to this achievement were the initial identification of a ligand-efficient isothiourea fragment through target-based NMR screening and the determination of its X-ray crystal structure in complex with BACE-1, which revealed an extensive H-bond network with the two active site aspartate residues. This detailed 3D structural information then enabled the design and validation of novel, chemically stable and accessible heterocyclic acylguanidines as aspartic acid protease inhibitor cores. Structure-assisted fragment hit-to-lead optimization yielded iminoheterocyclic BACE-1 inhibitors that possess desirable molecular properties as potential therapeutic agents to test the amyloid hypothesis of Alzheimer's disease in a clinical setting.

Synthesis and SAR studies of imidazo-[1,2-a]-pyrazine Aurora kinase inhibitors with improved off-target kinase selectivity.

The structure-activity relationships of new Aurora A/B kinase inhibitors derived from the previously identified kinase inhibitor 12 are described. Introduction of acetic acid amides onto the pyrazole of compound 12 was postulated to influence Aurora A/B selectivity and improve the profile against off-target kinases. The SAR of the acetic acid amides was explored and the effect of substitution on enzyme inhibition as well as mechanism-based cell activity was studied. Additionally, several of the more potent inhibitors were screened for their off-target kinase selectivity.

Structure based design of iminohydantoin BACE1 inhibitors: identification of an orally available, centrally active BACE1 inhibitor.

From an initial lead 1, a structure-based design approach led to identification of a novel, high-affinity iminohydantoin BACE1 inhibitor that lowers CNS-derived Aß following oral administration to rats. Herein we report SAR development in the S3 and F' subsites of BACE1 for this series, the synthetic approaches employed in this effort, and in vivo data for the optimized compound.

Comparison of random forest and Pipeline Pilot Naïve Bayes in prospective QSAR predictions.

Random forest is currently considered one of the best QSAR methods available in terms of accuracy of prediction. However, it is computationally intensive. Naïve Bayes is a simple, robust classification method. The Laplacian-modified Naïve Bayes implementation is the preferred QSAR method in the widely used commercial chemoinformatics platform Pipeline Pilot. We made a comparison of the ability of Pipeline Pilot Naïve Bayes (PLPNB) and random forest to make accurate predictions on 18 large, diverse in-house QSAR data sets. These include on-target and ADME-related activities. These data sets were set up as classification problems with either binary or multicategory activities. We used a time-split method of dividing training and test sets, as we feel this is a realistic way of simulating prospective prediction. PLPNB is computationally efficient. However, random forest predictions are at least as good and in many cases significantly better than those of PLPNB on our data sets. PLPNB performs better with ECFP4 and ECFP6 descriptors, which are native to Pipeline Pilot, and more poorly with other descriptors we tried.

Small-molecule inhibitors of FABP4/5 ameliorate dyslipidemia but not insulin resistance in mice with diet-induced obesity.

Fatty acid binding protein-4 (FABP4) and FABP5 are two closely related FA binding proteins expressed primarily in adipose tissue and/or macrophages. The small-molecule FABP4 inhibitor BMS309403 was previously reported to improve insulin sensitivity in leptin-deficient Lep(ob)/Lep(ob) (ob/ob) mice. However, this compound was not extensively characterized in the more physiologically relevant animal model of mice with diet-induced obesity (DIO). Here, we report the discovery and characterization of a novel series of FABP4/5 dual inhibitors represented by Compounds 1-3. Compared with BMS309403, the compounds had significant in vitro potency toward both FABP4 and FABP5. In cell-based assays, Compounds 2 and 3 were more potent than BMS309403 to inhibit lipolysis in 3T3-L1 adipocytes and in primary human adipocytes. They also inhibited MCP-1 release from THP-1 macrophages as well as from primary human macrophages. When chronically administered to DIO mice, BMS309403 and Compound 3 reduced plasma triglyceride and free FA levels. Compound 3 reduced plasma free FAs at a lower dose level than BMS309403. However, no significant change was observed in insulin, glucose, or glucose tolerance. Our results indicate that the FABP4/5 inhibitors ameliorate dyslipidemia but not insulin resistance in DIO mice.

Discovery of imidazo[1,2-a]pyrazine-based Aurora kinase inhibitors.

The synthesis and structure-activity relationships (SAR) of novel, potent imidazo[1,2-a]pyrazine-based Aurora kinase inhibitors are described. The X-ray crystal structure of imidazo[1,2-a]pyrazine Aurora inhibitor 1j is disclosed. Compound 10i was identified as lead compound with a promising overall profile.

Iminoheterocycles as gamma-secretase modulators.

The synthesis of a novel series of iminoheterocycles and their structure-activity relationship (SAR) as modulators of gamma-secretase activity will be detailed. Encouraging SAR generated from a monocyclic core led to a structurally unique bicyclic core. Selected compounds exhibit good potency as gamma-secretase modulators, excellent rat pharmacokinetics, and lowering of Abeta42 levels in various in vivo models.

Piperazine sulfonamide BACE1 inhibitors: design, synthesis, and in vivo characterization.

With collaboration between chemistry, X-ray crystallography, and molecular modeling, we designed and synthesized a series of novel piperazine sulfonamide BACE1 inhibitors. Iterative exploration of the non-prime side and S2' sub-pocket of the enzyme culminated in identification of an analog that potently lowers peripheral Abeta(40) in transgenic mice with a single subcutaneous dose.

Discovery of novel chemotypes to a G-protein-coupled receptor through ligand-steered homology modeling and structure-based virtual screening.

Melanin-concentrating hormone receptor 1 (MCH-R1) is a G-protein-coupled receptor (GPCR) and a target for the development of therapeutics for obesity. The structure-based development of MCH-R1 and other GPCR antagonists is hampered by the lack of an available experimentally determined atomic structure. A ligand-steered homology modeling approach has been developed (where information about existing ligands is used explicitly to shape and optimize the binding site) followed by docking-based virtual screening. Top scoring compounds identified virtually were tested experimentally in an MCH-R1 competitive binding assay, and six novel chemotypes as low micromolar affinity antagonist "hits" were identified. This success rate is more than a 10-fold improvement over random high-throughput screening, which supports our ligand-steered method. Clearly, the ligand-steered homology modeling method reduces the uncertainty of structure modeling for difficult targets like GPCRs.

Initial Assessment of a Prototype 3D Cone-Beam Computed Tomography System for Imaging of the Lumbar Spine, Evaluating Human Cadaveric Specimens in the Upright Position.

OBJECTIVES: The aims of this study were to assess feasibility, image quality, and radiation dose and to estimate the optimal dose protocol for the lumbar spine of cadaveric specimens with different body mass indices (BMIs) in the upright position using a prototype 3-dimensional cone-beam computed tomography (CT) software implemented on a robotic x-ray system and compare with CT. MATERIALS AND METHODS: The lumbar spine of 5 formalin-fixed human cadaveric specimens (BMI, 22-35 kg/m) was prospectively assessed in the upright position using prototype software for 3-dimensional tomography implemented on a robotic x-ray system. Specimens were scanned with varying kilovolt values (70, 81, 90, 100, 109, 121 kV) and thereafter with 80 kV (BMI ≤30 kg/m) and 121 kV (BMI >30 kg/m) and varying dose levels (DLs; 0.278, 0.435, 0.548, 0.696, 0.87, 1.09). Computed tomography data were acquired with a standard clinical protocol. Two independent readers rated visibility of the cortex, endplates, facet joints, trabeculae, neuroforamina, posterior alignment, and spinal canal as well as nerve roots. Radiation dose was measured with a cylindrical CTDI phantom. Descriptive statistics and analysis of variance were used (P < 0.05). RESULTS: Average intraclass correlation was excellent (0.94). The lowest technically possible kilovolt and the highest technically possible DL yielded the best image quality. In specimens with a BMI of 30 kg/m or less, depiction of all structures was good and comparable to CT, except for nerve roots. For specimens with a BMI greater than 30 kg/m, image quality was limited. CONCLUSIONS: Three-dimensional cone-beam CT of the lumbar spine in cadaveric specimens in the upright position is feasible. An optimal dose protocol was estimated. Depiction of osseous structures is comparable to CT in specimens with BMI of 30 kg/m or less. Image quality is limited for soft tissue structures and specimens with BMI greater than 30 kg/m.

Initial evaluation of image performance of a 3-D x-ray system: phantom-based comparison of 3-D tomography with conventional computed tomography.

Phantom-based initial performance assessment of a prototype three-dimensional (3-D) x-ray system and comparison of 3-D tomography with computed tomography (CT) were proposed. A 3-D image quality phantom was scanned with a prototype version of 3-D cone-beam CT imaging implemented on a twin robotic x-ray system using three trajectories (163 deg = table, 188 deg = upright, and 200 deg = side), six tube voltages (60, 70, 81, 90, 100, and 121 kV), and four detector doses (0.348, 0.696, 1.740, and [Formula: see text]). CT was obtained with a clinical protocol. Spatial resolution (line pairs/cm) and soft-tissue-contrast resolution were assessed by two independent readers. Radiation dose was assessed. Descriptive and analysis of variance (ANOVA) ([Formula: see text]) were performed. With 3-D tomography, a maximum of 16 lp/cm was visible and best soft-tissue-contrast resolution was 2 mm at 30 Hounsfield units (HU) for 160 projections. With CT, 10 lp/cm was visible and soft-tissue-contrast resolution was 4 mm at 20 HU. The upright trajectory yielded significantly better spatial resolution and soft tissue contrast, and the side trajectory yielded significantly higher soft tissue contrast than the table trajectory ([Formula: see text]). Radiation dose was higher in 3-D tomography (45 to 704 mGycm) than CT (44 mGycm). Three-dimensional tomography renders overall equal or higher spatial resolution and comparable soft tissue contrast to CT for medium- and high-dose protocols in the side and upright trajectories, but with higher radiation doses.

Identification of inhibitors to papillomavirus type 16 E6 protein based on three-dimensional structures of interacting proteins.

Human papillomaviruses (HPV) cause cutaneous and genital warts. A subset of HPV types is associated with a high-risk for progression to malignancy. The E6 protein from the high-risk HPV types represents an attractive target for intervention because of its roles in viral propagation and cellular transformation. E6 functions in part by interaction with human cellular proteins, several of which possess a helical E6-binding motif. The role for each amino acid in this motif for binding E6 has been tested through structure determination and site-directed mutagenesis. These structural and molecular biological approaches defined the spatial geometry of functional groups necessary for binding to E6. This E6-binding information (the E6-binding pharmacophore) was transferred into a three-dimensional query format suitable for computational screening of large chemical databases. Compounds were identified and tested using in vitro and cell culture-based assays. Several compounds selectively inhibited E6 interaction with the E6-binding protein E6AP and interfered with the ability of E6 to promote p53 degradation. Such compounds provide leads for the development of new pharmacologic agents to treat papillomavirus infections and their associated cancers.

Structure-Based Design of an Iminoheterocyclic ß-Site Amyloid Precursor Protein Cleaving Enzyme (BACE) Inhibitor that Lowers Central Aß in Nonhuman Primates.

We describe successful efforts to optimize the in vivo profile and address off-target liabilities of a series of BACE1 inhibitors represented by 6 that embodies the recently validated fused pyrrolidine iminopyrimidinone scaffold. Employing structure-based design, truncation of the cyanophenyl group of 6 that binds in the S3 pocket of BACE1 followed by modification of the thienyl group in S1 was pursued. Optimization of the pyrimidine substituent that binds in the S2'-S2″ pocket of BACE1 remediated time-dependent CYP3A4 inhibition of earlier analogues in this series and imparted high BACE1 affinity. These efforts resulted in the discovery of difluorophenyl analogue 9 (MBi-4), which robustly lowered CSF and cortex Aß40 in both rats and cynomolgus monkeys following a single oral dose. Compound 9 represents a unique molecular shape among BACE inhibitors reported to potently lower central Aß in nonrodent preclinical species.

Discovery of the 3-Imino-1,2,4-thiadiazinane 1,1-Dioxide Derivative Verubecestat (MK-8931)-A ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor for the Treatment of Alzheimer's Disease.

Verubecestat 3 (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-thiadiazinane 1,1-dioxide derivative, is a high-affinity ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor currently undergoing Phase 3 clinical evaluation for the treatment of mild to moderate and prodromal Alzheimer's disease. Although not selective over the closely related aspartyl protease BACE2, verubecestat has high selectivity for BACE1 over other key aspartyl proteases, notably cathepsin D, and profoundly lowers CSF and brain Aß levels in rats and nonhuman primates and CSF Aß levels in humans. In this annotation, we describe the discovery of 3, including design, validation, and selected SAR around the novel iminothiadiazinane dioxide core as well as aspects of its preclinical and Phase 1 clinical characterization.

Prosit, an online service to calculate pseudorotational parameters of nucleosides and nucleotides.

The online service PROSIT (Pseudo-Rotational Online Service and Interactive Tool) is a free service available at http://cactus.nci.nci.gov/prosit/ that performs pseudorotational analysis of nucleosides(tides). PROSIT reads the 3D coordinates of nucleosides and returns the pseudorotational phase angle P, puckering amplitude vmax and other related information. As examples, the sugar conformations in a parallel-stranded guanine tetraplex and a four-way Holliday junction are presented here.