Search NASA⌕ Search

SEARCH · Search NASA

Results for “binding modes”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 145 records · Page 8
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Mobile loop dynamics in adenosyltransferase control binding and reactivity of coenzyme B 12

Cobalamin is a complex organometallic cofactor that is processed and targeted via a network of chaperones to its dependent enzymes. AdoCbl (5'-deoxyadenosylcobalamin) is synthesized from cob(II)alamin in a reductive adenosylation reaction catalyzed by adenosyltransferase (ATR), which also serves as an escort, delivering AdoCbl to methylmalonyl-CoA mutase (MCM). The mechanism by which ATR signals that its cofactor cargo is ready (AdoCbl) or not [cob(II)alamin] for transfer to MCM, is not known. In this study, we have obtained crystallographic snapshots that reveal ligand-induced ordering of the N terminus of Mycobacterium tuberculosis ATR, which organizes a dynamic cobalamin binding site and exerts exquisite control over coordination geometry, reactivity, and solvent accessibility. Cob(II)alamin binds with its dimethylbenzimidazole tail splayed into a side pocket and its corrin ring buried. The cosubstrate, ATP, enforces a four-coordinate cob(II)alamin geometry, facilitating the unfavorable reduction to cob(I)alamin. The binding mode for AdoCbl is notably different from that of cob(II)alamin, with the dimethylbenzimidazole tail tucked under the corrin ring, displacing the N terminus of ATR, which is disordered. In this solvent-exposed conformation, AdoCbl undergoes facile transfer to MCM. The importance of the tail in cofactor handover from ATR to MCM is revealed by the failure of 5'-deoxyadenosylcobinamide, lacking the tail, to transfer. In the absence of MCM, ATR induces a sacrificial cobalt–carbon bond homolysis reaction in an unusual reversal of the heterolytic chemistry that was deployed to make the same bond. Finally, the data support an important role for the dimethylbenzimidazole tail in moving the cobalamin cofactor between active sites.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Tertiary folds of the SL5 RNA from the 5 ' proximal region of SARS-CoV-2 and related coronaviruses

Coronavirus genomes sequester their start codons within stem-loop 5 (SL5), a structured, 5' genomic RNA element. In most alpha- and betacoronaviruses, the secondary structure of SL5 is predicted to contain a four-way junction of helical stems, some of which are capped with UUYYGU hexaloops. Here, using cryogenic electron microscopy (cryo-EM) and computational modeling with biochemically determined secondary structures, we present three-dimensional structures of SL5 from six coronaviruses. The SL5 domain of betacoronavirus severe-acute-respiratory-syndrome-related coronavirus 2 (SARS-CoV-2), resolved at 4.7 Å resolution, exhibits a T-shaped structure, with its UUYYGU hexaloops at opposing ends of a coaxial stack, the T’s “arms.” Further analysis of SL5 domains from SARS-CoV-1 and MERS (7.1 and 6.4 to 6.9 Å resolution, respectively) indicate that the junction geometry and inter-hexaloop distances are conserved features across these human-infecting betacoronaviruses. The MERS SL5 domain displays an additional tertiary interaction, which is also observed in the non-human-infecting betacoronavirus BtCoV-HKU5 (5.9 to 8.0 Å resolution). SL5s from human-infecting alphacoronaviruses, HCoV-229E and HCoV-NL63 (6.5 and 8.4 to 9.0 Å resolution, respectively), exhibit the same coaxial stacks, including the UUYYGU-capped arms, but with a phylogenetically distinct crossing angle, an X-shape. As such, all SL5 domains studied herein fold into stable tertiary structures with cross-genus similarities and notable differences, with implications for potential protein-binding modes and therapeutic targets.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The crystal structures of a chloride-pumping microbial rhodopsin and its proton-pumping mutant illuminate proton transfer determinants

Microbial rhodopsins are versatile and ubiquitous retinal-binding proteins that function as light-driven ion pumps, light-gated ion channels, and photosensors, with potential utility as optogenetic tools for altering membrane potential in target cells. Insights from crystal structures have been central for understanding proton, sodium, and chloride transport mechanisms of microbial rhodopsins. Two of three known groups of anion pumps, the archaeal halorhodopsins (HRs) and bacterial chloride-pumping rhodopsins, have been structurally characterized. Here we report the structure of a representative of a recently discovered third group consisting of cyanobacterial chloride and sulfate ion-pumping rhodopsins, the Mastigocladopsis repens rhodopsin (MastR). Chloride-pumping MastR contains in its ion transport pathway a unique Thr-Ser-Asp (TSD) motif, which is involved in the binding of a chloride ion. The structure reveals that the chloride-binding mode is more similar to HRs than chloride-pumping rhodopsins, but the overall structure most closely resembles bacteriorhodopsin (BR), an archaeal proton pump. The MastR structure shows a trimer arrangement reminiscent of BR-like proton pumps and shows features at the extracellular side more similar to BR than the other chloride pumps. We further solved the structure of the MastR-T74D mutant, which contains a single amino acid replacement in the TSD motif. We provide insights into why this point mutation can convert the MastR chloride pump into a proton pump but cannot in HRs. Our study points at the importance of precise coordination and exact location of the water molecule in the active center of proton pumps, which serves as a bridge for the key proton transfer.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

A novel class of broad-spectrum active-site-directed 3C-like protease inhibitors with nanomolar antiviral activity against highly immune-evasive SARS-CoV-2 Omicron subvariants

Antivirals with broad coronavirus activity are important for treating high-risk individuals exposed to the constantly evolving SARS-CoV-2 variants of concern (VOCs) as well as emerging drug-resistant variants. We developed and characterized a novel class of active-site-directed 3-chymotrypsin-like protease (3CLpro) inhibitors (C2-C5a). Our lead direct-acting antiviral (DAA), C5a, is a non-covalent, non-peptide with a dissociation constant of 170 nM against recombinant SARS-CoV-2 3CLpro. The compounds C2-C5a exhibit broad-spectrum activity against Omicron subvariants (BA.5, BQ.1.1, and XBB.1.5) and seasonal human coronavirus-229E infection in human cells. Notably, C5a has median effective concentrations of 30-50 nM against BQ.1.1 and XBB.1.5 in two different human cell lines. X-ray crystallography has confirmed the unique binding modes of C2-C5a to the 3CLpro, which can limit virus cross-resistance to emerging Paxlovid-resistant variants. We tested the effect of C5a with two of our newly discovered host-directed antivirals (HDAs): N-0385, a TMPRSS2 inhibitor, and bafilomycin D (BafD), a human vacuolar H+-ATPase [V-ATPase] inhibitor. We demonstrated a synergistic action of C5a in combination with N-0385 and BafD against Omicron BA.5 infection in human Calu-3 lung cells. Our findings underscore that a SARS-CoV-2 multi-targeted treatment for circulating Omicron subvariants based on DAAs (C5a) and HDAs (N-0385 or BafD) can lead to therapeutic benefits by enhancing treatment efficacy. Furthermore, the high-resolution structures of SARS-CoV-2 3CLpro in complex with C2-C5a will facilitate future rational optimization of our novel broad-spectrum active-site-directed 3C-like protease inhibitors.

3CLpro inhibitor↗
DOE OSTI2023

Structure of the WYL-domain containing transcription activator, DriD, in complex with ssDNA effector and DNA target site

Transcription regulators play central roles in orchestrating responses to changing environmental conditions. Recently the Caulobacter crescentus transcription activator DriD, which belongs to the newly defined WYL-domain family, was shown to regulate DNA damage responses independent of the canonical SOS pathway. However, the molecular mechanisms by which DriD and other WYL-regulators sense environmental signals and recognize DNA are not well understood. We showed DriD DNA-binding is triggered by its interaction with ssDNA, which is produced during DNA damage. Here we describe the structure of the full-length C. crescentus DriD bound to both target DNA and effector ssDNA. DriD consists of an N-terminal winged-HTH (wHTH) domain, linker region, three-helix bundle, WYL-domain and C-terminal WCX-dimer domain. Strikingly, DriD binds DNA using a novel, asymmetric DNA-binding mechanism that results from different conformations adopted by the linker. Although the linker does not touch DNA, our data show that contacts it makes with the wHTH are key for specific DNA binding. The structure indicates how ssDNA-effector binding to the WYL-domain impacts wHTH DNA binding. In conclusion, we present the first structure of a WYL-activator bound to both effector and target DNA. The structure unveils a unique, asymmetric DNA binding mode that is likely conserved among WYL-activators.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Sparsomycin inhibits translation through a conserved mechanism across all domains of life

Abstract Sparsomycin (SPA) is a broad-spectrum inhibitor of protein synthesis with activity across all three domains of life. Although SPA has long been known to target the ribosomal peptidyl transferase center (PTC), previous structural studies suggested that SPA binds differently to bacterial ribosomes compared to their archaeal and eukaryotic counterparts—an unexpected conclusion given the high evolutionary conservation of the ribosomal catalytic center. Here, we show that SPA inhibits a majority of elongation-competent bacterial ribosomal complexes and present X-ray crystal structures of Thermus thermophilus 70S ribosomes stalled by SPA at the initiation and early elongation stages of translation. These structures reveal that SPA binds to the bacterial ribosome in a manner essentially identical to that observed in archaeal and eukaryotic ribosomes, establishing a unified structural mechanism of SPA action across all domains of life. In this conserved binding mode, SPA occupies the A-site cleft of the PTC and forms an extensive network of interactions with universally conserved ribosomal RNA nucleotides and the CCA-end of the P-site transfer RNA (tRNA), thereby stabilizing the peptidyl-tRNA substrate while sterically blocking accommodation of an incoming aminoacyl-tRNA. By clarifying the mode of action of SPA on the bacterial ribosome, our work provides a structural framework for the rational design of SPA derivatives with improved potency and bacterial specificity.

Paranjpe, Madhura N [Department of Biological Scie↗
DOE OSTI2022

Crystals of $\mathrm{TELSAM}$–target protein fusions that exhibit minimal crystal contacts and lack direct inter-$\mathrm{TELSAM}$ contacts

While conducting pilot studies into the usefulness of fusion to TELSAM polymers as a potential protein crystallization strategy, we observed novel properties in crystals of two TELSAM–target protein fusions, as follows. (i) A TELSAM–target protein fusion can crystallize more rapidly and with greater propensity than the same target protein alone. (ii) TELSAM–target protein fusions can be crystallized at low protein concentrations. This unprecedented observation suggests a route to crystallize proteins that can only be produced in microgram amounts. (iii) The TELSAM polymers themselves need not directly contact one another in the crystal lattice in order to form well-diffracting crystals. This novel observation is important because it suggests that TELSAM may be able to crystallize target proteins too large to allow direct inter-polymer contacts. (iv) Flexible TELSAM–target protein linkers can allow target proteins to find productive binding modes against the TELSAM polymer. (v) TELSAM polymers can adjust their helical rise to allow fused target proteins to make productive crystal contacts. (vi). Fusion to TELSAM polymers can stabilize weak inter-target protein crystal contacts. We report features of these TELSAM–target protein crystal structures and outline future work needed to validate TELSAM as a crystallization chaperone and determine best practices for its use.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Increasing the bulk of the 1TEL–target linker and retaining the 10×His tag in a 1TEL–CMG2-vWa construct improves crystal order and diffraction limits

TELSAM-fusion crystallization has the potential to become a revolutionary tool for the facile crystallization of proteins. TELSAM fusion can increase the crystallization rate and enable crystallization at low protein concentrations, in some cases with minimal crystal contacts [Nawarathnage et al. (2022), Open Biol. 12 , 210271]. Here, requirements for the linker composition between 1TEL and a fused CMG2 vWa domain were investigated. Ala-Ala, Ala-Val, Thr-Val and Thr-Thr linkers were evaluated, comparing metrics for crystallization propensity and crystal order. The effect on crystallization of removing or retaining the purification tag was then tested. It was discovered that increasing the linker bulk and retaining the 10×His purification tag improved the diffraction resolution, likely by decreasing the number of possible vWa-domain orientations in the crystal. Additionally, it was discovered that some vWa-domain binding modes are correlated with scrambling of the 1TEL polymer orientation in crystals and an effective mitigation strategy for this pathology is presented.

1TEL↗
DOE OSTI2019

Structure-based mechanism of cysteinyl leukotriene receptor inhibition by antiasthmatic drugs

The G protein–coupled cysteinyl leukotriene receptor CysLT 1 R mediates inflammatory processes and plays a major role in numerous disorders, including asthma, allergic rhinitis, cardiovascular disease, and cancer. Selective CysLT 1 R antagonists are widely prescribed as antiasthmatic drugs; however, these drugs demonstrate low effectiveness in some patients and exhibit a variety of side effects. To gain deeper understanding into the functional mechanisms of CysLTRs, we determined the crystal structures of CysLT 1 R bound to two chemically distinct antagonists, zafirlukast and pranlukast. The structures reveal unique ligand-binding modes and signaling mechanisms, including lateral ligand access to the orthosteric pocket between transmembrane helices TM4 and TM5, an atypical pattern of microswitches, and a distinct four-residue–coordinated sodium site. These results provide important insights and structural templates for rational discovery of safer and more effective drugs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

Heme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERBα (NR1D1) and REV-ERBβ (NR1D2), but how heme regulates REV-ERB activity remains unclear. Cellular studies indicate that heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription. However, fluorescence-based biochemical assays suggest that heme displaces NCoR; here, we show that this is due to a heme-dependent artifact. Using ITC and NMR spectroscopy, we show that heme binding remodels the thermodynamic interaction profile of NCoR receptor interaction domain (RID) binding to REV-ERBβ ligand-binding domain (LBD). We solved two crystal structures of REV-ERBβ LBD cobound to heme and NCoR peptides, revealing the heme-dependent NCoR binding mode. ITC and chemical cross-linking mass spectrometry reveals a 2:1 LBD:RID stoichiometry, consistent with cellular studies showing that NCoR-dependent repression of REV-ERB transcription occurs on dimeric DNA response elements. Our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structure of cytoplasmic ring of nuclear pore complex by integrative cryo-EM and AlphaFold

The nuclear pore complex (NPC) is the conduit for bidirectional cargo traffic between the cytoplasm and the nucleus. We determined a near-complete structure of the cytoplasmic ring of the NPC from Xenopus oocytes using single-particle cryo–electron microscopy and AlphaFold prediction. Structures of nucleoporins were predicted with AlphaFold and fit into the medium-resolution map by using the prominent secondary structural density as a guide. Certain molecular interactions were further built or confirmed by complex prediction by using AlphaFold. Here, we identified the binding modes of five copies of Nup358, the largest NPC subunit with Phe-Gly repeats for cargo transport, and predicted it to contain a coiled-coil domain that may provide avidity to assist its role as a nucleation center for NPC formation under certain conditions

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Evolution of carbapenemase activity in the class C β-lactamase ADC-1

Antibiotic resistance in bacteria poses a significant threat to public health. Among dozens of available antimicrobial agents, carbapenems are used as drugs of choice for the treatment of serious infections caused by pathogens resistant to other antibiotics. However, their usefulness has been severely compromised due to the emergence and wide spread of carbapenem-resistant clinical isolates worldwide. High-level resistance to carbapenems in bacteria is mediated by the production of β-lactamases from three molecular classes, A, B, and D, but not by class C enzymes. In this study, we selected a triple mutant of the intrinsic class C Acinetobacter-derived cephalosporinase ADC-1 (ADC-1 TM ) that confers high-level resistance to the carbapenems meropenem, ertapenem, and doripenem. Kinetic experiments demonstrated that the apparent binding affinity, along with the acylation and deacylation rates, were all improved for the mutant enzyme. X-ray crystallography, molecular docking, and molecular dynamics simulations revealed that the amino acid substitutions in ADC-1 TM produce significant changes in the enzyme active site architecture and binding mode of the carbapenem ertapenem. These changes allow for better positioning of a deacylating water for nucleophilic attack, thus explaining the significantly improved rate of ertapenem deacylation by ADC-1 TM . In this study, we showed for the first time that a class C β-lactamase can produce high-level resistance to carbapenem antibiotics, which underlines the potential for enzymes of this class to evolve such resistance and could further exacerbate the problem of antibiotic resistance in bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Surface Functionalized Barium Titanate Nanoparticles: A Combined Experimental and Computational Study

Barium titanate (BTO) nanoparticles show great potential for use in electrostatic capacitors with high energy density. This includes both polymer composite and sintered capacitors. However, questions about the nanoparticles' size distribution, amount of agglomeration, and surface ligand effect on performance properties remain. Reducing particle agglomeration is a crucial step to understanding the properties of nanoscale particles, as agglomeration has significant effects on the composite dielectric constant. BTO surface functionalization using phosphonic acids is known reduce BTO nanoparticle agglomeration. We explore solution synthesized 10 nm BTO particles with tert-butylphosphonic acid ligands. Recent methods to quantifying agglomeration using an epoxy matrix before imaging shows that tert-butylphosphonic acid ligands reduce BTO agglomeration by 33%. Thermometric, spectroscopic, and computational methods provide confirmation of ligand binding and provide evidence of multiple ligand binding modes on the BTO particle surface.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Structure of BTK kinase domain with the second-generation inhibitors acalabrutinib and tirabrutinib

Bruton’s tyrosine kinase (BTK) is the target of the therapeutic agent, Ibrutinib, that treats chronic lymphocyte leukemia (CLL), mantle cell lymphoma (MCL) and other B cell malignancies. Ibrutinib is a first in class, covalent BTK inhibitor that limits B-cell survival and proliferation. Designing new inhibitors of BTK has been an important objective for advancing development of improved therapeutic agents against cancer and autoimmune disorders. Based on the success of Ibrutinib, several second-generation irreversible BTK inhibitors have been developed that exhibit fewer off-target effects. However, the binding-mode and their interaction with Btk have not been experimentally determined and evaluated at atomic resolution. Here we determined the first crystal structure of the BTK kinase domain in complex with acalabrutinib. In addition, we report a structure of the BTK/tirabrutinib complex and compare these structures with previously solved structures. The structures provide insight in the superior selectivity reported for acalabrutinb and guide future BTK inhibitor development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

DataSet for Elucidating molecular level interfacial interactions between a de novo protein and nucleated calcite with solid-state NMR

Biomineralization is the process by which organisms use biomolecules to produce hierarchically structured organic-inorganic composites. Using biology as inspiration, a protein construct (FD31) was previously designed to accelerate formation of nano-calcite with an unconventional {110} face. To understand the molecular interactions essential for protein aided calcite nucleation, solid-state nuclear magnetic resonance (ssNMR) spectroscopy was used in this work to characterize the FD31-calcite interface at the atomic level. Glutamic acid side chains designed to interact directly with calcium ions on the surface were found to have dynamics on the sub-millisecond timescale, indicating possible interactions between the protein and surface waters that were not included in the original model. Dipolar ssNMR recoupling techniques also showed that the protein backbone is ~2 Å closer to the surface than in the original docking model. Refined molecular simulations were done in the presence of explicit waters, which resulted in the protein backbone closer to the surface than in the original docking structure, providing better agreement with experiment and highlighting the important role played by water in FD31-calcite interactions. These studies provide the first experimental evidence to confirm that FD31 interactions with calcite are localized to the surface of the protein designed to serve as a template. However, these studies do indicate a more dynamic binding and closer binding mode between FD31 and the nucleated surface than originally proposed. In all, this enhanced molecular insight into the FD31-calcite interface has advanced our fundamental understanding of the atomic interactions at the organic-inorganic interface and will aid in the design of biological templates for the nucleation of inorganic crystals.

Saccuzzo Close, Emily Grace [Pacific Northwest Nat↗
DOE OSTI2020

In Silico Insights into Protein–Protein Interaction Disruptive Mutations in the PCSK9-LDLR Complex

Gain-of-function mutations in PCSK9 (proprotein convertase subtilisin/kexin type 9) lead to reduced uptake of LDL (low density lipoprotein) cholesterol and, therefore, increased plasma LDL levels. However, the mechanism by which these mutants reduce LDL reuptake is not fully understood. Here, we have used molecular dynamics simulations, MM/PBSA (Molecular Mechanics/Poisson–Boltzmann Surface Area) binding affinity calculations, and residue interaction networks, to investigate the protein–protein interaction (PPI) disruptive effects of two of PCSK9's gain-of-function mutations, Ser127Arg and Asp374Tyr on the PCSK9 and LDL receptor complex. In addition to these PPI disruptive mutants, a third, non-interface mutation (Arg496Trp) is included as a positive control. Our results indicate that Ser127Arg and Asp374Tyr confer significantly improved binding affinity, as well as different binding modes, when compared to the wild-type. These PPI disruptive mutations lie between the EGF(A) (epidermal growth factor precursor homology domain A) of the LDL receptor and the catalytic domain of PCSK9 (Asp374Tyr) and between the prodomain of PCSK9 and the β-propeller of the LDL receptor (Ser127Arg). The interactions involved in these two interfaces result in an LDL receptor that is sterically inhibited from entering its closed conformation. This could potentially implicate the prodomain as a target for small molecule inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Fyn–Saracatinib Complex Structure Reveals an Active State-like Conformation

Fyn is a Src-family tyrosine kinase implicated in synaptic dysfunction and neuroinflammation across multiple neurodegenerative disorders, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Saracatinib (AZD0530) is a potent Src-family inhibitor that has been explored as a repurposed therapeutic; however, its clinical utility is limited by poor kinase selectivity caused by high sequence conservation within Src-family ATP-binding sites. Here, we combine surface plasmon resonance (SPR) and X-ray crystallography to define saracatinib recognition by the Fyn kinase domain (KD). SPR single-cycle kinetics shows that saracatinib binds the isolated Fyn KD and full-length Fyn with low-nanomolar affinity, whereas dasatinib binds with subnanomolar affinity and markedly slower dissociation. We determined the crystal structure of the Fyn KD-saracatinib complex at 2.22 Å resolution. The kinase adopts an active-like conformation with the DFG motif and αC-helix in the ‘in’ state and a conserved β3 αC Lys-Glu salt bridge. Saracatinib occupies the adenine and ribose pockets, and engages the hinge through direct and water-mediated hydrogen bonding while complementing a hydrophobic back pocket by van der Waals contacts. Comparison with reported saracatinib-bound structures of other kinases suggests that the active-state geometry observed for Fyn creates a pocket not observed in inactive-like complexes, providing a structural handle for designing Fyn-selective inhibitors. Comparison with all saracatinib-bound kinase co-structures currently available in the PDB (ALK2 and PKMYT1) indicates a conserved monodentate hinge binding mode but kinase-dependent αC-helix conformations, providing a structural rationale for designing Fyn-selective analogues.

AZD0530↗
DOE OSTI2023

Development of Novel Small-Molecule Activators of Pyruvate Kinase Muscle Isozyme 2, PKM2, to Reduce Photoreceptor Apoptosis

Treatment options are lacking to prevent photoreceptor death and subsequent vision loss. Previously, we demonstrated that reprogramming metabolism via the pharmacologic activation of PKM2 is a novel photoreceptor neuroprotective strategy. However, the features of the tool compound used in those studies, ML-265, preclude its advancement as an intraocular, clinical candidate. This study sought to develop the next generation of small-molecule PKM2 activators, aimed specifically for delivery into the eye. Compounds were developed that replaced the thienopyrrolopyridazinone core of ML-265 and modified the aniline and methyl sulfoxide functional groups. Compound 2 demonstrated that structural changes to the ML-265 scaffold are tolerated from a potency and efficacy standpoint, allow for a similar binding mode to the target, and circumvent apoptosis in models of outer retinal stress. To overcome the low solubility and problematic functional groups of ML-265, compound 2’s efficacious and versatile core structure for the incorporation of diverse functional groups was then utilized to develop novel PKM2 activators with improved solubility, lack of structural alerts, and retained potency. No other molecules are in the pharmaceutical pipeline for the metabolic reprogramming of photoreceptors. Thus, this study is the first to cultivate the next generation of novel, structurally diverse, small-molecule PKM2 activators for delivery into the eye.

59 BASIC BIOLOGICAL SCIENCES↗