Amino Acid Insertion Research Articles

Side chain inset of neurogenerative amino acids to metalloproteins: a therapeutic signature for huntingtin protein in Huntington's disease.

Huntington's disease is a dominant autosomal inherited neurodegenerative disease that results in progressive impairment, characterized by dementia, chorea, and behavioral and cognitive decline. The objective of this study was to investigate the potential activity of metalloproteins against the huntingtin protein using various insertion-based engineering computational methods. Metalloproteins, metal protein complexes involved in important biochemical and physiological processes, were explored as potential drug candidates for Huntington's disease. A total of 18 metalloproteins were selected as drug candidates and studied to assess their potential inhibitory effects on the huntingtin protein. The screening process was based on the lowest binding energy. The metalloprotein with the lowest docking score was chosen for side chain insertion of neurogenerative amino acids. The engineered metalloprotein was then evaluated based on physiochemical properties, allergenicity, toxicity, and surface accessibility. Cloning and expression analysis was performed to further investigate its potential as a therapeutic agent. The metalloprotein chosen for side chain insertion, cytochrome C oxidase, showed promising results. It was computed as a probable non-allergen and exhibited no toxic domains, indicating its non-toxic nature. Additionally, it demonstrated a strong binding affinity with the huntingtin protein, with a binding energy of -1,253.3 Kcal/mol. Metal-based proteins, when engineered with additional neurogenerative amino acids, hold potential as drug candidates for treating neurodegenerative diseases such as Huntington's disease. The successful development of these engineered metalloproteins could offer therapeutic advantages. Further testing, both in vitro and in vivo, is necessary to evaluate their efficacy and validate their potential activity as novel drugs for the treatment of neurodegenerative diseases.

A Porcine DNMT1 Variant: Molecular Cloning and Generation of Specific Polyclonal Antibody.

DNA methyltransferase 1 (DNMT1), the first-identified DNA methyltransferase in mammals, has been well studied in the control of embryo development and somatic homeostasis in mice and humans. Accumulating reports have demonstrated that DNMT1 plays an important role in the regulation of differentiation and the activation of immune cells. However, little is known about the effects of porcine DNMT1 on such functional regulation, especially the regulation of the biological functions of immune cells. In this study, we report the cloning of DNMT1 (4833 bp in length) from porcine alveolar macrophages (PAMs). According to the sequence of the cloned DNMT1 gene, the deduced protein sequence contains a total of 1611 amino acids with a 2 amino acid insertion, a 1 amino acid deletion, and 12 single amino acid mutations in comparison to the reported DNMT1 protein. A polyclonal antibody based on a synthetic peptide was generated to study the expression of the porcine DNMT1. The polyclonal antibody only recognized the cloned porcine DNMT1 and not the previously reported protein due to a single amino acid difference in the antigenic peptide region. However, the polyclonal antibody recognized the endogenous DNMT1 in several porcine cells (PAM, PK15, ST, and PIEC) and the cells of other species (HEK-293T, Marc-145, MDBK, and MDCK cells). Moreover, our results demonstrated that all the detected tissues of piglet express DNMT1, which is the same as that in porcine alveolar macrophages. In summary, we have identified a porcine DNMT1 variant with sequence and expression analyses.

EGFR exon 19 insertion EGFR-K745_E746insIPVAIK and others with rare XPVAIK amino-acid insertions: Preclinical and clinical characterization of the favorable therapeutic window to all classes of approved EGFR kinase inhibitors

BackgroundThe epidermal growth factor receptor (EGFR)-K745_E746insIPVAIK and others with XPVAIK amino-acid insertions are exon 19 insertion mutations, which, at the structural modeling level, resemble EGFR tyrosine kinase inhibitor (TKI)-sensitizing mutants. An important unmet need is the characterization of therapeutic windows plus clinical outcomes of exon 19 XPVAIK amino-acid insertion mutations to available EGFR TKIs. MethodsWe used preclinical models of EGFR-K745_E746insIPVAIK and more typical EGFR mutations (exon 19 deletion, L858R, L861Q, G719S, A763_Y764insFQEA, other exon 20 insertion mutations) to probe representative 1st (erlotinib), 2nd (afatinib), 3rd generation (osimertinib), and EGFR exon 20 insertion active (mobocertinib) TKIs. We also compiled outcomes of EGFR exon 19 insertion mutated lung cancers—from our institution plus the literature—treated with EGFR TKIs. ResultsExon 19 insertions represented 0.3–0.8% of all EGFR kinase domain mutation in two cohorts (n = 1772). Cells driven by EGFR-K745_E746insIPVAIK had sensitivity to all classes of approved EGFR TKIs when compared to cells driven by EGFR-WT in proliferation assays and at the protein level. However, the therapeutic window of EGFR-K745_E746insIPVAIK driven cells was most akin to those of cells driven by EGFR-L861Q and EGFR-A763_Y764insFQEA than the more sensitive patterns seen with cells driven by an EGFR exon 19 deletion or EGFR-L858R. The majority (69.2%, n = 26) of patients with lung cancers harboring EGFR-K745_E746insIPVAIK and other mutations with rare XPVAIK amino-acid insertions responded to clinically available EGFR TKIs (including icotinib, gefitinib, erlotinib, afatinib and osimertinib), with heterogeneous periods of progression-free survival. Mechanisms of acquired EGFR TKI resistance of this mutant remained underreported. ConclusionsThis is the largest preclinical/clinical report to highlight that EGFR-K745_E746insIPVAIK and other mutations with exon 19 XPVAIK amino-acid insertions are rare but sensitive to clinically available 1st, 2nd, and 3rd generation as well as EGFR exon 20 active TKIs; in a pattern that mostly resembles the outcomes of models with EGFR-L861Q and EGFR-A763_Y764insFQEA mutations. These data may help with the off-label selection of EGFR TKIs and clinical expectations of outcomes when targeted therapy is deployed for these EGFR mutated lung cancers.

Structural Dynamics Predominantly Determine the Adaptability of Proteins to Amino Acid Deletions.

The insertion or deletion (indel) of amino acids has a variety of effects on protein function, ranging from disease-forming changes to gaining new functions. Despite their importance, indels have not been systematically characterized towards protein engineering or modification goals. In the present work, we focus on deletions composed of multiple contiguous amino acids (mAA-dels) and their effects on the protein (mutant) folding ability. Our analysis reveals that the mutant retains the native fold when the mAA-del obeys well-defined structural dynamics properties: localization in intrinsically flexible regions, showing low resistance to mechanical stress, and separation from allosteric signaling paths. Motivated by the possibility of distinguishing the features that underlie the adaptability of proteins to mAA-dels, and by the rapid evaluation of these features using elastic network models, we developed a positive-unlabeled learning-based classifier that can be adopted for protein design purposes. Trained on a consolidated set of features, including those reflecting the intrinsic dynamics of the regions where the mAA-dels occur, the new classifier yields a high recall of 84.3% for identifying mAA-dels that are stably tolerated by the protein. The comparative examination of the relative contribution of different features to the prediction reveals the dominant role of structural dynamics in enabling the adaptation of the mutant to mAA-del without disrupting the native fold.

Improvement of the UV-resistance capability of fish gelatin-oxidized starch film via inserting mycosporine-like amino acids.

Mycosporine-like amino acids (MAAs) are known as the strongest solar guardians in nature. In the present study, the extraction of MAAs from dried Pyropia haitanensis was achieved. Fish gelatin and oxidized starch composite films embedded with MAAs (0-0.3% w/w) were fabricated. The maximum absorption wavelength of the composite film appeared at 334 nm, which was consistent with MAA solution. Furthermore, the UV absorption intensity of the composite film was highly dependent on the concentration of MAAs. The composite film exhibited excellent stability during the 7-day storage period. The physicochemical features of composite film were demonstrated by the measurement of water content, water vapor transmission rate, oil transmission, and visual characteristics. Furthermore, in the actual anti-UV effect investigation, the increase in peroxide value and the acid value of grease under the films coverage was delayed. In the meantime, the decrease in ascorbic acid content in dates was postponed, and survivability of Escherichia coli was increased. Our results suggest that fish gelatin-oxidized starch-mycosporine-like amino acids film (FOM film) with biodegradable and anti-ultraviolet properties has a high potential for usage in food packaging materials. © 2023 Society of Chemical Industry.

Human Papillomavirus Infection and EGFR Exon 20 Insertions in Sinonasal Inverted Papilloma and Squamous Cell Carcinoma.

This study aimed to clarify the roles of high-risk human papillomavirus (HR-HPV) infection and epidermal growth factor receptor (EGFR) exon 20 mutations in sinonasal inverted papilloma (IP) and sinonasal squamous cell carcinoma (SNSCC). Samples were collected from 20 cases with IP, 7 with IP and squamous cell carcinoma (IP-SCC), and 20 with SNSCC and examined for HPV infection and EGFR exon 20 mutations. Low- or high-risk HPV DNA was observed in 25% of IP, 57.1% of IP-SCC, and 35% of SNSCC cases. Transcriptionally active HR-HPV infections in IP-SCC and SNSCC, accompanied by p16 overexpression, were observed in 28.5% and 25% of cases, respectively. Heterozygous EGFR exon 20 amino acid insertions (ex20ins), located between amino acids 768-774, were observed in 45% of IP, 28.5% of IP-SCC, and 0% of SNSCC and chronic sinusitis cases. EGFR phosphorylation sites were located at tyrosine (Y) 845, Y1068, Y1086, and Y1197 and induced PI3K/AKT/mTOR activation. The phosphorylation pattern of EGFR with ex20ins resembled that of HPV-related SNSCC and oropharyngeal cancer. The transcriptionally active HR-HPV infection and ex20ins might be responsible for the pathogenesis of IP-SCC cases with different fashions. Since IP-SCC might be a multifactorial disease, further investigation is needed to understand IP-SCC etiology.

A novel recombinant porcine sapovirus infection in piglets with diarrhea in Shandong Province, China, 2022.

Sapporo virus (SaV) is an emerging enteric virus causing acute gastroenteritis in animals. Here, we found a novel porcine SaV (PoSaV) strain (named SD2202) from the piglets with diarrhea in China in 2022. The highest nucleotide homology of SD2202 with other PoSaV strains is only 90.67%, and there are four amino acids insertion in the viral capsid protein and minor structural protein compared to other PoSaV; furthermore, we found that SD2202 belongs to a new GIII genogroup clade (GIII-6 clade). Interestingly, we found that SD2202 may be an intra-genogroup recombinant strain. Taken together, we found a novel PoSaV implicated in the piglet diarrhea epidemic and emphasized the importance of continuous surveillance of PoSaV.

Using the Noncanonical Metallo-Amino Acid [Cu(II)(2,2'-Bipyridin-5-yl)]-alanine to Study the Structures of Proteins.

Genetic code expansion allows modification of the physical and chemical properties of proteins by the site-directed insertion of noncanonical amino acids. Here we exploit this technology for measuring nanometer-scale distances in proteins. (2,2'-Bipyridin-5-yl)alanine was incorporated into the green fluorescent protein (GFP) and used as an anchoring point for Cu(II) to create a spin-label. The incorporation of (2,2'-bipyridin-5-yl)alanine directly into the protein resulted in a high-affinity binding site for Cu(II) capable of outcompeting other binding positions in the protein. The resulting Cu(II)-spin label is very compact and not larger than a conventional amino acid. By using 94 GHz electron paramagnetic resonance (EPR) pulse dipolar spectroscopy we have been able to determine accurately the distance between two such spin-labels. Our measurements revealed that GFP dimers can adopt different quaternary conformations. The combination of spin-labeling using a paramagnetic nonconventional amino acid with high-frequency EPR techniques resulted in a sensitive method for studying the structures of proteins.

The mutation of CaCKI1 causes seedless fruits in chili pepper (Capsicum annuum)

The seedless mutant tn-1 in chili pepper is caused by a mutation in CaCKI1 (CA12g21620), which encodes histidine kinase involving female gametophyte development. An amino acid insertion in the receiver domain of CaCKI1 may be the mutation responsible for tn-1. Seedlessness is a desirable trait in fruit crops because the removal of seeds is a troublesome step for consumers and processing industries. However, little knowledge is available to develop seedless chili peppers. In a previous study, a chili pepper mutant tn-1, which stably produces seedless fruits, was isolated. In this study, we report characterization of tn-1 and identification of the causative gene. Although pollen germination was normal, confocal laser microscopy observations revealed deficiency in embryo sac development in tn-1. By marker analysis, the tn-1 locus was narrowed down to a 313kb region on chromosome 12. Further analysis combined with mapping-by-sequencing identified CA12g21620, which encodes histidine kinase as a candidate gene. Phylogenetic analysis revealed CA12g21620 was the homolog of Arabidopsis CKI1 (Cytokinin Independent 1), which plays an important role in female gametophyte development, and CA12g21620 was designated as CaCKI1. Sequence analysis revealed that tn-1 has a 3-bp insertion in the 6th exon resulting in one lysine (K) residue insertion in receiver domain of CaCKI1, and the sequence nearby the insertion is widely conserved among CKI1 orthologs in various plants. This suggested that one K residue insertion may reduce the phosphorylation relay downstream of CaCKI1 and impair normal development of female gametophyte, resulting in seedless fruits production in tn-1. Furthermore, we demonstrated that virus-induced gene silencing of CaCKI1 reducednormally developed female gametophyte in chili pepper. This study describes the significant role of CaCKI1 in seed development in chili pepper and the possibility of developing seedless cultivars using its mutation.

In vitro activity of cefepime/zidebactam and cefepime/taniborbactam against aztreonam/avibactam-resistant NDM-like-producing Escherichia coli clinical isolates.

Aztreonam/avibactam is one of the last therapeutic options for treating infections caused by NDM-like-producing Enterobacterales. However, PBP3-modified and NDM-producing Escherichia coli strains that co-produce CMY-42 have been shown to be resistant to this drug combination. The aim of our study was to assess the in vitro activity of cefepime/taniborbactam and cefepime/zidebactam against such aztreonam/avibactam-resistant E. coli strains. MIC values of aztreonam, aztreonam/avibactam, cefepime, cefepime/taniborbactam, cefepime/zidebactam and zidebactam alone were determined for 28 clinical aztreonam/avibactam-resistant E. coli isolates. Those isolates produced either NDM-5 (n = 24), NDM-4 (n = 2) or NDM-1 (n = 2), and they all co-produced CMY-42 (n = 28). They all harboured a four amino acid insertion in PBP-3 (Tyr-Arg-Ile-Asn or Tyr-Arg-Ile-Lys). All strains were resistant to aztreonam/avibactam and cefepime, as expected. The resistance rate to cefepime/taniborbactam was 100%, with MIC50 and MIC90 being at 16 mg/L and 64 mg/L, respectively. Conversely, all strains were susceptible to cefepime/zidebactam, with both MIC50 and MIC90 at 0.25 mg/L. Notably, all strains showed low MICs for zidebactam alone, with MIC50 and MIC90 at 0.5 mg/L and 1 mg/L. Our data highlighted the excellent in vitro performance of the newly developed β-lactam/β-lactamase inhibitor combination cefepime/zidebactam against aztreonam/avibactam-resistant E. coli strains, suggesting that this combination could be considered as an efficient therapeutic option in this context. Our study also highlights the cross-resistance between acquired resistance to aztreonam/avibactam and the cefepime/taniborbactam combination.

Evaluation of in silico pathogenicity prediction tools for the classification of small in-frame indels

BackgroundThe use of in silico pathogenicity predictions as evidence when interpreting genetic variants is widely accepted as part of standard variant classification guidelines. Although numerous algorithms have been developed and evaluated for classifying missense variants, in-frame insertions/deletions (indels) have been much less well studied.MethodsWe created a dataset of 3964 small (< 100 bp) indels predicted to result in in-frame amino acid insertions or deletions using data from gnomAD v3.1 (minor allele frequency of 1–5%), ClinVar and the Deciphering Developmental Disorders (DDD) study. We used this dataset to evaluate the performance of nine pathogenicity predictor tools: CADD, CAPICE, FATHMM-indel, MutPred-Indel, MutationTaster2021, PROVEAN, SIFT-indel, VEST-indel and VVP.ResultsOur dataset consisted of 2224 benign/likely benign and 1740 pathogenic/likely pathogenic variants from gnomAD (n = 809), ClinVar (n = 2882) and, DDD (n = 273). We were able to generate scores across all tools for 91% of the variants, with areas under the ROC curve (AUC) of 0.81–0.96 based on the published recommended thresholds. To avoid biases caused by inclusion of our dataset in the tools’ training data, we also evaluated just DDD variants not present in either gnomAD or ClinVar (70 pathogenic and 81 benign). Using this subset, the AUC of all tools decreased substantially to 0.64–0.87. Several of the tools performed similarly however, VEST-indel had the highest AUCs of 0.93 (full dataset) and 0.87 (DDD subset).ConclusionsAlgorithms designed for predicting the pathogenicity of in-frame indels perform well enough to aid clinical variant classification in a similar manner to missense prediction tools.

Different Variants of SARS-CoV-2: A Comprehensive Review on Mutation Patterns and Pathogenicity

Abstract: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a single-stranded and positive- sense RNA virus, is exhibiting a great degree of mutation acclimated to variable environments, posing a great threat to human life, with numerous SARS-CoV-2 variants. This review aims to analyze emerging strains of SARS-CoV-2, their mutational pattern, their impact on viral pathogenicity, and the identification of the targets for the development of vaccines and therapeutics against the virus. SARSCoV- 2 consists of four structural protein codes viz, peplomer protein (S), an envelope protein (E), nucleoprotein capsid (N), and matrix protein (M), that are involved in the ingression of the viral genome into the host cell by interacting with the host cell receptor, i.e., human angiotensin-converting enzyme 2 (ACE2) (only S protein is shown to bind to ACE-2 receptor). The review includes consequences of mutations- D614G (pre-dominant mutation), E484K, N501Y, K417N, L452R, P681H, del69/70, del157/158, Ins214EPE, and so forth, in alpha, beta, gamma, delta, omicron including other strains of SARS-CoV-2. Mutations include substitution, deletion, and insertion of amino acids in the spike glycoprotein of the SARS-CoV-2, resulting in antibody neutralization, immune escape, etc. As a consequence of modifications in the nucleotide sequence of the viral genome code pushed on by exposure to various environments, these new strains influence the pathogenicity of the virus.

Characterization of binding interaction between S100A1 and titin's UN2A region.

Titin, the giant sarcomere protein, consists of multiple domains and regions of repeated structural elements that are essential for sarcomeric integrity of the myofibril. The N2A region within titin has been identified as a signaling hub in skeletal muscle that is responsible for various mechanics and interactions in the muscle. The N2A region is located at the junction between the proximal tandem Ig region and the PEVK region and is comprised of four Ig domains and a unique 117 amino acid insertion sequence (UN2A). The insertion sequence resides between the Ig80 and Ig81 domains, and exists in skeletal muscle, as well as the N2AB isoform of cardiac muscle. An analysis of the insertion region sequence revealed a potential binding site for the Ca2+-binding protein S100A1. A combination of circular dichroism and FRET experiments demonstrated the conformational change in UN2A that is induced by S100A1, and can be affected differently due to pH changes. Surface plasmon resonance was used to study the binding kinetics and affinities of this interaction at different pH and pCa values. S100A1's binding to UN2A is calcium-dependent, and the strength of this interaction is affected by pH. The S100A1/UN2A binding interaction has been shown to perturb the structure of UN2A potentially at its stable helical core. The results will provide insight on the nature of this interaction and how it may have a functional role in the muscle.

The C5C7 domains of cMyBP-C do not alter myofilament calcium sensitivity of tension.

Cardiac myosin binding protein-C (cMyBP-C) regulates muscle contraction and relaxation. Most of the functional effects of cMyBP-C have been attributed to its N’-terminal domains C0-C2. Yet, specific functional roles of its central domains (C3, C4, C5, C6, and C7), if any, are unknown. Structural studies show that C5 contains of a 28 amino acid insertion that is unstructured and hypothesized to contribute to protein-protein interactions. In this study, we aimed to determine the functional effects of C5, C6 and C7 on myofilament force mechanics. We used the novel Spy-C3 “cut and paste” mouse model that allows for the replacement of recombinant cMyBP-C protein within its location in the sarcomere. The homozygous Spy-C3 mouse has a 20 amino acid cassette inserted between C7 and C8 with a tobacco etch virus protease (TEVp) recognition site followed by a “SpyTag.” Detergent-permeabilized cardiomyocytes were attached to a force transducer and exposed to TEVp to cut and wash out the C0-C7 domains. Recombinant C5-C7 domains expressing SpyCatcher (sc) were then ligated to SpyTag at the position of endogenous cMyBP-C. Results showed that cutting cMyBP-C with TEVp led to a significant rightward shift in the tension-pCa curve (repeated measures one-way ANOVA, p < 0.0001), an increase in the rate of cross-bridge detachment determined by stretch activation (Krel) (p = 0.0003), and the presence of spontaneous oscillatory contractions (SPOC) compared to before TEVp treatment. However, replacement with recombinant protein C5C7sc did not rescue the functional effects following loss of C0-C7. These data may indicate that domains C5-C7 alone do not contribute to cMyBP-C's role in sensitizing the myofilament and regulating rates of cross-bridge cycling. This work was supported by NIH HL080367 (SPH), AHA Predoctoral Fellowship (NE), and an AHA Postdoctoral Fellowship (AG).

N-terminal domain influences steroid activation of the Atlantic sea lamprey corticoid receptor

Lampreys are jawless fish that evolved about 550 million years ago at the base of the vertebrate line. Modern lampreys contain a corticoid receptor (CR), the common ancestor of the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), which first appear in cartilaginous fish, such as sharks. Until recently, 344 amino acids at the amino terminus of adult lamprey CR were not present in the lamprey CR sequence in GenBank. A search of the recently sequenced lamprey germline genome identified two CR sequences, CR1 and CR2, containing the 344 previously un-identified amino acids. CR1 also contains a novel four amino acid insertion in the DNA-binding domain (DBD). We studied corticosteroid and progesterone activation of CR1 and CR2 and found their strongest response was to 11-deoxycorticosterone and 11-deoxycortisol, the two circulating corticosteroids in lamprey. Based on steroid specificity, both CRs are close to elephant shark MR and distant from elephant shark GR. HEK293 cells that were transfected with full-length CR1 or CR2 and the MMTV promoter have about 3-fold higher steroid-mediated activation compared to HEK293 cells transfected with these CRs and the TAT3 promoter. Deletion of the amino-terminal domain (NTD) of lamprey CR1 and CR2 to form truncated CRs decreased transcriptional activation by about 70% in HEK293 cells that were transfected with MMTV, but increased transcription by about 6-fold in cells transfected with TAT3. This indicated that the promoter has an important effect on NTD regulation of transcriptional activation of the CR by steroids. Our results also indicate that the entire lamprey CR sequence is needed for an accurate determination of steroid-mediated transcription.

Insertion of ten amino acids into 13S globulin zero-repeat subunit improves trypsin digestibility in common buckwheat (Fagopyrum esculentum Moench) seeds

The 13S globulin zero-repeat subunit is resistant to trypsin and may have higher allergenicity than the 1–6 tandem repeat subunits in common buckwheat (Fagopyrum esculentum Moench). To explore alleles useful for lowering allergenicity, amplicon deep sequencing targeting the zero-repeat subunit gene was conducted in bulked genomic DNA from eight cultivars and landraces. The analysis identified a unique allele encoding a zero-repeat subunit with 10 amino acid insertion (10aa) at a position equivalent to the tandem repeat insertion. Prediction of its 3-D structure suggested that 10aa changes the β-hairpin structure in the non-10aa (native) subunit to a random coil, which is also found in 1- and 3- repeat subunits. Homozygotes of the 10aa allele were developed and showed that the 10aa subunit was more digestible than the native subunit. However, the 10aa subunit was still less digestible than the 1–6 repeat subunits, suggesting needs to explore unfunctional alleles.

1160. Treatment-Emergent Viral Variants in the Phase 3 TACKLE Trial Investigating Efficacy and Safety of AZD7442 (Tixagevimab/Cilgavimab) for the Treatment of Mild to Moderate COVID-19 in Adults

Abstract Background AZD7442 (tixagevimab/cilgavimab) is a combination of neutralizing monoclonal antibodies (mAbs) that bind to distinct epitopes on the SARS-CoV-2 spike protein, with neutralization activity against variants including Omicron. In the Phase 3 TACKLE study, AZD7442 significantly reduced severe disease progression or death and was well-tolerated through Day 29. Viral evolution during treatment has the potential for resistance selection, such as variants exhibiting reduced mAb binding. We report genotypic analysis and phenotypic characterization of variants identified over 15 days after AZD7442 treatment in TACKLE. Methods In TACKLE (NCT04723394), non-hospitalized adults with mild to moderate COVID19 were randomized and dosed ≤7 days from symptom onset with a single 600-mg AZD7442 dose (2 consecutive intramuscular injections, 300 mg of each antibody; n=452) or placebo (n=451). Next-generation sequencing of the spike gene was performed on SARS-CoV-2 reverse-transcription polymerase chain reaction–positive nasal swabs (at baseline and Days 3, 6, and 15). SARS-CoV-2 lineages were assigned using spike nucleotide sequences. Amino acid substitutions, insertions, and deletions were analyzed at allele fractions (AF, % of sequence reads represented by mutation) ≥25% and 3–25%. Results Baseline spike sequences were available from 744 participants (82.4%) (AZD7442, n=380; placebo, n=364); 87% of sequences corresponded to variants of concern/interest; these were balanced between AZD7442 and placebo groups (Table 1). Treatment-emergent (post-dosing) viral variants were rare, with 11 (4.5%) AZD7442 and 3 (1.3%) placebo participants showing the emergence of ≥1 mutation at tixagevimab/cilgavimab binding sites, with an AF ≥25% (Table 2). At AF 3–25%, treatment-emergent viral variants in the AZD7442 binding site were observed in 16 (6.6%) AZD7442 and 15 (6.5%) placebo participants. Conclusion Following AZD7442 treatment, low levels of SARS-CoV-2 variants bearing mutations at tixagevimab/cilgavimab binding sites were identified. These data indicate that combination of two antibodies creates a high genetic barrier for resistance, supporting the use of mAb combinations that bind to distinct epitopes for the treatment of COVID-19. Disclosures Gustavo H. Kijak, PharmD PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Bahar Ahani, BSC, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Douglas Arbetter, MPH, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Tyler Brady, MS, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Fernando Chuecos, BS, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Vancheswaran Gopalakrishnan, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Tiffany L. Roe, B.S., AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Nicolette Schuko, MS, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds F.D. Richard Hobbs, FMedSci, AstraZeneca: Grant/Research Support|AstraZeneca: Principal investigator|NIHR Applied Research Collaboration, Oxford Thames Valley: Director|Oxford BRC and NIHR MedTech: Investigator|UKRI and NIHR: Grant/Research Support Francisco Padilla, MD, Amgen: Grant/Research Support|Amgen: Personal fees|AstraZeneca: Grant/Research Support|AstraZeneca: Personal fees|Boehringer Ingelheim: Grant/Research Support|Boehringer Ingelheim: Personal fees|Ferrer: Grant/Research Support|Ferrer: Personal fees|Kowa: Grant/Research Support|Kowa: Personal fees|Medix: Grant/Research Support|Medix: Personal fees|Merck Sharp and Dohme: Grant/Research Support|Merck Sharp and Dohme: Personal fees|Novartis: Grant/Research Support|Novartis: Personal fees|Pfizer: Grant/Research Support|Pfizer: Personal fees|Sanofi: Grant/Research Support|Sanofi: Personal fees|Servier: Grant/Research Support|Servier: Personal fees|Silanes: Grant/Research Support|Silanes: Personal fees Elizabeth J. Kelly, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Hugh Montgomery, MD, AstraZeneca: Advisor/Consultant|Millfield Medical Ltd: Advisor/Consultant|UK National Institute for Health Research's Comprehensive Biomedical Research Centre at University College London Hospitals: Grant/Research Support Katie Streicher, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds.

Epistatic Effects Between Amino Acid Insertions and Substitutions Mediate Toxin resistance of Vertebrate Na+,K+-ATPases.

The recurrent evolution of resistance to cardiotonic steroids (CTS) across diverse animals most frequently involves convergent amino acid substitutions in the H1-H2 extracellular loop of Na+,K+-ATPase (NKA). Previous work revealed that hystricognath rodents (e.g., chinchilla) and pterocliform birds (sandgrouse) have convergently evolved amino acid insertions in the H1-H2 loop, but their functional significance was not known. Using protein engineering, we show that these insertions have distinct effects on CTS resistance in homologs of each of the two species that strongly depend on intramolecular interactions with other residues. Removing the insertion in the chinchilla NKA unexpectedly increases CTS resistance and decreases NKA activity. In the sandgrouse NKA, the amino acid insertion and substitution Q111R both contribute to an augmented CTS resistance without compromising ATPase activity levels. Molecular docking simulations provide additional insight into the biophysical mechanisms responsible for the context-specific mutational effects on CTS insensitivity of the enzyme. Our results highlight the diversity of genetic substrates that underlie CTS insensitivity in vertebrate NKA and reveal how amino acid insertions can alter the phenotypic effects of point mutations at key sites in the same protein domain.

Trypanosoma brucei Mitochondrial DNA Polymerase POLIB Contains a Novel Polymerase Domain Insertion That Confers Dominant Exonuclease Activity.

Trypanosoma brucei and related parasites contain an unusual catenated mitochondrial genome known as kinetoplast DNA (kDNA) composed of maxicircles and minicircles. The kDNA structure and replication mechanism are divergent and essential for parasite survival. POLIB is one of three Family A DNA polymerases independently essential to maintain the kDNA network. However, the division of labor among the paralogs, particularly which might be a replicative, proofreading enzyme, remains enigmatic. De novo modeling of POLIB suggested a structure that is divergent from all other Family A polymerases, in which the thumb subdomain contains a 369 amino acid insertion with homology to DEDDh DnaQ family 3'-5' exonucleases. Here we demonstrate recombinant POLIB 3'-5' exonuclease prefers DNA vs RNA substrates and degrades single- and double-stranded DNA nonprocessively. Exonuclease activity prevails over polymerase activity on DNA substrates at pH 8.0, while DNA primer extension is favored at pH 6.0. Mutations that ablate POLIB polymerase activity slow the exonuclease rate suggesting crosstalk between the domains. We show that POLIB extends an RNA primer more efficiently than a DNA primer in the presence of dNTPs but does not incorporate rNTPs efficiently using either primer. Immunoprecipitation of Pol I-like paralogs from T. brucei corroborates the pH selectivity and RNA primer preferences of POLIB and revealed that the other paralogs efficiently extend a DNA primer. The enzymatic properties of POLIB suggest this paralog is not a replicative kDNA polymerase, and the noncanonical polymerase domain provides another example of exquisite diversity among DNA polymerases for specialized function.

Resistance to aztreonam-avibactam due to CTX-M-15 in the presence of penicillin-binding protein 3 with extra amino acids in Escherichia coli.

Aztreonam-avibactam is a promising combination to treat carbapenem-resistant Enterobacterales including coverage for metallo-β-lactamases. Escherichia coli strains resistant to aztreonam-avibactam have emerged but resistance mechanisms remain to be elucidated. We performed a study to investigate the mechanism for aztreonam-avibactam in a carbapenem-resistant Escherichia coli clinical strain. This strain was resistant to aztreonam-avibactam (aztreonam MIC, 16 mg/L in the presence of 4 mg/L avibactam). Whole genome sequencing revealed that the strain carried metallo-β-lactamase gene bla NDM-4 and the extended-spectrum β-lactamase (ESBL) gene bla CTX-M-15 and had a YRIK four amino acid insertion in penicillin-binding protein 3 (PBP3). bla CTX-M-15 was cloned into pET-28a(+), followed by the transformation, with the gene, of E. coli strain 035125∆pCMY42 possessing the YRIK insertion in PBP3 and strain BL21 with the wildtype PBP3. bla CTX-M-14, another common ESBL gene, and bla CTX-M-199, a hybrid of bla CTX-M-14 and bla CTX-M-15 were also individually cloned into both E. coli strains for comparison. Aztreonam-avibactam resistance was only observed in the E. coli strains with the YRIK insertion in PBP3 that produced CTX-M-15 or its hybrid enzyme CTX-M-199. Checkerboard titration assays were performed to determine the synergistic effects between aztreonam-avibactam and ceftazidime or meropenem. Doubling avibactam concentration in vitro reversed aztreonam-avibactam resistance, while the combination of aztreonam-avibactam and ceftazidime or meropenem did not. In conclusion, CTX-M enzymes with activity against aztreonam, (e.g., CTX-M-15 and CTX-M-199), can confer resistance in the combination of PBP3 with YRIK insertions in metallo-β-lactamase-producing carbapenem-resistant E. coli. Doubling the concentration of avibactam may overcome such resistance.