Generation of T-cell-redirecting bispecific antibodies with differentiated profiles of cytokine release and biodistribution by CD3 affinity tuning.

T-cell-redirecting bispecific antibodies have emerged as a new class of therapeutic agents designed to simultaneously bind to T cells via CD3 and to tumor cells via tumor-cell-specific antigens (TSA), inducing T-cell-mediated killing of tumor cells. The promising preclinical and clinical efficacy of TSAxCD3 antibodies is often accompanied by toxicities such as cytokine release syndrome due to T-cell activation. How the efficacy and toxicity profile of the TSAxCD3 bispecific antibodies depends on the binding affinity to CD3 remains unclear. Here, we evaluate bispecific antibodies that were engineered to have a range of CD3 affinities, while retaining the same binding affinity for the selected tumor antigen. These agents were tested for their ability to kill tumor cells in vitro, and their biodistribution, serum half-life, and anti-tumor activity in vivo. Remarkably, by altering the binding affinity for CD3 alone, we can generate bispecific antibodies that maintain potent killing of TSA + tumor cells but display differential patterns of cytokine release, pharmacokinetics, and biodistribution. Therefore, tuning CD3 affinity is a promising method to improve the therapeutic index of T-cell-engaging bispecific antibodies.

Bioelectronic control of a microbial community using surface-assembled electrogenetic cells to route signals.

We developed a bioelectronic communication system that is enabled by a redox signal transduction modality to exchange information between a living cell-embedded bioelectronics interface and an engineered microbial network. A naturally communicating three-member microbial network is 'plugged into' an external electronic system that interrogates and controls biological function in real time. First, electrode-generated redox molecules are programmed to activate gene expression in an engineered population of electrode-attached bacterial cells, effectively creating a living transducer electrode. These cells interpret and translate electronic signals and then transmit this information biologically by producing quorum sensing molecules that are, in turn, interpreted by a planktonic coculture. The propagated molecular communication drives expression and secretion of a therapeutic peptide from one strain and simultaneously enables direct electronic feedback from the second strain, thus enabling real-time electronic verification of biological signal propagation. Overall, we show how this multifunctional bioelectronic platform, termed a BioLAN, reliably facilitates on-demand bioelectronic communication and concurrently performs programmed tasks.

NMR as a "Gold Standard" Method in Drug Design and Discovery.

Studying disease models at the molecular level is vital for drug development in order to improve treatment and prevent a wide range of human pathologies. Microbial infections are still a major challenge because pathogens rapidly and continually evolve developing drug resistance. Cancer cells also change genetically, and current therapeutic techniques may be (or may become) ineffective in many cases. The pathology of many neurological diseases remains an enigma, and the exact etiology and underlying mechanisms are still largely unknown. Viral infections spread and develop much more quickly than does the corresponding research needed to prevent and combat these infections; the present and most relevant outbreak of SARS-CoV-2, which originated in Wuhan, China, illustrates the critical and immediate need to improve drug design and development techniques. Modern day drug discovery is a time-consuming, expensive process. Each new drug takes in excess of 10 years to develop and costs on average more than a billion US dollars. This demonstrates the need of a complete redesign or novel strategies. Nuclear Magnetic Resonance (NMR) has played a critical role in drug discovery ever since its introduction several decades ago. In just three decades, NMR has become a "gold standard" platform technology in medical and pharmacology studies. In this review, we present the major applications of NMR spectroscopy in medical drug discovery and development. The basic concepts, theories, and applications of the most commonly used NMR techniques are presented. We also summarize the advantages and limitations of the primary NMR methods in drug development.

Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication.

The main protease, Mpro (or 3CLpro) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide. Feline infectious peritonitis, a fatal coronavirus infection in cats, was successfully treated previously with a prodrug GC376, a dipeptide-based protease inhibitor. Here, we show the prodrug and its parent GC373, are effective inhibitors of the Mpro from both SARS-CoV and SARS-CoV-2 with IC50 values in the nanomolar range. Crystal structures of SARS-CoV-2 Mpro with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 replication in cell culture. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals. The work here lays the framework for their use in human trials for the treatment of COVID-19.

Diverse residues of intracellular loop 5 of the Na+/H+ exchanger modulate proton sensing, expression, activity and targeting.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pHi) by removing a single intracellular proton in exchange for one extracellular sodium ion. It is involved in cardiac hypertrophy and ischemia reperfusion damage to the heart and elevation of its activity is a trigger for breast cancer metastasis. NHE1 has an extensive 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments connected by intracellular and extracellular loops. Intracellular loops are hypothesized to modulate the sensitivity to pHi. In this study, we characterized the structure and function of intracellular loop 5 (IL5), specifically amino acids 431-443. Mutation of eleven residues to alanine caused partial or nearly complete inhibition of transport; notably, mutation of residues L432, T433, I436, N437, R440 and K443 demonstrated these residues had critical roles in NHE1 function independent of effects on targeting or expression. The nuclear magnetic resonance (NMR) solution spectra of the IL5 peptide in a membrane mimetic sodium dodecyl sulfate solution revealed that IL5 has a stable three-dimensional structure with substantial alpha helical character. NMR chemical shifts indicated that K438 was in close proximity with W434. Overall, our results show that IL5 is a critical, intracellular loop with a propensity to form an alpha helix, and many residues of this intracellular loop are critical to proton sensing and ion transport.

Development of Cell-Based Sentinels for Nitric Oxide: Ensuring Marker Expression and Unimodality.

We generated "sentinel" bacteria that respond to the biomarker nitric oxide (NO) and produce a homogeneous and strong fluorescent response. Our dual-plasmid system consists of a signal "relay" vector that employs an NO-responsive promoter that amplifies the native signal (via expression of T7 Polymerase (T7Pol)) to a second vector responsible for GFP expression. Importantly, to achieve an optimal "sentinel" response, we developed strategies that balance the transcriptional load within cells by altering (i) translation and (ii) activity of the T7Pol. Our optimized genetic circuitry was then used to transform commensal E. coli Nissle, as a proof-of-concept toward an ingestible cell-based sensor for Crohn's disease (CD) that, in turn, is marked by elevated levels of intestinal NO. Thus, the "biosensors" demonstrated here may serve as a simple diagnostic tool, contrasting the standard of care including colonoscopies or biopsies.

Long TE STEAM and PRESS for estimating fat olefinic/methyl ratios and relative ω-3 fat content at 3T.

BACKGROUND: Fat olefinic/methyl ratios provide a measure of fat unsaturation. The methyl resonance linewidth is altered with the presence of ω-3 fat. PURPOSE: To optimize stimulated echo acquisition mode (STEAM) and point resolved spectroscopy (PRESS) echo times (TE) at 3T to 1) improve olefinic/methyl ratios and 2) enable relative ω-3 fat content assessment. STUDY TYPE: Technical development on phantoms and healthy volunteers. POPULATION: Nine edible oils and four healthy volunteers (tibial bone marrow). FIELD STRENGTH/SEQUENCE: STEAM (mixing time = 20 msec) and PRESS sequences at 3T. High-resolution oil spectra at 16.5T. ASSESSMENT: 3T STEAM and PRESS olefinic/methyl ratios as a function of TE were compared to 16.5T measures for the oils, and to a literature-deduced value for tibial bone marrow. Oil methyl linewidths were calculated at each TE to investigate correlation with expected ω-3 fatty acid content. STATISTICAL TESTS: Percent differences were calculated between oil olefinic/methyl ratios obtained at 3T and 16.5T. Linear regression R2 values measured correlation of methyl linewidth to ω-3 content. RESULTS: STEAM, TE = 120 msec, resulted in average oil olefinic/methyl ratios that differ by about -4.8% compared to high-resolution ratios. Tibial bone marrow olefinic/methyl ratios differ by -1.8% compared with literature-obtained ratios. PRESS, TE = 180 msec, resulted in oil ratios that differ by 7.8% and tibial bone marrow ratios that differ by 0.2%. A TE of 160 msec for both STEAM and PRESS enabled relative levels of oil ω-3 fatty acid content to be estimated (R2 values ≥0.9). DATA CONCLUSION: STEAM, TE = 120 msec (mixing time = 20 msec), and PRESS, TE = 180 msec, optimally estimated olefinic/methyl ratios. STEAM and PRESS, TE = 160 msec, enable relative oil ω-3 fatty acid estimation from methyl linewidths. LEVEL OF EVIDENCE: 1 Technical Efficacy Stage 1 J. Magn. Reson. Imaging 2017.

The 3D structure of thuricin CD, a two-component bacteriocin with cysteine sulfur to α-carbon cross-links.

Thuricin CD is an antimicrobial factor that consists of two peptides, Trn-α and Trn-ß, that exhibit synergistic activity against drug resistant strains of Clostridium difficile. Trn-α and Trn-ß each possess three sulfur to α-carbon thioether bridges for which the stereochemistry is unknown. This report presents the three-dimensional solution structures of Trn-α and Trn-ß. Structure calculations were performed for the eight possible stereoisomers of each peptide based on the same NMR data. The structure of the stereoisomer that best fit the experimental data was chosen as the representative structure for each peptide. It was determined that Trn-α has L-stereochemistry at Ser21 (α-R), L-stereochemistry at Thr25 (α-R), and D-stereochemistry at Thr28 (α-S) (an LLD isomer). Trn-ß was also found to be the LLD isomer, with L-stereochemistry at Thr21 (α-R), L-stereochemistry at Ala25 (α-R), and D-stereochemistry at Tyr28 (α-S).

Public compliance with mass prophylaxis guidance.

In 2008, the New York City Department of Health and Mental Hygiene (NYC DOHMH) conducted a series of 8 focus groups to determine what improvements could be made to existing plans to ensure that the public would adhere to instructions issued during an emergency that required mass antibiotic distribution following an aerosolized anthrax attack. Discussion focused on perceptions surrounding public health emergencies, overall point-of dispensing (POD) strategy, willingness to pick up medications for others, and additional information that participants would need before and during an emergency. Participation in each group ranged from 7 to 10 members. Most participants indicated a willingness to actively participate in emergency response and to follow directions issued by authorities. Some said they would wait to see how others reacted to medication being provided before taking theirs. Participants expressed a universal desire for education on both dispensing plans and diseases before an incident occurs. They expressed concerns about anxiety levels among the public and maintaining adequate security at dispensing sites, though they felt that NYC's plans were generally realistic. The most trusted sources identified to disseminate information were the mayor, the city health commissioner, and a local cable news channel. While many participants indicated they would use the internet to find information during an emergency, multiple delivery methods must be used to ensure the broadest reach within the community, as not everyone has internet access. Health authorities must partner with the public before, during, and after an emergency to achieve the best possible outcomes from a response effort that relies greatly on public cooperation.

Solution structure of Cu6 metallothionein from the fungus Neurospora crassa.

The 3D-solution structure of Neurospora crassa Cu(6)-metallothionein (NcMT) polypeptide backbone was determined using homonuclear, multidimensional (1)H-NMR spectroscopy. It represents a new metallothionein (MT) fold with a protein chain where the N-terminal half is left-handed and the C-terminal half right-handedly folded around a copper(I)-sulfur cluster. As seen with other MTs, the protein lacks definable secondary structural elements; however, the polypeptide fold is unique. The metal coordination and the cysteine spacing defines this unique fold. NcMT is only the second MT in the copper-bound form to be structurally characterized and the first containing the -CxCxxxxxCxC- motif. This motif is found in a variety of mammalian MTs and metalloregulatory proteins. The in vitro formation of the Cu(6)NcMT identical to the native Cu(6)NcMT was dependent upon the prior formation of the Zn(3)NcMT and its titration with Cu(I). The enhanced sensitivity and resolution of the 800 MHz (1)H-NMR spectral data permitted the 3D structure determination of the polypeptide backbone without the substitution and utilization of the NMR active spin 1/2 metals such as (113)Cd and (109)Ag. These restraints have been necessary to establish specific metal to cysteine restraints in 3D structural studies on this family of proteins when using lower field, less sensitive (1)H-NMR spectral data. The accuracy of the structure calculated without these constraints is, however, supported by the similarities of the 800 MHz structures of the alpha-domain of mouse MT1 compared to the one recalculated without metal-cysteine connectivities.

Structure of subtilosin A, a cyclic antimicrobial peptide from Bacillus subtilis with unusual sulfur to alpha-carbon cross-links: formation and reduction of alpha-thio-alpha-amino acid derivatives.

The complete primary and three-dimensional solution structures of subtilosin A (1), a bacteriocin from Bacillus subtilis, were determined by multidimensional NMR studies on peptide produced using isotopically labeled [(13)C,(15)N]medium derived from Anabaena sp. grown on sodium [(13)C]bicarbonate and [(15)N]nitrate. Additional samples of 1 were also generated by separate incorporations of [U-(13)C,(15)N]-L-phenylalanine and [U-(13)C,(15)N]-L-threonine using otherwise unlabeled media. The results demonstrate that in addition to having a cyclized peptide backbone (amide between N and C termini), three cross-links are formed between the sulfurs of Cys13, Cys7, and Cys4 and the alpha-positions of Phe22, Thr28, and Phe31, respectively. The stereochemistry of all residues in 1 except for the three modified ones was confirmed to be L by complete desulfurization with nickel boride, acid hydrolysis to the constituent amino acids, and conversion of these to the corresponding pentafluoropropanamide isopropyl esters for chiral GC MS analysis. The stereochemistry at the modified residues was determined by subjecting each of the eight possible stereoisomers of 1 to eight rounds of ARIA structure calculations, starting with the same NMR peak files and assignments. The stereoisomer with the l stereochemistry at Phe22 (alpha-R) and d stereochemistry at Thr28 (alpha-S) and Phe31 (alpha-S) (LDD isomer) fit the NMR data, giving the lowest energy family of structures with the best rmsd. Thus, biochemical formation of the unusual thio links proceeds with net retention of configuration at Phe22, and inversion at Thr28 and Phe31. Model amino acid derivatives bearing a sulfide moiety at the alpha-carbon were synthesized by reaction of the corresponding alpha-alkoxy compounds with benzyl thiol and SnCl(4). Separation of their pure stereoisomers and desulfurization with nickel boride demonstrated that the reduction of such compounds proceeds with epimerization, in contrast to the previously reported retention of stereochemistry for analogous reaction of steroidal sulfides. However, desulfurization of subtilosin A to cyclic peptide 14, which is inactive as an antimicrobial agent, occurs with inversion of stereochemistry at the alpha-carbons of Phe22 and Thr28 and with 4:1 retention at Phe31. This indicates that the desulfurization reaction proceeds via an N-acyl imine and that the structure of the surrounding peptide controls the geometry of reduction. Posttranslational linkage of a thiol to the alpha-carbon of an amino acid residue is unprecedented in ribosomally synthesized peptides or proteins, and very rare in secondary metabolites. Subtilosin A (1) represents a new class of bacteriocins.

Structure of subtilosin A, an antimicrobial peptide from Bacillus subtilis with unusual posttranslational modifications linking cysteine sulfurs to alpha-carbons of phenylalanine and threonine.

The complete primary and three-dimensional solution structures of subtilosin A (1), a bacteriocin from Bacillus subtilis, were determined by multidimensional NMR studies on peptide produced using isotopically labeled [(13)C,(15)N]medium derived from Anabaena sp. grown on sodium [(13)C]bicarbonate and [(15)N]nitrate. Additional samples of 1 were also generated by separate incorporations of [U-(13)C,(15)N]phenylalanine and [U-(13)C,(15)N]threonine using otherwise unlabeled media. The results demonstrate that in addition to having a cyclized peptide backbone (N and C termini), three cross-links are formed between the sulfurs of Cys13, Cys7, and Cys4 and the alpha-positions of Phe22, Thr28, and Phe31, respectively. Such posttranslational linkage of a thiol to the alpha-carbon of an amino acid residue is very unusual in natural peptides or proteins. Subtilosin A (1) belongs to a new class of bacteriocins.