Structure Guided Discovery of Novel Pan Metallo-ß-Lactamase Inhibitors with Improved Gram-Negative Bacterial Cell Penetration.

The use of ß-lactam (BL) and ß-lactamase inhibitor combination to overcome BL antibiotic resistance has been validated through clinically approved drug products. However, unmet medical needs still exist for the treatment of infections caused by Gram-negative (GN) bacteria expressing metallo-ß-lactamases. Previously, we reported our effort to discover pan inhibitors of three main families in this class: IMP, VIM, and NDM. Herein, we describe our work to improve the GN coverage spectrum in combination with imipenem and relebactam. This was achieved through structure- and property-based optimization to tackle the GN cell penetration and efflux challenges. A significant discovery was made that inhibition of both VIM alleles, VIM-1 and VIM-2, is essential for broad GN coverage, especially against VIM-producing P. aeruginosa. In addition, pharmacokinetics and nonclinical safety profiles were investigated for select compounds. Key findings from this drug discovery campaign laid the foundation for further lead optimization toward identification of preclinical candidates.

Orally Bioavailable Macrocyclic Peptide That Inhibits Binding of PCSK9 to the Low Density Lipoprotein Receptor.

BACKGROUND: Inhibition of PCSK9 (proprotein convertase subtilisin/kexin type 9)-low density lipoprotein receptor interaction with injectable monoclonal antibodies or small interfering RNA lowers plasma low density lipoprotein-cholesterol, but despite nearly 2 decades of effort, an oral inhibitor of PCSK9 is not available. Macrocyclic peptides represent a novel approach to target proteins traditionally considered intractable to small-molecule drug design. METHODS: Novel mRNA display screening technology was used to identify lead chemical matter, which was then optimized by applying structure-based drug design enabled by novel synthetic chemistry to identify macrocyclic peptide (MK-0616) with exquisite potency and selectivity for PCSK9. Following completion of nonclinical safety studies, MK-0616 was administered to healthy adult participants in a single rising-dose Phase 1 clinical trial designed to evaluate its safety, pharmacokinetics, and pharmacodynamics. In a multiple-dose trial in participants taking statins, MK-0616 was administered once daily for 14 days to characterize the safety, pharmacokinetics, and pharmacodynamics (change in low density lipoprotein cholesterol). RESULTS: MK-0616 displayed high affinity (Ki = 5pM) for PCSK9 in vitro and sufficient safety and oral bioavailability preclinically to enable advancement into the clinic. In Phase 1 clinical studies in healthy adults, single oral doses of MK-0616 were associated with >93% geometric mean reduction (95% CI, 84-103) of free, unbound plasma PCSK9; in participants on statin therapy, multiple-oral-dose regimens provided a maximum 61% geometric mean reduction (95% CI, 43-85) in low density lipoprotein cholesterol from baseline after 14 days of once-daily dosing of 20 mg MK-0616. CONCLUSIONS: This work validates the use of mRNA display technology for identification of novel oral therapeutic agents, exemplified by the identification of an oral PCSK9 inhibitor, which has the potential to be a highly effective cholesterol lowering therapy for patients in need.

IGTCM: An integrative genome database of traditional Chinese medicine plants.

Fully understanding traditional Chinese medicines (TCMs) is still challenging because of the extreme complexity of their chemical components and mechanisms of action. The TCM Plant Genome Project aimed to obtain genetic information, determine gene functions, discover regulatory networks of herbal species, and elucidate the molecular mechanisms involved in the disease prevention and treatment, thereby accelerating the modernization of TCMs. A comprehensive database that contains TCM-related information will provide a vital resource. Here, we present an integrative genome database of TCM plants (IGTCM) that contains 14,711,220 records of 83 annotated TCM-related herb genomes, including 3,610,350 genes, 3,534,314 proteins and corresponding coding sequences, and 4,032,242 RNAs, as well as 1033 non-redundant component records for 68 herbs, downloaded and integrated from the GenBank and RefSeq databases. For minimal interconnectivity, each gene, protein, and component was annotated using the eggNOG-mapper tool and Kyoto Encyclopedia of Genes and Genomes database to acquire pathway information and enzyme classifications. These features can be linked across several species and different components. The IGTCM database also provides visualization and sequence similarity search tools for data analyses. These annotated herb genome sequences in IGTCM database are a necessary resource for systematically exploring genes related to the biosynthesis of compounds that have significant medicinal activities and excellent agronomic traits that can be used to improve TCM-related varieties through molecular breeding. It also provides valuable data and tools for future research on drug discovery and the protection and rational use of TCM plant resources. The IGTCM database is freely available at http://yeyn.group:96/.

Emerging Flexible Thermally Conductive Films: Mechanism, Fabrication, Application.

Effective thermal management is quite urgent for electronics owing to their ever-growing integration degree, operation frequency and power density, and the main strategy of thermal management is to remove excess energy from electronics to outside by thermal conductive materials. Compared to the conventional thermal management materials, flexible thermally conductive films with high in-plane thermal conductivity, as emerging candidates, have aroused greater interest in the last decade, which show great potential in thermal management applications of next-generation devices. However, a comprehensive review of flexible thermally conductive films is rarely reported. Thus, we review recent advances of both intrinsic polymer films and polymer-based composite films with ultrahigh in-plane thermal conductivity, with deep understandings of heat transfer mechanism, processing methods to enhance thermal conductivity, optimization strategies to reduce interface thermal resistance and their potential applications. Lastly, challenges and opportunities for the future development of flexible thermally conductive films are also discussed.

Discovery of MK-8153, a Potent and Selective ROMK Inhibitor and Novel Diuretic/Natriuretic.

A renal outer medullary potassium channel (ROMK, Kir1.1) is a putative drug target for a novel class of diuretics with potential for treating hypertension and heart failure. Our first disclosed clinical ROMK compound, 2 (MK-7145), demonstrated robust diuresis, natriuresis, and blood pressure lowering in preclinical models, with reduced urinary potassium excretion compared to the standard of care diuretics. However, 2 projected to a short human half-life (∼5 h) that could necessitate more frequent than once a day dosing. In addition, a short half-life would confer a high peak-to-trough ratio which could evoke an excessive peak diuretic effect, a common liability associated with loop diuretics such as furosemide. This report describes the discovery of a new ROMK inhibitor 22e (MK-8153), with a longer projected human half-life (∼14 h), which should lead to a reduced peak-to-trough ratio, potentially extrapolating to more extended and better tolerated diuretic effects.

Behavior of an Advanced Bolted Shear Connector in Prefabricated Steel-Concrete Composite Beams.

The high-strength bolt shear connector in prefabricated concrete slab has advantages in applications as it reduces time during the construction of steel-concrete composite building structures and bridges. In this research, an innovative and advanced bolt shear connector in steel-concrete composite structures is proposed. To investigate the fundamental mechanical behavior and the damage form, 22 static push-off tests were conducted with consideration of different bolt dimensions, the reserved hole constraint condition, and the dimension of slab holes. A finite element (FE) model was established and verified by using test results, and then the model was utilized to investigate the influence of concrete strength, bolt dimension, yield strength, bolt pretension, as well as length-to-diameter ratio of high strength bolts on the performances of shear connectors. On the basis of FE simulation and test results, new design formulas for the calculation of shear resistance behavior were proposed, and comparisons were made with current standards, including AISC, EN 1994-1-1, GB 50017-2017, and relevant references, to check the calculation efficiency. It is confirmed that the proposed equation is in better agreement with the experimental results.

Highly (00l)-oriented Bi2Te3/Te heterostructure thin films with enhanced power factor.

Introducing nanoscale heterostructure interfaces into material matrix is an effective strategy to optimize the thermoelectric performance by energy-dependent carrier filtering effect. In this study, highly (00l)-oriented Bi2Te3/Te heterostructure thin films have been fabricated on single-crystal MgO substrates using a facile magnetron co-sputtering method. Bi2Te3/Te heterostructure thin films with Te contents of 63.8 at% show an optimized thermoelectric performance, which possess a Seebeck coefficient of -157.7 µV K-1 and an electrical conductivity of 9.72 × 104 S m-1, leading to a high power factor approaching 25 µW cm-1 K-2. The partially decoupled behavior of the Seebeck coefficient and electrical conductivity is contributed to Bi2Te3/Te heterostructure interfaces, which causes interfacial barrier filtering and scattering effects; thus, a high level of the Seebeck coefficient is obtained. Meanwhile, carrier transport in a-b plane can benefit from the highly preferred orientation, which guarantees a remarkably high electrical conductivity. We anticipate that our strategy may guide the way for preparing high-performance thermoelectric materials by microstructure design and regulation.

Arterial Stiffness and Cerebral Small Vessel Disease.

Background and Objective: Studies on relations between arterial stiffness and full spectrum of radiological features of cerebral small vessel disease (CSVD) are scarce. We aim to investigate the association of arterial stiffness with lacunes, white matter hyperintensities (WMH), microbleeds (CMBs), dilated perivascular spaces (PVS), and brain atrophy in a community-based sample. Methods: A total of 953 participants (55.7 ± 9.4 years) who underwent brachial-ankle pulse wave velocity (baPWV) and brain magnetic resonance imaging were included. Lacunes, CMBs, and PVS were visually rated. Brain structure and WMH were automatically segmented. Brain parenchyma fraction (BPF), a surrogate index of brain atrophy, was calculated as a ratio of brain parenchyma volume to total intracranial volume. Multivariable logistic and linear regressions were used to investigate the associations between baPWV and CSVD. Subsequently, we explored these associations in strata of age. Results: Increased baPWV was associated with severe PVS in white matter (OR, 1.09; 95%CI, 1.01-1.17; p = 0.022), larger WMH volume (ß, 0.08; 95%CI, 0.04-0.12; p < 0.001), lower BPF (ß, -0.09; 95%CI, -0.15- -0.03; p = 0.007), and marginally associated with strictly lobar CMBs (OR, 1.11; 95%CI, 1.00-1.23; p = 0.055), but not with lacunes. WMH volume mediated the relation between baPWV and BPF. In age subgroup analysis, the association of baPWV with PVS in white matter was stronger among those aged <55 years, whereas the association with brain atrophy was more prominent among those aged ≥55 years. Increased baPWV was associated with larger WMH volume in both younger and older individuals. Conclusions: Increased arterial stiffness was associated with most of imaging markers of CSVD, including PVS in white matter, larger WMH volume, strictly lobar CMBs, and brain atrophy, but not lacunes. The mechanisms underlying these associations and their potential clinical significances warrant further investigations.

Relationship between Neutrophil-to-Lymphocyte Ratio and Pulse Wave Velocity in Young Patients with Systemic Lupus Erythematosus.

BACKGROUND: The incidence of atherosclerosis-related myocardial infarction can be as much as 50-fold greater in young patients with systemic lupus erythematosus (SLE) than in age-matched controls. There are several explanations for this phenomenon, all of which result in a chronic state of low-grade inflammation. Recently, the neutrophil-to-lymphocyte ratio (NLR) has been proposed as a useful biomarker of inflammation. Pulse wave velocity (PWV) is a reliable indicator of vascular damage and atherosclerosis. There is a paucity of data concerning the relationship between NLR and atherosclerosis as measured by PWV in patients with SLE. This study aimed to verify whether there is a positive correlation between NLR and PWV and to explore factors that influence PWV in young SLE patients. METHODS: A total of 90 female patients with SLE were enrolled in this cross-sectional investigation. Traditional and nontraditional cardiovascular risk factors were assessed on the same day that brachial-ankle PWV (baPWV) was examined. The patients were divided into three groups according to their mean baPWV values: patients whose mean baPWV value was lower than the first tertile were placed in Group 1; patients whose mean baPWV value was between the first tertile and the second tertile were placed in Group 2; and patients whose mean baPWV value was higher than the second tertile were placed in Group 3. SPSS 20.0 was used to perform all statistical analyses in this study. Both univariate linear regression and multivariate regression models were utilized to analyze the association between NLR and arterial stiffness. RESULTS: Systolic blood pressure, diastolic blood pressure (DBP), and triglycerides were all significantly different among Groups 1, 2, and 3 (111.90 ± 12.85 mmHg vs. 114.60 ± 12.88 mmHg vs. 129.43 ± 16.21 mmHg, P < 0.001; 68.77 ± 8.63 mmHg vs. 71.87 ± 9.77 mmHg vs. 82.57 ± 14.89 mmHg, P < 0.001; and 1.44 [0.91-2.47] mmol/L vs. 0.98 [0.78-1.26] mmol/L vs. 2.20 [0.94-3.66] mmol/L, P = 0.030; respectively), as were creatinine (57.50 [52.00-69.00] µmol/L vs. 55.50 [49.00-64.00] µmol/L vs. 64.00 [56.00-86.00] µmol/L, P = 0.045) and blood urea nitrogen (4.27 [3.79-6.22] mmol/L vs. 4.16 [3.47-4.84] mmol/L vs. 5.88 [4.04-8.19] mmol/L, P = 0.011). NLRs were significantly different among Groups 1, 2, and 3 (2.16 [1.56-3.42] vs. 3.12 [1.91-4.19] vs. 5.29 [2.63-7.25], P = 0.001). NLR, together with DBP and the SLE disease activity index, independently predicts PWV. CONCLUSIONS: This study demonstrated that there was a positive correlation between NLR and PWV. Moreover, we found that disease activity and DBP were also positively correlated with PWV.

Pin1 and secondary hyperparathyroidism of chronic kidney disease: gene polymorphisms and protein levels.

BACKGROUND: Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is a key regulator of PTH mRNA stability. Secondary hyperparathyroidism (SHPT), which is characterized by elevated serum PTH levels, is a common complication of CKD. We investigated the possible associations between CKD with SHPT (CKD SHPT) and single-nucleotide polymorphisms of the Pin1 gene and compared the levels of the Pin1 protein in the CKD SHPT patients with those of the controls. METHODS: The study group included 251 CKD SHPT patients and 61 controls. One putative functional SNP (single nucleotide polymorphism) in the Pin1 promoter (rs2233679C > T: c.-667C > T) is the main object. Genotyping was performed on purified DNA using polymerase chain reaction-restriction (PCR) and restriction fragment length polymorphisms (RFLP). The levels of Pin1 were measured in serum using an enzyme-linked immunosorbent assay. RESULTS: Genotyping showed that CT + TT in the Pin1 promoter was significantly more common in the CKD SHPT group than in the control group (p<.05). The correlation analysis demonstrated that a significant difference in the C to T transition in the Pin1 promoter contributed to CKD SHPT (χ2=12.47, p<.05; Odds ratios (OR) = 1.26, 95% confidence (CI) intervals =1.06-1.49). The multivariate logistic regression analysis reported that the OR and 95%CI were 12.693 and 2.029-75.819 (p<.05), respectively, in the Pin1 gene promoter -667T variant genotypes (CT + TT) after adjusting for other factors, and those values in Pin1 were 0.310 and 0.122-0.792 (p<.05). CONCLUSION: The -667T genetic variants in the Pin1 promoter contribute to an increased risk of CKD SHPT and may be biomarkers of susceptibility to CKD SHPT.

Discovery of a potent and selective ROMK inhibitor with improved pharmacokinetic properties based on an octahydropyrazino[2,1-c][1,4]oxazine scaffold.

Following the discovery of small molecule acyl piperazine ROMK inhibitors, the acyl octahydropyrazino[2,1-c][1,4]oxazine series was identified. This series displays improved ROMK/hERG selectivity, and as a consequence, the resulting ROMK inhibitors do not evoke QTc prolongation in an in vivo cardiovascular dog model. Further efforts in this series led to the discovery of analogs with improved pharmacokinetic profiles. This new series also retained comparable ROMK potency compared to earlier leads.

Accelerated Age-Related Arterial Stiffness in Systemic Lupus Erythematosus Patients.

BACKGROUND: An increased prevalence of arterial stiffness in systemic lupus erythematosus (SLE) patients has been established, but the mechanisms of progression of arterial stiffness with increasing age have not been fully explored. OBJECTIVES: To investigate age-related progression of arterial stiffness among SLE patients relative to healthy controls. METHODS: A total of 161 female SLE patients who were enrolled in the Chinese SLE Treatment and Research group (CSTAR) registry and 135 age-matched healthy control subjects participated in this cross-sectional investigation. Traditional cardiovascular risk factors and SLE-related parameters were assessed on the day that brachial-ankle pulse wave velocity (baPWV) was examined. SAS 9.3 was used to perform all statistical analyses in this study. Linear regression and curvilinear regression models were utilized to analyze the association between age and arterial stiffness. RESULTS: Arterial stiffness based on baPWV significantly differed between the SLE patients and controls in the different age groups, and within the SLE group. The baPWV increments for each age group (<25, 25-34, 35-45, and >45) were 30 cm/s, 52 cm/s, and 121 cm/s for the controls and 61 cm/s, 132 cm/s, and 155 cm/s for the SLE patients, respectively. Curvilinear regression and linear regression revealed various trends of increased arterial stiffness among the SLE patients compared with the healthy controls. The correlation coefficients between age and arterial stiffness significantly differed among the SLE patients relative to the healthy controls (correlation coefficients of 0.46478 and 0.52612, respectively; t = 2.05; P = 0.0409). Multivariate analysis revealed that age, mean blood pressure (BP) (P < 0.0001), erythrocyte sedimentation rate (ESR) (P = 0.0073), prednisone course (P = 0.0144), and SLE disease activity (P = 0.0405) were associated with arterial stiffness among the SLE patients. Further, these patients exhibited earlier exposure to and higher frequencies of several risk factors compared with the controls in each age group (<25 years: OR = 6.3253; 25-34 years: OR = 3.1043; 35-45 years: OR = 3.1316; >45 years: OR = 3.6020). CONCLUSIONS: The mechanisms of the age-related progression of arterial stiffness differed among the SLE patients relative to the healthy controls. Furthermore, accelerated arterial stiffness was observed among the SLE patients relative to the healthy controls with advancing age.

The importin α subunit PsIMPA1 mediates the oxidative stress response and is required for the pathogenicity of Phytophthora sojae.

The sensing of extracellular signals and their transduction into an appropriate response are crucial for the survival and virulence of plant pathogens. Eukaryotic plant pathogens must overcome the obstacles posed by nuclear membranes to manipulate gene expression to adapt to the host challenge. A highly sophisticated mechanism is the use of importins to transport proteins into the nucleus. In this study, we identified a conserved importin α gene, PsIMPA1, in Phytophthora sojae that was differentially expressed during the life cycle of this soybean pathogen. PsIMPA1 expression was lowest in zoospores and cysts but relatively consistent during the other life cycle stages, except for a slight increase at 6h post infection. Silenced mutants Psimpa1 had a decreased growth rate, an aberrant mycelial morphology, and a severely impaired ability to form oospores and sporangia. In addition, the Psimpa1 mutants exhibited reduced pathogenicity compared to the wild type. 3,3-Diaminobenzidine (DAB) staining and in vitro hydrogen peroxide tolerance assays showed that the scavenging of reactive oxygen species by these mutants was significantly impaired. Taken together, these results indicate that PsIMPA1 regulates multiple processes during the life cycle of P. sojae.

Guided reconstitution of membrane protein fragments.

Structural analysis by NMR of G protein-coupled receptors (GPCRs) has proven to be extremely challenging. To reduce the number of peaks in the NMR spectra by segmentally labeling a GPCR, we have developed a Guided Reconstitution method that includes the use of charged residues and Cys activation to drive heterodimeric disulfide bond formation. Three different cysteine-activating reagents: 5-5'-dithiobis(2-nitrobenzoic acid) [DTNB], 2,2'-dithiobis(5-nitropyridine) [DTNP], and 4,4'-dipyridyl disulfide [4-PDS] were analyzed to determine their efficiency in heterodimer formation at different pHs. Short peptides representing the N-terminal (NT) and C-terminal (CT) regions of the first extracellular loop (EL1) of Ste2p, the Saccharomyces cerevisiae alpha-factor mating receptor, were activated using these reagents and the efficiencies of activation and rates of heterodimerization were analyzed. Activation of NT peptides with DTNP and 4-PDS resulted in about 60% yield, but heterodimerization was rapid and nearly quantitative. Double transmembrane domain protein fragments were biosynthesized and used in Guided Reconstitution reactions. A 102-residue fragment, 2TM-tail [Ste2p(G31-I120C)], was heterodimerized with CT-EL1-tail(DTNP) at pH 4.6 with a yield of ∼75%. A 132-residue fragment, 2TMlong-tail [Ste2p(M1-I120C)], was expressed in both unlabeled and (15)N-labeled forms and used with a peptide comprising the third transmembrane domain, to generate a 180-residue segmentally labeled 3TM protein that was found to be segmentally labeled using [(15)N,(1)H]-HSQC analysis. Our data indicate that the Guided Reconstitution method would be applicable to the segmental labeling of a membrane protein with 3 transmembrane domains and may prove useful in the preparation of an intact reconstituted GPCR for use in biophysical analysis and structure determination.

NMR mapping of RANTES surfaces interacting with CCR5 using linked extracellular domains.

Chemokines constitute a large family of small proteins that regulate leukocyte trafficking to the site of inflammation by binding to specific cell-surface receptors belonging to the G-protein-coupled receptor (GPCR) superfamily. The interactions between N-terminal (Nt-) peptides of these GPCRs and chemokines have been studied extensively using NMR spectroscopy. However, because of the lower affinities of peptides representing the three extracellular loops (ECLs) of chemokine receptors to their respective chemokine ligands, information concerning these interactions is scarce. To overcome the low affinity of ECL peptides to chemokines, we linked two or three CC chemokine receptor 5 (CCR5) extracellular domains using either biosynthesis in Escherichia coli or chemical synthesis. Using such chimeras, CCR5 binding to RANTES was followed using (1)H-(15)N-HSQC spectra to monitor titration of the chemokine with peptides corresponding to the extracellular surface of the receptor. Nt-CCR5 and ECL2 were found to be the major contributors to CCR5 binding to RANTES, creating an almost closed ring around this protein by interacting with opposing faces of the chemokine. A RANTES positively charged surface involved in Nt-CCR5 binding resembles the positively charged surface in HIV-1 gp120 formed by the C4 and the base of the third variable loop of gp120 (V3). The opposing surface on RANTES, composed primarily of ß2-ß3 hairpin residues, binds ECL2 and was found to be analogous to a surface in the crown of the gp120 V3. The chemical and biosynthetic approaches for linking GPCR surface regions discussed herein should be widely applicable to the investigation of interactions of extracellular segments of chemokine receptors with their respective ligands.

Functional fusions of T4 lysozyme in the third intracellular loop of a G protein-coupled receptor identified by a random screening approach in yeast.

The insertion of a stable soluble protein into loops of transmembrane proteins has proved to be a successful approach for enhancing their stabilities and crystallization, and may also be useful in contexts where the inserted proteins can modulate or report on the activities of membrane proteins. While the use of T4 lysozyme to replace portions of the third intracellular loops of G protein-coupled receptors (GPCRs) has allowed determination of the structures of members of this important class of receptors, the creation of such fusion proteins generally leads to loss of signaling function of the resulting fusion protein, since the third intracellular loops of GPCRs play critical roles in their interactions with G proteins. We describe here a random screening approach allowing insertion of T4 lysozyme into diverse positions in the third loop of the yeast α-pheromone receptor, a GPCR encoded by the yeast STE2 gene. Insertions were accompanied by varying extents of deletion or duplication of the loop. A set of phenotypic screens allow detection of potentially rare variant receptors that are expressed, bind to agonist and are capable of signal transduction via activation of the cognate G protein. A large fraction of screened full-length receptor variants containing at least partial duplications of the loop on either side of the inserted T4 lysozyme retain the ability to activate the downstream signaling pathway in response to binding of ligand. However, we were unable to identify any receptors with truncated C-termini that retain significant signaling function in the presence of inserted T4 lysozyme. Our results establish the feasibility of creating functional receptors containing insertions of T4 lysozyme in their third intracellular loops.

Synergism between a CD4-mimic peptide and antibodies elicited by a constrained V3 peptide.

Due to the different mechanisms HIV-1 has evolved to escape from a neutralizing antibody response it has been extremely challenging to develop an effective anti-HIV-1 vaccine. The V3 region of the gp120 HIV-1 envelope glycoprotein has been considered as one of the possible targets for an anti-HIV vaccine. It is well known that the V3 region of gp120 is at least partially masked in circulating strains and becomes exposed only after CD4 binding. However, when the virus is bound to surface CD4, steric hindrance prevents effective neutralization by V3-directed antibodies. Here we have used a 27-residue CD4-mimetic peptide in combination with immune sera elicited by an optimally constrained V3 peptide to enhance neutralization of a panel of clade B viruses. We observed strong synergism between the immune sera and the CD4-mimetic in the neutralization of tier 1 and a representative tier 2 clade B virus suggesting that the constrained V3 peptide immunogen correctly mimics the V3 conformation even in tier 2 clade B viruses. This synergy should improve the potential of CD4-mimetic compounds for preexposure prophylaxis and in the treatment of HIV-1-infected patients who usually manifest high titers of V3-directed antibodies. Moreover, constrained V3 immunogens elicit immune sera that may neutralize HIV in synergy with CD4 binding site antibodies that expose V3 and the coreceptor binding site.

Discovery of benzodihydroisofurans as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase inhibitors.

A series of benzodihydroisofurans were discovered as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase (PrCP) inhibitors. The structure-activity relationship (SAR) is focused on improving PrCP activity and metabolic stability, and reducing plasma protein binding. In the established diet-induced obese (eDIO) mouse model, compound ent-3a displayed target engagement both in plasma and in brain. However, this compound failed to induce significant body weight loss in eDIO mice in a five-day study.

The conformation and orientation of a 27-residue CCR5 peptide in a ternary complex with HIV-1 gp120 and a CD4-mimic peptide.

Interaction of CC chemokine receptor 5 (CCR5) with the human immunodeficiency virus type 1 (HIV-1) gp120/CD4 complex involves its amino-terminal domain (Nt-CCR5) and requires sulfation of two to four tyrosine residues in Nt-CCR5. The conformation of a 27-residue Nt-CCR5 peptide, sulfated at Y10 and Y14, was studied both in its free form and in a ternary complex with deglycosylated gp120 and a CD4-mimic peptide. NMR experiments revealed a helical conformation at the center of Nt-CCR5(1-27), which is induced upon gp120 binding, as well as a helical propensity for the free peptide. A well-defined structure for the bound peptide was determined for residues 7-23, increasing by 2-fold the length of Nt-CCR5's known structure. Two-dimensional saturation transfer experiments and measurement of relaxation times highlighted Nt-CCR5 residues Y3, V5, P8-T16, E18, I23 and possibly D2 as the main binding determinant. A calculated docking model for Nt-CCR5(1-27) suggests that residues 2-22 of Nt-CCR5 interact with the bases of V3 and C4, while the C-terminal segment of Nt-CCR5(1-27) points toward the target cell membrane, reflecting an Nt-CCR5 orientation that differs by 180° from that of a previous model. A gp120 site that could accommodate (CCR5)Y3 in a sulfated form has been identified. The present model attributes a structural basis for binding interactions to all gp120 residues previously implicated in Nt-CCR5 binding. Moreover, the strong interaction of sulfated (CCR5)Tyr14 with (gp120)Arg440 revealed by the model and the previously found correlation between E322 and R440 mutations shed light on the role of these residues in HIV-1 phenotype conversion, furthering our understanding of CCR5 recognition by HIV-1.

Differential interactions of fluorescent agonists and antagonists with the yeast G protein coupled receptor Ste2p.

We describe a rapid method to probe for mutations in cell surface ligand-binding proteins that affect the environment of bound ligand. The method uses fluorescence-activated cell sorting to screen randomly mutated receptors for substitutions that alter the fluorescence emission spectrum of environmentally sensitive fluorescent ligands. When applied to the yeast α-factor receptor Ste2p, a G protein-coupled receptor, the procedure identified 22 substitutions that red shift the emission of a fluorescent agonist, including substitutions at residues previously implicated in ligand binding and at additional sites. A separate set of substitutions, identified in a screen for mutations that alter the emission of a fluorescent α-factor antagonist, occurs at sites that are unlikely to contact the ligand directly. Instead, these mutations alter receptor conformation to increase ligand-binding affinity and provide signaling in response to antagonists of normal receptors. These results suggest that receptor--agonist interactions involve at least two sites, of which only one is specific for the activated conformation of the receptor.