Showing papers by "Robert McKenna published in 2022"

The three-tails approach as a new strategy to improve selectivity of action of sulphonamide inhibitors against tumour-associated carbonic anhydrase IX and XII

Abstract: Abstract Human (h) carbonic anhydrase (CAs, EC 4.2.1.1) isoforms IX and XII were recently confirmed as anticancer targets against solid hypoxic tumours. The “three-tails approach” has been proposed as an extension of the forerunner “tail” and “dual-tail approach” to fully exploit the amino acid differences at the medium/outer active site rims among different hCAs and to obtain more isoform-selective inhibitors. Many three-tailed inhibitors (TTIs) showed higher selectivity against the tumour-associated isoforms hCA IX and XII with respect to the off-targets hCA I and II. X-ray crystallography studies were performed to investigate the binding mode of four TTIs in complex with a hCA IX mimic. The ability of the most potent and selective TTIs to reduce in vitro the viability of colon cancer (HT29), prostate adenocarcinoma (PC3), and breast cancer (ZR75-1) cell lines was evaluated in normoxic (21% O2) and hypoxic (3% O2) conditions demonstrating relevant anti-proliferative effects.

Semirational bioengineering of AAV vectors with increased potency and specificity for systemic gene therapy of muscle disorders

Abstract: Bioengineering of viral vectors for therapeutic gene delivery is a pivotal strategy to reduce doses, facilitate manufacturing, and improve efficacy and patient safety. Here, we engineered myotropic adeno-associated viral (AAV) vectors via a semirational, combinatorial approach that merges AAV capsid and peptide library screens. We first identified shuffled AAVs with increased specificity in the murine skeletal muscle, diaphragm, and heart, concurrent with liver detargeting. Next, we boosted muscle specificity by displaying a myotropic peptide on the capsid surface. In a mouse model of X-linked myotubular myopathy, the best vectors—AAVMYO2 and AAVMYO3—prolonged survival, corrected growth, restored strength, and ameliorated muscle fiber size and centronucleation. In a mouse model of Duchenne muscular dystrophy, our lead capsid induced robust microdystrophin expression and improved muscle function. Our pipeline is compatible with complementary AAV genome bioengineering strategies, as demonstrated here with two promoters, and could benefit many clinical applications beyond muscle gene therapy.

Structures of LIG1 that engage with mutagenic mismatches inserted by polβ in base excision repair

Abstract: Abstract DNA ligase I (LIG1) catalyzes the ligation of the nick repair intermediate after gap filling by DNA polymerase (pol) β during downstream steps of the base excision repair (BER) pathway. However, how LIG1 discriminates against the mutagenic 3′-mismatches incorporated by polβ at atomic resolution remains undefined. Here, we determine the X-ray structures of LIG1/nick DNA complexes with G:T and A:C mismatches and uncover the ligase strategies that favor or deter the ligation of base substitution errors. Our structures reveal that the LIG1 active site can accommodate a G:T mismatch in the wobble conformation, where an adenylate (AMP) is transferred to the 5′-phosphate of a nick (DNA-AMP), while it stays in the LIG1-AMP intermediate during the initial step of the ligation reaction in the presence of an A:C mismatch at the 3′-strand. Moreover, we show mutagenic ligation and aberrant nick sealing of dG:T and dA:C mismatches, respectively. Finally, we demonstrate that AP-endonuclease 1 (APE1), as a compensatory proofreading enzyme, removes the mismatched bases and interacts with LIG1 at the final BER steps. Our overall findings provide the features of accurate versus mutagenic outcomes coordinated by a multiprotein complex including polβ, LIG1, and APE1 to maintain efficient repair.

Structural Dynamics and Activity of B19V VP1u during the pHs of Cell Entry and Endosomal Trafficking

Abstract: Parvovirus B19 (B19V) is a human pathogen that is the causative agent of fifth disease in children. It is also known to cause hydrops in fetuses, anemia in AIDS patients, and transient aplastic crisis in patients with sickle cell disease. The unique N-terminus of Viral Protein 1 (VP1u) of parvoviruses, including B19V, exhibits phospholipase A2 (PLA2) activity, which is required for endosomal escape. Presented is the structural dynamics of B19V VP1u under conditions that mimic the pHs of cell entry and endosomal trafficking to the nucleus. Using circular dichroism spectroscopy, the receptor-binding domain of B19V VP1u is shown to exhibit an α-helical fold, whereas the PLA2 domain exhibits a probable molten globule state, both of which are pH invariant. Differential scanning calorimetry performed at endosomal pHs shows that the melting temperature (Tm) of VP1u PLA2 domain is tuned to body temperature (37 °C) at pH 7.4. In addition, PLA2 assays performed at temperatures ranging from 25–45 °C show both a temperature and pH-dependent change in activity. We hypothesize that VP1u PLA2 domain differences in Tm at differing pHs have enabled the virus to “switch on/off” the phospholipase activity during capsid trafficking. Furthermore, we propose the environment of the early endosome as the optimal condition for endosomal escape leading to B19V infection.

Small Molecule Alkoxy Oriented Selectiveness on Human Carbonic Anhydrase II and IX Inhibition

Abstract: We report aryl sulfonamide inhibitors of human carbonic anhydrase (hCA; EC 4.2.1.1) enzymes containing short ureido alkoxy tails. The inhibition potency of such compounds was investigated in vitro on the major hCA isoforms (i.e. I, II, IX, and XII). A selection of the most potent inhibitory derivatives against the hCA IX isoform (i.e. 5a, 5c, and 6c) was studied, and their binding modes on either hCA II and IX‐mimic isoform were assessed by X‐ray crystallography on the corresponding ligand/protein adducts. This study adds to the field of developing hCA inhibitors at molecular level the critical interactions governing ligand selectivity.

Characterization of the Serpentine Adeno-Associated Virus (SAAV) Capsid Structure: Receptor Interactions and Antigenicity

Abstract: AAVs are widely studied therapeutic gene delivery vectors. However, preexisting antibodies and their detrimental effect on therapeutic efficacy are a primary challenge encountered during clinical trials. ABSTRACT Adeno-associated viruses (AAVs) are being developed as clinical gene therapy vectors. One issue undermining their broad use in the clinical setting is the high prevalence of circulating antibodies in the general population capable of neutralizing AAV vectors. Hence, there is a need for AAV vectors that can evade the preexisting immune response. One possible source of human naive vectors are AAVs that do not disseminate in the primate population, and one such example is serpentine AAV (SAAV). This study characterizes the structural and biophysical properties of the SAAV capsid and its receptor interactions and antigenicity. Single particle cryo-electron microscopy (cryo-EM) and thermal stability studies were conducted to characterize the SAAV capsid structure at pH 7.4, 6.0, 5.5, and 4.0, conditions experienced during cellular trafficking. Cell binding assays using Chinese hamster ovary (CHO) cell lines identified terminal sialic acid as the primary attachment receptor for SAAV similar to AAV1, 4, 5, and 6. The binding site of sialic acid to the SAAV capsid was mapped near the 2-fold axis toward the 2/5-fold wall, in a different location than AAV1, 4, 5, and 6. Towards determining the SAAV capsid antigenicity native immunodot blots showed that SAAV evades AAV serotype-specific mouse monoclonal antibodies. However, despite its reptilian origin, it was recognized by ~25% of 50 human sera tested, likely due to the presence of cross-reactive antibodies. These findings will inform future gene delivery applications using SAAV-based vectors and further aid the structural characterization and annotation of the repertoire of available AAV capsids. IMPORTANCE AAVs are widely studied therapeutic gene delivery vectors. However, preexisting antibodies and their detrimental effect on therapeutic efficacy are a primary challenge encountered during clinical trials. In order to circumvent preexisting neutralizing antibodies targeting mammalian AAV capsids, serpentine AAV (SAAV) was evaluated as a potential alternative to existing mammalian therapeutic vectors. The SAAV capsid was found to be thermostable at a wide range of environmental pH conditions, and its structure showed conservation of the core capsid topology but displays high structural variability on the surface. At the same time, it binds to a common receptor, sialic acid, that is also utilized by other AAVs already being utilized in gene therapy trials. Contrary to the initial hypothesis, SAAV capsids were recognized by one in four human sera tested, pointing to conserved amino acids around the 5-fold region as epitopes for cross-reacting antibodies.

Safety and Efficacy Outcomes From a Single-Center Study of Image-Guided Percutaneous Microwave Ablation for Primary and Metastatic Lung Malignancy

Abstract: Image-guided percutaneous microwave ablation (MWA) is becoming a more common treatment option for patients with primary and metastatic lung malignancies. Nevertheless, there is limited literature on the safety and efficacy of MWA compared with standard-of-care therapy, including surgical resection and radiation. This study will report the long-term outcomes after MWA for pulmonary malignancies and investigate the factors related to efficacy, including lesion size, location, and ablation power.Retrospective single-center study analyzing 93 patients who underwent percutaneous MWA for primary or metastatic lung malignancies. Outcomes included immediate technical success, local tumor recurrence, overall survival, disease-specific survival, and complications.At a single institution, 190 lesions (81 primary and 109 metastatic) were treated in 93 patients. Immediate technical success was achieved in all cases. Freedom from local recurrence was 87.6%, 75.3%, and 69.2% and overall survival was 87.7%, 76.2%, and 74.3% at 1 year, 2 years, and 3 years, respectively. Disease-specific survival was 92.6%, 81.8%, and 81.8%. The most common complication was pneumothorax, which occurred in 54.7% (104 of 190) of procedures, with 35.2% (67 of 190) requiring a chest tube. No life-threatening complications occurred.Percutaneous MWA seems safe and effective for treatment of primary and metastatic lung malignancies and should be considered for patients with limited metastatic burden and lesions less than 3 cm in size.

Ibuprofen: a weak inhibitor of carbonic anhydrase II.

Abstract: Carbonic anhydrases (CAs) are drug targets for a variety of diseases. While many clinically relevant CA inhibitors are sulfonamide-based, novel CA inhibitors are being developed that incorporate alternative zinc-binding groups, such as carboxylic acid moieties, to develop CA isoform-specific inhibitors. Here, the X-ray crystal structure of human CA II (hCA II) in complex with the carboxylic acid ibuprofen [2-(4-isobutylphenyl)propanoic acid, a common over-the-counter nonsteroidal anti-inflammatory drug] is reported to 1.54 Å resolution. The binding of ibuprofen is overlaid with the structures of other carboxylic acids in complex with hCA II to compare their inhibition mechanisms by direct or indirect (via a water) binding to the active-site zinc. Additionally, enzyme-inhibition assays using ibuprofen, nicotinic acid and ferulic acid were performed with hCA II to determine their IC50 values and were compared with those of other carboxylic acid binders. This study discusses the potential development of CA inhibitors utilizing the carboxylic acid moiety.

Structural and molecular biology of Acheta domesticus segmented densovirus, the first parvovirus to harbor a bipartite genome

Abstract: Parvoviruses (family Parvoviridae) are defined by their linear monopartite ssDNA genome, T=1 icosahedral capsid, and distinct structural (VP) and non-structural (NS) protein expression cassettes within their genome. Here, we report the first parvovirus with a segmented genome, Acheta domesticus segmented densovirus (AdSDV), a house cricket (Acheta domesticus) pathogen. The AdSDV harbors its NS and VP cassettes on two separate segmented genomes. Its VP segment acquired a phospholipase A2-encoding gene via inter-subfamily recombination, which is absent from its capsid. The AdSDV evolved a transcription profile in response to its multipartite replication strategy that has diverged from its Brevihamaparvovirus ancestors. Furthermore, AdSDV assembles three capsid populations, which package one genome segment per particle. The cryoEM structures of these three capsids (2.3 to 3.3 Å resolution) reveal a genome packaging mechanism, which differs from other parvoviruses. This study provides a new perspective on ssDNA genome segmentation and on the plasticity of parvovirus biology.

Cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to asparagine synthetase de fi ciency

Abstract: Asparagine synthetase (ASNS) catalyzes synthesis of asparagine (Asn) and Glu from Asp and Gln in an ATP-dependent reaction. Asparagine synthetase de fi ciency (ASNSD) results from biallelic mutations in the ASNS gene. Affected children exhibit congenital microcephaly, continued brain atrophy, seizures, and often premature mortality. However, the under-lying mechanisms are unclear. This report describes a compound heterozygotic ASNSD child with two novel mutations in the ASNS gene, c.1118G > T (paternal) and c.1556G > A (maternal), that lead to G373V or R519H ASNS variants. Structural mapping suggested that neither variant participates directly in catalysis. Growth of cultured fi broblasts from either parent was unaffected in Asn-free medium, whereas growth of the child ’ s cells was suppressed by about 50%. Analysis of Asn levels unexpectedly revealed that extracellular rather than intracellular Asn correlated with the reduced proliferation during incubation of the child ’ s cells in Asn-free medium. Our attempts to ectopically express the G373V variant in either

Abstract 4055: First in class multifunctional non classical antifolates inhibits thymidylate synthase and extends survival in pancreatic cancer model

Abstract: Thymidylate synthase (TS) inhibitors are an integral component of chemotherapy regimens for difficult to treat cancer subtypes. Despite initial therapeutic benefit, current inhibitors induce TS overexpression or alter folate transport metabolism feedback pathways that tumor cells exploit for drug resistance. Here we report a small molecule TS inhibitor that exhibits i) enhanced antitumor activity as compared to current fluoropyrimidines and antifolates without inducing TS overexpression, ii) is structurally distinct from classical antifolates, iii) extends survival in a pancreatic tumor mouse model, iv) and is well tolerated with equal efficacy using either intraperitoneal or oral administration. Mechanistically, we confirm the compound is a multifunctional non classical antifolate and through a series of analogues identify structural features allowing direct TS inhibition while also maintaining the ability to inhibit dihydrofolate reductase (DHFR). Collectively, this work identifies new non classical antifolate inhibitors that optimize inhibition of thymidylate biosynthesis with a favorable safety profile highlighting potential for enhanced cancer therapy. Citation Format: Maria V. Guijarro, Patrick C. Kellish, Peter E. Dib, Nicholas G. Paciaroni, Akbar Nawab, Jacob Andring, Lidia Kulemina, Nicholas V. Borrero, Carlos Modenutti, Richard L. Bennett, Daniil Shabashvili, Jonathan D. Licht, Robert McKenna, Adrian Roitberg, Robert W. Huigens, Frederic J. Kaye, Maria Zajac-Kaye. First in class multifunctional non classical antifolates inhibits thymidylate synthase and extends survival in pancreatic cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4055.

Structures of LIG1 engaging with mutagenic mismatches inserted by polβ in base excision repair

Abstract: DNA ligase I (LIG1) catalyzes final ligation step following DNA polymerase (pol) β gap filling and an incorrect nucleotide insertion by polβ creates a nick repair intermediate with mismatched end at the downstream steps of base excision repair (BER) pathway. Yet, how LIG1 discriminates against the mutagenic 3′-mismatches at atomic resolution remains undefined. Here, we determined X-ray structures of LIG1/nick DNA complexes with G:T and A:C mismatches and uncovered the ligase strategies that favor or deter ligation of base substitution errors. Our structures revealed that LIG1 active site can accommodate G:T mismatch in a similar conformation with A:T base pairing, while it stays in the LIG1-adenylate intermediate during initial step of ligation reaction in the presence of A:C mismatch at 3′-strand. Moreover, we showed mutagenic ligation and aberrant nick sealing of the nick DNA substrates with 3′-preinserted dG:T and dA:C mismatches, respectively. Finally, we demonstrated that AP-Endonuclease 1 (APE1), as a compensatory proofreading enzyme, interacts and coordinates with LIG1 during mismatch removal and DNA ligation. Our overall findings and ligase/nick DNA structures provide the features of accurate versus mutagenic outcomes at the final BER steps where a multi-protein complex including polβ, LIG1, and APE1 can maintain accurate repair.

Capsid Structure of Aleutian Mink Disease Virus and Human Parvovirus 4: New Faces in the Parvovirus Family Portrait

Abstract: Parvoviruses are small, single-stranded DNA viruses with non-enveloped capsids. Determining the capsid structures provides a framework for annotating regions important to the viral life cycle. Aleutian mink disease virus (AMDV), a pathogen in minks, and human parvovirus 4 (PARV4), infecting humans, are parvoviruses belonging to the genera Amdoparvovirus and Tetraparvovirus, respectively. While Aleutian mink disease caused by AMDV is a major threat to mink farming, no clear clinical manifestations have been established following infection with PARV4 in humans. Here, the capsid structures of AMDV and PARV4 were determined via cryo-electron microscopy at 2.37 and 3.12 Å resolutions, respectively. Despite low amino acid sequence identities (10–30%) both viruses share the icosahedral nature of parvovirus capsids, with 60 viral proteins (VPs) assembling the capsid via two-, three-, and five-fold symmetry VP-related interactions, but display major structural variabilities in the surface loops when the capsid structures are superposed onto other parvoviruses. The capsid structures of AMDV and PARV4 will add to current knowledge of the structural platform for parvoviruses and permit future functional annotation of these viruses, which will help in understanding their infection mechanisms at a molecular level for the development of diagnostics and therapeutics.