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The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has led to over 760 million cases and >6.8 million deaths worldwide. We developed a panel of human neutralizing monoclonal antibodies (mAbs) targeting the SARS-CoV-2 Spike protein using Harbour H2L2 transgenic mice immunized with Spike receptor binding domain (RBD) (1). Representative antibodies from genetically-distinct families were evaluated for inhibition of replication-competent VSV expressing SARS-CoV-2 Spike (rcVSV-S) in place of VSV-G. One mAb (denoted FG-10A3) inhibited infection of all rcVSV-S variants; its therapeutically-modified version, STI-9167, inhibited infection of all tested SARS-CoV-2 variants, including Omicron BA.1 and BA.2, and limited virus proliferationin vivo(1). To characterize the binding specificity and epitope of FG-10A3, we generated mAb-resistant rcVSV-S virions and performed structural analysis of the antibody/antigen complex using cryo-EM. FG-10A3/STI-9167 is a Class 1 antibody that prevents Spike-ACE2 binding by engaging a region within the Spike receptor binding motif (RBM). Sequencing of mAb-resistant rcVSV-S virions identified F486 as a critical residue for mAb neutralization, with structural analysis revealing that both the variable heavy and light chains of STI-9167 bound the disulfide-stabilized 470-490 loop at the Spike RBD tip. Interestingly, substitutions at position 486 were later observed in emerging variants of concern BA.2.75.2 and XBB. This work provides a predictive modeling strategy to define the neutralizing capacity and limitations of mAb therapeutics against emerging SARS-CoV-2 variants.ImportanceThe COVID-19 pandemic remains a significant public health concern for the global population; development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as SARS-CoV-2 variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread with the caveat that they interact with the circulating variants. The epitope and binding specificity of a broadly neutralizing anti-SARS-CoV-2 Spike RBD antibody clone against many SARS-CoV-2 VOC was characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

1. Accurate estimates of wildlife distributions and population persistence are essential for conservation programs. Occupancy models that account for detection probability have been used to characterize the occupancy and habitat selection of imperilled species. However, failure to distinguish between true-presence and pseudo-presence associated with territorial behaviour can result in the overestimation of the occupancy probability of target species in unsuitable habitats, and this can have major implications for the development of conservation strategies.2. For highly territorial wildlife species requiring high-quality habitat for survival, occasional ‘Presence’ in unsuitable areas might be related to dispersal or migration, but this does not reflect actual occupancy and habitat use. ‘True-Presence’ and ‘Pseudo-Presence’ should be distinguished for target species to better reflect their actual occupancy and habitat use.3. To investigate the effect of ‘True-Presence’ and ‘Pseudo-Presence’ on wildlife occupancy estimation, we developed a modified model (Mm) that considers the territorial behaviour of the Amur tiger in northeast China, which distinguished between ‘True-Presence’ and ‘Pseudo-Presence’. We compared two models, Mmand M0(conventional occupancy model), and assessed model performance using goodness-of-fit evaluation, detection and occupancy probability, and favourable variable selection.4. We found that Mm, which has fewer favourable variables, is more powerful than M0for estimating detection and occupancy probability, as well as characterizing the effect of various factors on the habitat use of Amur tigers. Furthermore, Mmsignificantly reduced the overestimation of occupancy probability outside the home range compared with M0, and detection probability estimates did not significantly differ between M0and Mm. Finally, Mmprovided more empirical habitat selection variables for the Amur tiger.5. Our results revealed how ‘True-Presence’ and ‘Pseudo-Presence’ affect the occupancy probability and habitat selection of Amur tigers. Our method improves the accuracy of occupancy models; it can also be used to characterize the habitat selection and distribution of wildlife species and be applied to other territorial species.

The development of "three-dimensional ecology" reveals refreshing phenomena and challenges us to use three-dimensional information for studying animal perception. We created a new processing framework to quantify the shielding effect using a reconstructed environmental structure. The framework achieves three objectives: 1) the observed is introduced, 2) the observed space size can be flexibly dealt with, and 3) three-dimensional attributes are assigned to the environmental structure. Our processing framework is an applicable method to "three-dimensional ecology" based on the three-dimensional attributes of physical structures. We advocate for greater emphasis on "three-dimensional ecology" to recreate realistic animal living conditions and better reveal their behaviors.

The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant and its BA.X lineages, has rendered ineffective a number of previously FDA emergency use authorized SARS-CoV-2 neutralizing antibody therapies. Furthermore, those approved antibodies with neutralizing activity against Omicron BA.1 are reportedly ineffective against the subset of Omicron subvariants that contain a R346K substitution, BA.1.1, and the more recently emergent BA.2, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern.Following a campaign of antibody discovery based on the vaccination of Harbor H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167.STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against each of the tested Omicron subvariants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice.With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for intravenous or intranasal use in human clinical trials.Funded by CRIPT (no. 75N93021R00014), DARPA (HR0011-19-2-0020), and NCI Seronet (U54CA260560).

The continual emergence of SARS-CoV-2 variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant, has rendered ineffective a number of previously EUA approved SARS-CoV-2 neutralizing antibody therapies. Furthermore, even those approved antibodies with neutralizing activity against Omicron are reportedly ineffective against the subset of Omicron variants that contain a R346K substitution, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern. Following a campaign of antibody discovery based on the vaccination of Harbour H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of Spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167. STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against the Omicron and Omicron + R346K variants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice. With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for use in human clinical trials.

We have previously reported that the SARS-CoV-2 neutralizing antibody, STI-2020, potently inhibits cytopathic effects of infection by genetically diverse clinical SARS-CoV-2 pandemic isolates in vitro, and has demonstrated efficacy in a hamster model of COVID-19 when administered by the intravenous route immediately following infection. We now have extended our in vivo studies of STI-2020 to include disease treatment efficacy, profiling of biodistribution of STI-2020 in mice when antibody is delivered intranasally (IN) or intravenously (IV), as well as pharmacokinetics in mice following IN antibody administration. Importantly, SARS-CoV-2-infected hamsters were treated with STI-2020 using these routes, and treatment effects on severity and duration of COVID-19-like disease in this model were evaluated. In SARS-CoV-2 infected hamsters, treatment with STI-2020 12 hours post-infection using the IN route led to a decrease in severity of clinical disease signs and a more robust recovery during 9 days of infection as compared to animals treated with an isotype control antibody. Treatment via the IV route using the same dose and timing regimen resulted in a decrease in the average number of consecutive days that infected animals experienced weight loss, shortening the duration of disease and allowing recovery to begin more rapidly in STI-2020 treated animals. Following IN administration in mice, STI-2020 was detected within 10 minutes in both lung tissue and lung lavage. The half-life of STI-2020 in lung tissue is approximately 25 hours. We are currently investigating the minimum effective dose of IN-delivered STI-2020 in the hamster model as well as establishing the relative benefit of delivering neutralizing antibodies by both IV and IN routes.

Bioconjugate formats provide alternative strategies for antigen targeting with bispecific antibodies. Here, PSMA-targeted Fab conjugates were generated using different bispecific formats. Interchain disulfide bridging of an αCD3 Fab enabled installation of either the PSMA-targeting small molecule DUPA (SynFab) or the attachment of an αPSMA Fab (BisFab) by covalent linkage. Optimization of the reducing conditions was critical for selective interchain disulfide reduction and good bioconjugate yield. Activity of αPSMA/CD3 Fab conjugates was tested by in vitro cytotoxicity assays using prostate cancer cell lines. Both bispecific formats demonstrated excellent potency and antigen selectivity.

Bispecific antibodies (BsAbs) are designed to engage two antigens simultaneously, thus, effectively expanding the ability of antibody-based therapeutics to target multiple pathways within the same cell, engage two separate soluble antigens, bind the same antigen with distinct paratopes, or crosslink two different cell types. Many recombinant BsAb formats have emerged, however, expression and purification of such constructs can often be challenging. To this end, we have developed a chemical strategy for generating BsAbs using native IgG2 architecture. Full-length antibodies can be conjugated via disulfide bridging with linkers bearing orthogonal groups to produce BsAbs. We report that an αHER2/EGFR BsAb was successfully generated by this approach and retained the ability to bind both antigens with no significant loss of potency.

High-throughput experiment can significantly accelerate the materials research efficiency. Thanks to national efforts, the Materials Genome Initiative further promotes the development of high-throughput experimental technology. A multi-channel fiber optical spectrometer has been designed and developed by us for high-throughput characterization of photoluminescence (PL) properties. It can quickly and automatically detect the PL spectrum, Commission International de l'Eclairage chromaticity, and PL intensity over time for luminescent materials under a given condition. The multi-channel fiber optical spectrometer synergistically combines a sample library holder, multiple modular excitation sources, multiple spectrometers, and Coral software, so it can measure and analyze multiple samples simultaneously. The number of channels in the multi-channel fiber optical spectrometer can be added or subtracted as required. Various modular light-emitting diode or laser diode excitation sources with the wavelength from 370 nm to 980 nm and corresponding filters can be provided according to the measurement need of different luminescent materials. The monitoring wavelength of the currently used fiber optical spectrometer is from 300 nm to 1000 nm. For example, the PL spectral measurement of 54 samples in a {6 × 9} array is completed in only about 30 min by using a representative triple-channel fiber optical spectrometer. The designed multi-channel fiber optical spectrometer facility not only makes PL measurements faster and more intuitive but is also easy to popularize for wide users.

All of the currently approved therapeutic anti-cancer antibodies are monospecific and therefore only capable of interfering with the biological function of a single molecular target. However, breast cancers mostly involve crosstalk of often synergistic signal transduction pathways, and thus, isolated blockade of a single signal transduction pathway is frequently met by escape mechanisms, such as upregulation of redundant pathways, rendering the monospecific immunotherapy less effective. Using chemical biology and molecular biology, Sorrento has developed new approaches to generate IgG-like bi-specific antibodies (BsAbs) targeting either two compensating signal transduction pathways, such as HER family members or a breast cancer specific antigen and an immuno-regulatory molecule such as PDL1 or PD1. The chemical biology method involves specific hetero-dimerization of two half antibody molecules using bio-orthogonal chemistry. The molecular biology approach utilizes phage display by either discovering common light chains for the two relevant antigens in combination with two distinct heavy chains or by selecting dual specific antibodies, namely a single antibody that can recognize two distinct antigens. Progress will be presented on bi-specific as well as dual specific antibodies against Her3 and Her4 as well as immunomodulatory BsAbs with data on in vitro characterization, cell-based functional assays, and in vivo models. Citation Format: Yanliang Zhang, Edwige Gros, Sarabjit Chagar, Jian Cao, Heyue Zhou, Kouros Motamed, Gunnar F Kaufmann, Yanwen Fu. Bi-specific antibodies targeting signaling pathway crosstalk are a new breast cancer immunotherapeutic strategy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-16-02.

C-Met, a transmembrane receptor tyrosine kinase, plays a key role in malignant transformation of epithelial cells by activating signal transduction pathways essential for cellular proliferation, survival, migration and invasion. C-Met overexpression, with or without gene amplification, has been reported in primary breast cancers and correlate with poor prognosis. C-Met signaling inhibition, such as tyrosine kinase inhibitors (TKIs), usually not sufficient for sustained treatment efficacy, we believe that antibody drug conjugates (ADCs) offer the promise and potential of delivering potent anti tumor activity with the advantage of reduced side effects. We generated antibody drug conjugates containing a proprietary human anti-c-Met antibody (STI-D0606) with either a tubulin inhibitor or DNA damaging agent, such as doxorubicin analogs. STI-D0606, a fully human antibody (IgG1) selected from phage library was conjugated with the cytotoxin via site specific bioconjugation. The conjugates retained binding affinity and showed potent cell killing in a variety of c-Met positive cell lines. Progress will be reported in terms of in vivo efficacy in c-Met-positive human breast tumor xenograft in preclinical models. Citation Format: Yanwen Fu, Edwige Gros, Alice Lee, Kimberly Johnson, Hong Zhang, Silpa Yalamanchili, Kouros Motamed, Gary Chen, Bryan Jones, David Miao, Gunnar F Kaufmann. C-Met is a potential therapeutic target for antibody-drug conjugates in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-16-03.

Cα,α-disubstituted amino acids (ααAAs) are widely used to conformationally constrain peptides. A series of pentapeptides containing dipropylglycine (Dpg) at alternating positions and their α-amino acid counterpart L-norvaline (Nva) analogues were synthesized to fully investigate the impact of Dpg on peptide backbone structure in aqueous solution. CD, VCD, and NMR spectral analysis suggest that Dpg containing peptides adopt more ordered structures relative to their Nva containing analogues. The central residues (Ala, Thr, Tyr, Val) and the charged side-chains of Glu and Lys play important roles in the degree of peptide folding. Hydrophobic and branched residues (Val, Tyr) at the central position of the peptide produce greater folding as judged by CD and NMR. Variation of the chemical shift with temperature (Δδ/ΔT NH) of Ac-Glu-Dpg-Tyr-Dpg-Lys-NH2 suggests a series of i → i + 3 hydrogen bonds between the N-terminal acetyl carbonyl and the Tyr3 NH, and the Glu1 carbonyl and the Dpg4 NH. The solution conformation of Ac-Glu-Dpg-Tyr-Dpg-Lys-NH2 calculated from NMR-derived constraints shows a 310-helical structure (two repetitive type-III β-turns) at residues 1-4, which is supported by 2D NMR, CD, and VCD spectra. Analysis of NMR-derived models of these peptides suggest that there is a strong hydrophobic interaction of the pro-S propyl side chain of Dpg2 and the Tyr3 side-chain that may be a strong stabilizing force of the peptide folding in water. © 2009 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 92: 452–464, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Amyloid beta (Abeta) peptide amyloidogenesis, involving the formation of numerous distinct quaternary structures, appears to cause Alzheimer's disease. However, the precise identification of the toxic structure(s) and the neurotoxicity mechanism(s) remains elusive. Mutating the Abeta 1-40 Phe19-Phe20 backbone amide bond to an isostructural E-olefin bond enables formation of spherical aggregates to the exclusion of detectable amyloid fibrils. Herein, the fibrillization and toxicity of amide-to-ester mutants of Abeta 1-40 at the 19-20 position and surrounding backbone amide bonds are compared to the fibrillization and toxicity of the 19-20 E-olefin Abeta analogue and wild type Abeta. Whereas isostructural amide-to-E-olefin mutations eliminate both the H-bond donor and acceptor capabilities, isostructural amide-to-ester mutations eliminate the donor while retaining the ester carbonyl as a weakened acceptor. None of the amide-to-ester Abeta 1-40 mutants prevent fibrillization; in fact several exhibit hastened amyloidogenesis. The 18-19 amide-to-ester substitution is the only backbone mutation within the hydrophobic core region of the fibril (residues 17-21) that significantly slows fibrillization. Despite forming different morphologies, the 19-20 E-olefin mutant, the 18-19 amide-to-ester mutant, and WT Abeta 1-40 fibrils all exhibit similar toxicities when applied to PC12 cells at 18 h into the aggregation reactions, as assessed by MTT metabolic activity measurements. This result suggests that a common but low abundance aggregate morphology, that is accessible to these Abeta analogues, mediates toxicity, or that several different aggregate morphologies are similarly toxic.

Molecular structure of a Boc-protected piperidine-spiro-hydantoin has been studied by solution state NMR and X-ray crystallography. Complete assignments of the 1 H and 13 C spectra have been performed using homo- and heteronuclear 2D NMR techniques. The 1 H NMR indicates that the molecule is asymmetric at low temperature and exhibits a C–N bond rotational process of the carbamate fragment attached to the piperidine ring. Solution conformation of the molecule was derived from NOE constraints, in which the piperidine ring exhibits a single half-chair conformation. The crystal structure of the piperidine-spiro-hydantoin determined by X-ray crystallography showed that the piperidine ring adopts the same conformation as in solution state and the exocyclic carbamate group is twisted from the potential symmetrical plane of the piperidine ring.

The preparation of sterically hindered and polyfunctional Cα,α-disubstituted α-amino acids (ααAAs) via alkylation of ethyl nitroacetate and transformation into derivatives ready for incorporation into peptides are described. Treatment of ethyl nitroacetate with N,N-diisopropylethylamine (DIEA) in the presence of a catalytic amount of tetraalkylammonium salt, followed by the addition of an activated alkyl halide or Michael acceptor, gives the doubly C-alkylated product in good to excellent yields. Selective nitro reduction with Zn in acetic acid or hydrogen over Raney Ni gives the corresponding amino ester that, upon saponification, can be protected with the fluorenylmethyloxycarbonyl (Fmoc) group. The first synthesis of an orthogonally protected, tetrafunctional Cα,α-disubstituted analogue of aspartic acid, 2,2-bis(tert-butylcarboxymethyl)glycine (Bcmg), is described. Also, the sterically demanding Cα,α-dibenzylglycine (Dbg) has been incorporated into a peptide using solid-phase synthesis. It was found th...

c-Met, the proto-oncogene transmembrane receptor tyrosine kinase, is involved in cell proliferation, survival, motility, and invasion in normal and tumor cells. It is widely expressed and associated with poor prognosis in breast, lung, liver, kidney and brain cancers. Hepatocyte growth factor (HGF) is the only known ligand of c-Met, also found in many tumors and cancers where activity is driven by the c-Met receptor. c-Met is accepted as an attractive anti-cancer target, but the development of cancer therapy targeting c-Met receptor or kinase remain very challenging. Clinical development of c-Met/HGF targeting antibodies has shown initial evidence of clinical efficacy but failed in phase III. Small molecule inhibitors often suffer from selectivity issues. Antibody drug conjugates (ADCs) which combine an antibody with highly potent cytotoxic agents, becomes an attractive approach against c-Met overexpressing cancers such NSCLC. We have identified an anti-c-Met antibody STI-0602 and generated ADCs from a panel of cytotoxic agents such as microtubulin inhibitor and DNA damaging agents. ADCs retained the binding to c-Met receptor and showed potent cell killing in a variety of c-Met-positive cell lines. ADCs also showed in vivo efficacy in a panel of c-Met-positive human NSCLC xenograft models like A549, H292, EBC-1, HCC827 and H1993, which suggest a potential therapy for c-Met overexpressing lung cancer and other cancers. Citation Format: Lingna Li, Cathrine Fells, Julia Guo, Pia Muyot, Edwige Gros, Yanliang Zhang, Yingqing Sun, Hong Zhang, Yanwen Fu, Tong Zhu, Jian Cao, Gunnar Kaufmann, Gang Chen, Zhenwei Miao. A novel c-Met targeting antibody drug conjugate for NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3897.

Both amide-to-ester and amide-to-E-olefin backbone amide mutation methods were employed to perturb the same H-bond (formed by the NH of F23 and the CO of R14) in the Pin WW domain. Comparison of the thermodynamic folding energies of the ester mutant and the E-olefin mutant, accounting for the transfer free energy differences measured on relevant model compounds, yielded an estimated value of 0.3 kcal/mol for the O-O repulsion term (DeltaGO-Orep) in a beta-sheet context. The value of DeltaGO-Orep enabled us to calculate the intrinsic F23-R14 H-bond free energy to be 1.3 kcal/mol.

DMAP(4-Dimethylamino)pyridine 1-tert-Butoxycarbonylpiperidine-4-spiro-5′-(1′,3′-bis(tert-butoxycarbonyl)hydantoin 4-Amino1-tert-butoxycarbonylpiperidine-4-carboxylic acid 1-tert-Butyloxycarbonyl-4-(9-fluorenylmethyloxyamino)piperidine-4-carboxylic acid 9-Fluorenylmethyl chlorformate 4-Piperidone monohydrate hydrochloride Piperidine-4-spiro-5′-hydantoin Keywords: amino acid analogs; lysine; waste disposal; potassium cyanide; disubstituted amino acids

[reaction: see text] Fmoc amino acid symmetrical anhydrides are efficient and readily available reagents for acylation of the N-terminus of highly hindered C(alpha)(alpha)-dialkylated alpha-amino acids. Comparison of a variety of coupling protocols showed that the symmetrical anhydride method always provided the superior results. This method was successfully applied to the solid-phase synthesis of a peptide containing three alpha(alpha)AAs at alternating positions.

Non-proteinogenic Cαα-disubstituted amino acid (ααAA) residues greatly restrict the number of conformations of peptides and thus induce distinct secondary structures, even in short peptides [1]. In this work several pentapeptides containing Cαα-dipropyl-glycine (Dpg) and dibenzylglycine (Dbg) at alternating sequence positions and their L-norvaline (Nva) analogs were prepared. The design of these peptides is based on our models of amyloid β-sheet blocker peptides. Dpg and Dbg could accommodate energetically favored extended conformation [2] and thus induce the formation of β-sheet by self-aggregation.

The discovery of Cα, α-disubstituted amino acids (ααAAs) and their propensity to induce secondary structure even into short peptides has resulted in an increased interest in novel methods for their synthesis [1, 2]. Herein ethyl nitroacetate, a useful synthetic intermediate for a variety of biologically significant compounds [3], has been employed for the preparation of sterically hindered and polyfunctional ααAAs which are suitable for incorporation into peptides.

We have prepared two peptides based on the hydrophobic core (Lys-Leu-Val-Phe-Phe) of amyloid beta-protein (Abeta) that contain alpha,alpha-disubstituted amino acids at alternating positions, but differ in the positioning of the oligolysine chain (AMY-1, C-terminus; AMY-2, N-terminus). We have studied the effects of AMY-1 and AMY-2 on the aggregation of Abeta and find that, at stoichiometric concentrations, both peptides completely stop Abeta fibril growth. Equimolar mixtures of AMY-1 and Abeta form only globular aggregates as imaged by scanning force microscopy and transmission electron microscopy. These samples show no signs of protofibrillar or fibrillar material even after prolonged periods of time (4.5 months). Also, 10 mol % of AMY-1 prevents Abeta self-assembly for long periods of time; aged samples (4.5 months) show only a few protofibrillar or fibrillar aggregates. Circular dichroism spectroscopy of equimolar mixtures of AMY-1 and Abeta show that the secondary structure of the mixture changes over time and progresses to a predominantly beta-sheet structure, which is consistent with the design of these inhibitors preferring a sheet-like conformation. Changing the position of the charged tail on the peptide, AMY-2 interacts with Abeta differently in that equimolar mixtures form large ( approximately 1 mum) globular aggregates which do not progress to fibrils, but precipitate out of solution. The differences in the aggregation mediated by the two peptides is discussed in terms of a model where the inhibitors act as cosurfactants that interfere with the native ability of Abeta to self-assemble by disrupting hydrophobic interactions either at the C-terminus or N-terminus of Abeta.

Herein, we report a stereospecific E-olefin dipeptide isostere synthesis that can be used to make gram quantities of the Phe-Phe isostere desired for eliminating a specific backbone H-bond donor and acceptor in the Alzheimer's disease related Abeta peptide. The Phe19-Phe20 E-olefin analogue of Abeta(1-40) was prepared by solid-phase peptide synthesis and was subjected to amyloidogenesis conditions. This analogue can aggregate into spherical morphologies but does not progress on to form protofibrils or fibrils as is the case for the all-amide sequence, providing insight into the structural requirements for amyloidogenesis.

The SDF1α / CXCR4 axis contributes to cancer metastasis, proliferation, angiogenesis and resistance to standard of care drugs. Here we describe novel phage display derived peptide SDF1α antagonists. Phage displayed peptides were optimized through a multistep process that resulted in a 400-fold improvement in activity, from 6.4 uM IC50 inhibition of SDF1α -driven Calcium flux to 16 nM IC50. First, peptides were affinity matured via panning of focused phage libraries. Peptides exhibiting enhanced potency were bioconjugated to the CovX antibody scaffold IgG (CVX-2000), generating bivalent CovX-Bodies with 5-fold improved potency in SDF1α driven Calcium flux assays. N and C-terminal extension of the peptide and introduction of unnatural amino acids produced an additional 11-fold improvement in activity to 16 nM. Constructs with optimal pharmacokinetic stability were identified, and demonstrated single agent tumor inhibition in a Ramos B cell lymphoma xenograft model. Citation Format: Julia A. Coronella, Yanwen Fu, Kimberly Johnson, Jingping Zhong, Lioudmila Campbell, Gang Chen, Dorian Willhite, Joselyn Del Rosario, Abhijit Bhat, Gary Woodnutt, Nancy Levin. Discovery and optimization of potent SDF1α antagonist peptides. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4113. doi:10.1158/1538-7445.AM2013-4113

Here we describe a series of CXCR4 antagonist CovX-Bodies, produced by covalently attaching CXCR4 antagonist peptides to a humanized IgG1 molecule with the goal of producing molecules with the potency and specificity of peptides and the pharmacokinetic parameters of an antibody. Extensive SAR was performed on peptides derived from T140, a highly charged 14 amino acid cyclic peptide containing 4 Arginine residues. Both the free peptides and the associated CovX-bodies were potent antagonists of CXCR4 in vitro, and SAR studies were consistent with a model of both allosteric and orthosteric inhibition. However, in vivo pharmacokinetic (PK) studies revealed an unexpected and CXCR4-independent disappearance of the CovX-Bodies from circulation, such that approximately 10% of the injected CovX-Body remained in circulation 1 hour after i.v. injection into mice. Based on the known nonspecific cell-penetrating properties of Arginine-rich peptides, we hypothesized that Arginine replacement and charge neutralization would improve the pharmacokinetics of the CovX-Bodies. We identified substitutions that could replace up to 3 of the 4 Arg residues while retaining potent CXCR4 binding and antagonism, but Arg2 was obligate. However, these CovX-Bodies were only modestly improved in PK, indicating that that the anomalous in vivo behavior was not solely a function of charge or the net number of Arg residues. Only the replacement of Arg2 significantly improved the pharmacokinetics of the CovX-Bodies, but also resulted in a loss of CXCR4 antagonism. Extensive SAR failed to identify a suitable replacement residue for Arg2, nor did alternative structural forms such as 5 member, bicyclic, or reversed ring molecules have significantly improved pharmacokinetics. Based on these data, we conclude that the specific property of the peptide that allows potent inhibition of CXCR4, of which Arg2 is an integral component, also mediates binding to moieties other than CXCR4, leading to the depletion of these molecules from circulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2849. doi:1538-7445.AM2012-2849

The title compound, C18H19NO4, is the key synthetic intermediate in the preparation of α,α-di­benzyl-α-amino acid (di­benzyl­glycine, Dbg), the disubstituted homologue of phenyl­alanine, following the di­alkyl­ation of ethyl nitro­acetate. The mol­ecule does not have its potential mirror symmetry in the crystal, with the two benzyl groups forming N—C—C—C torsion angles of 60.31 (13) and 79.89 (13)°.