Multi-omic Mitoprotease Profiling Defines a Role for Oct1p in Coenzyme Q Production.

Mitoproteases are becoming recognized as key regulators of diverse mitochondrial functions, although their direct substrates are often difficult to discern. Through multi-omic profiling of diverse Saccharomyces cerevisiae mitoprotease deletion strains, we predicted numerous associations between mitoproteases and distinct mitochondrial processes. These include a strong association between the mitochondrial matrix octapeptidase Oct1p and coenzyme Q (CoQ) biosynthesis-a pathway essential for mitochondrial respiration. Through Edman sequencing and in vitro and in vivo biochemistry, we demonstrated that Oct1p directly processes the N terminus of the CoQ-related methyltransferase, Coq5p, which markedly improves its stability. A single mutation to the Oct1p recognition motif in Coq5p disrupted its processing in vivo, leading to CoQ deficiency and respiratory incompetence. This work defines the Oct1p processing of Coq5p as an essential post-translational event for proper CoQ production. Additionally, our data visualization tool enables efficient exploration of mitoprotease profiles that can serve as the basis for future mechanistic investigations.

Cerebellar Ataxia and Coenzyme Q Deficiency through Loss of Unorthodox Kinase Activity.

The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.

Mitochondrial ADCK3 employs an atypical protein kinase-like fold to enable coenzyme Q biosynthesis.

The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. Here, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flips this coenzyme selectivity and enables autophosphorylation but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways.

Machine Learning on DNA-Encoded Library Count Data Using an Uncertainty-Aware Probabilistic Loss Function.

DNA-encoded library (DEL) screening and quantitative structure-activity relationship (QSAR) modeling are two techniques used in drug discovery to find novel small molecules that bind a protein target. Applying QSAR modeling to DEL selection data can facilitate the selection of compounds for off-DNA synthesis and evaluation. Such a combined approach has been done recently by training binary classifiers to learn DEL enrichments of aggregated "disynthons" in order to accommodate the sparse and noisy nature of DEL data. However, a binary classification model cannot distinguish between different levels of enrichment, and information is potentially lost during disynthon aggregation. Here, we demonstrate a regression approach to learning DEL enrichments of individual molecules, using a custom negative-log-likelihood loss function that effectively denoises DEL data and introduces opportunities for visualization of learned structure-activity relationships. Our approach explicitly models the Poisson statistics of the sequencing process used in the DEL experimental workflow under a frequentist view. We illustrate this approach on a DEL dataset of 108,528 compounds screened against carbonic anhydrase (CAIX), and a dataset of 5,655,000 compounds screened against soluble epoxide hydrolase (sEH) and SIRT2. Due to the treatment of uncertainty in the data through the negative-log-likelihood loss used during training, the models can ignore low-confidence outliers. While our approach does not demonstrate a benefit for extrapolation to novel structures, we expect our denoising and visualization pipeline to be useful in identifying structure-activity trends and highly enriched pharmacophores in DEL data. Further, this approach to uncertainty-aware regression modeling is applicable to other sparse or noisy datasets where the nature of stochasticity is known or can be modeled; in particular, the Poisson enrichment ratio metric we use can apply to other settings that compare sequencing count data between two experimental conditions.

Multimodal small-molecule screening for human prion protein binders.

Prion disease is a rapidly progressive neurodegenerative disorder caused by misfolding and aggregation of the prion protein (PrP), and there are currently no therapeutic options. PrP ligands could theoretically antagonize prion formation by protecting the native protein from misfolding or by targeting it for degradation, but no validated small-molecule binders have been discovered to date. We deployed a variety of screening methods in an effort to discover binders of PrP, including 19F-observed and saturation transfer difference (STD) NMR spectroscopy, differential scanning fluorimetry (DSF), DNA-encoded library selection, and in silico screening. A single benzimidazole compound was confirmed in concentration-response, but affinity was very weak (Kd > 1 mm), and it could not be advanced further. The exceptionally low hit rate observed here suggests that PrP is a difficult target for small-molecule binders. Whereas orthogonal binder discovery methods could yield high-affinity compounds, non-small-molecule modalities may offer independent paths forward against prion disease.

Domain-specific Quantification of Prion Protein in Cerebrospinal Fluid by Targeted Mass Spectrometry.

Therapies currently in preclinical development for prion disease seek to lower prion protein (PrP) expression in the brain. Trials of such therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint. Studies using PrP ELISA kits have shown that CSF PrP is lowered in the symptomatic phase of disease, a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. Because misfolding or proteolytic cleavage could potentially render PrP invisible to ELISA even if its concentration were constant or increasing in disease, we sought to establish an orthogonal method for CSF PrP quantification. We developed a multi-species targeted mass spectrometry method based on multiple reaction monitoring (MRM) of nine PrP tryptic peptides quantified relative to an isotopically labeled recombinant protein standard for human samples, or isotopically labeled synthetic peptides for nonhuman species. Analytical validation experiments showed process replicate coefficients of variation below 15%, good dilution linearity and recovery, and suitable performance for both CSF and brain homogenate and across humans as well as preclinical species of interest. In n = 55 CSF samples from individuals referred to prion surveillance centers with rapidly progressive dementia, all six human PrP peptides, spanning the N- and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared with individuals with nonprion diagnoses. Thus, lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not an artifact of ELISA-based measurement. As a result, dose-finding studies for PrP lowering drugs may need to be conducted in presymptomatic at-risk individuals rather than in symptomatic patients. We provide a targeted mass spectrometry-based method suitable for preclinical quantification of CSF PrP as a tool for drug development.

Evaluation of a cyclopentane-based γ-amino acid for the ability to promote α/γ-peptide secondary structure.

We report the asymmetric synthesis of the γ-amino acid (1R,2R)-2-aminomethyl-1-cyclopentane carboxylic acid (AMCP) and an evaluation of this residue's potential to promote secondary structure in α/γ-peptides. Simulated annealing calculations using NMR-derived distance restraints obtained for α/γ-peptides in chloroform reveal that AMCP-containing oligomers are conformationally flexible. However, additional evidence suggests that an internally hydrogen-bonded helical conformation is partially populated in solution. From these data, we propose characteristic NOE patterns for the formation of the α/γ-peptide 12/10-helix and discuss the apparent conformational frustration of AMCP-containing oligomers.

Diversity-oriented synthesis encoded by deoxyoligonucleotides.

Diversity-oriented synthesis (DOS) is a powerful strategy to prepare molecules with underrepresented features in commercial screening collections, resulting in the elucidation of novel biological mechanisms. In parallel to the development of DOS, DNA-encoded libraries (DELs) have emerged as an effective, efficient screening strategy to identify protein binders. Despite recent advancements in this field, most DEL syntheses are limited by the presence of sensitive DNA-based constructs. Here, we describe the design, synthesis, and validation experiments performed for a 3.7 million-member DEL, generated using diverse skeleton architectures with varying exit vectors and derived from DOS, to achieve structural diversity beyond what is possible by varying appendages alone. We also show screening results for three diverse protein targets. We will make this DEL available to the academic scientific community to increase access to novel structural features and accelerate early-phase drug discovery.

Regional variability and genotypic and pharmacodynamic effects on PrP concentration in the CNS.

Prion protein (PrP) concentration controls the kinetics of prion replication and is a genetically and pharmacologically validated therapeutic target for prion disease. In order to evaluate PrP concentration as a pharmacodynamic biomarker and assess its contribution to known prion disease risk factors, we developed and validated a plate-based immunoassay reactive for PrP across 6 species of interest and applicable to brain and cerebrospinal fluid (CSF). PrP concentration varied dramatically across different brain regions in mice, cynomolgus macaques, and humans. PrP expression did not appear to contribute to the known risk factors of age, sex, or common PRNP genetic variants. CSF PrP was lowered in the presence of rare pathogenic PRNP variants, with heterozygous carriers of P102L displaying 55%, and D178N just 31%, of the CSF PrP concentration of mutation-negative controls. In rodents, pharmacologic reduction of brain Prnp RNA was reflected in brain parenchyma PrP and, in turn in CSF PrP, validating CSF as a sampling compartment for the effect of PrP-lowering therapy. Our findings support the use of CSF PrP as a pharmacodynamic biomarker for PrP-lowering drugs and suggest that relative reduction from individual baseline CSF PrP concentration may be an appropriate marker for target engagement.

Evolution of amide stacking in larger γ-peptides: triamide H-bonded cycles.

The single-conformation spectroscopy of two model γ-peptides has been studied under jet-cooled conditions in the gas phase. The methyl-capped triamides, Ac-γ(2)-hPhe-γ(2)-hAla-NHMe and Ac-γ(2)-hAla-γ(2)-hPhe-NHMe, were probed by resonant two-photon ionization (R2PI) and resonant ion-dip infrared (RIDIR) spectroscopies. Four conformers of Ac-γ(2)-hPhe-γ(2)-hAla-NHMe and three of Ac-γ(2)-hAla-γ(2)-hPhe-NHMe were observed and spectroscopically interrogated. On the basis of comparison with the predictions of density functional theory calculations employing a dispersion-corrected functional (ωB97X-D/6-311++G(d,p)), all seven conformers have been assigned to particular conformational families. The preference for formation of nine-membered rings (C9) observed in a previous study [James, W. H., III et al., J. Am. Chem. Soc. 2009, 131, 14243] of the smaller analog, Ac-γ(2)-hPhe-NHMe, carries over to these triamides, with four of the seven conformers forming C9/C9 sequential double-ring structures, and one conformer a C9/C14 bifurcated double ring. The remaining two conformers form C7/C7/C14 H-bonded cycles involving all three amide NH groups, unprecedented in other peptides and peptidomimetics. The amide groups in these structures form a H-bonded triangle with the two trimethylene bridges forming loops above and below the molecule's midsection. The structure is a natural extension of amide stacking, with the two terminal amides blocked from forming the amide tristack by formation of the C14 H-bond. Pair interaction energy decomposition analysis based on the fragment molecular orbital method (FMO-PIEDA) is used to determine the nonbonded contributions to the stabilization of these conformers. Natural bond orbital (NBO) analysis identifies amide stacking with a pair of n → π* interactions between the nitrogen lone pairs and π* orbitals on the carbonyl of the opposing amide groups.

Novel quaternary structures of the human prion protein globular domain.

Prion disease is caused by the misfolding of the cellular prion protein, PrPC, into a self-templating conformer, PrPSc. Nuclear magnetic resonance (NMR) and X-ray crystallography revealed the 3D structure of the globular domain of PrPC and the possibility of its dimerization via an interchain disulfide bridge that forms due to domain swap or by non-covalent association of two monomers. On the contrary, PrPSc is composed by a complex and heterogeneous ensemble of poorly defined conformations and quaternary arrangements that are related to different patterns of neurotoxicity. Targeting PrPC with molecules that stabilize the native conformation of its globular domain emerged as a promising approach to develop anti-prion therapies. One of the advantages of this approach is employing structure-based drug discovery methods to PrPC. Thus, it is essential to expand our structural knowledge about PrPC as much as possible to aid such drug discovery efforts. In this work, we report a crystallographic structure of the globular domain of human PrPC that shows a novel dimeric form and a novel oligomeric arrangement. We use molecular dynamics simulations to explore its structural dynamics and stability and discuss potential implications of these new quaternary structures to the conversion process.

Helix formation in preorganized beta/gamma-peptide foldamers: hydrogen-bond analogy to the alpha-helix without alpha-amino acid residues.

We report the first high-resolution structural data for the beta/gamma-peptide 13-helix (i,i+3 C=O...H-N H-bonds), a secondary structure that is formed by oligomers with a 1:1 alternation of beta- and gamma-amino acid residues. Our characterization includes both crystallographic and 2D NMR data. Previous studies suggested that beta/gamma-peptides constructed from conformationally flexible residues adopt a different helical secondary structure in solution. Our design features preorganized beta- and gamma-residues, which strongly promote 13-helical folding by the 1:1 beta/gamma backbone.

Characterization of the Prion Protein Binding Properties of Antisense Oligonucleotides.

Antisense oligonucleotides (ASOs) designed to lower prion protein (PrP) expression in the brain through RNase H1-mediated degradation of PrP RNA are in development as prion disease therapeutics. ASOs were previously reported to sequence-independently interact with PrP and inhibit prion accumulation in cell culture, yet in vivo studies using a new generation of ASOs found that only PrP-lowering sequences were effective at extending survival. Cerebrospinal fluid (CSF) PrP has been proposed as a pharmacodynamic biomarker for trials of such ASOs, but is only interpretable if PrP lowering is indeed the relevant mechanism of action in vivo and if measurement of PrP is unconfounded by any PrP-ASO interaction. Here, we examine the PrP-binding and antiprion properties of ASOs in vitro and in cell culture. Binding parameters determined by isothermal titration calorimetry were similar across all ASOs tested, indicating that ASOs of various chemistries bind full-length recombinant PrP with low- to mid-nanomolar affinity in a sequence-independent manner. Nuclear magnetic resonance, dynamic light scattering, and visual inspection of ASO-PrP mixtures suggested, however, that this interaction is characterized by the formation of large aggregates, a conclusion further supported by the salt dependence of the affinity measured by isothermal titration calorimetry. Sequence-independent inhibition of prion accumulation in cell culture was observed. The inefficacy of non-PrP-lowering ASOs against prion disease in vivo may be because their apparent activity in vitro is an artifact of aggregation, or because the concentration of ASOs in relevant compartments within the central nervous system (CNS) quickly drops below the effective concentration for sequence-independent antiprion activity after bolus dosing into CSF. Measurements of PrP concentration in human CSF were not impacted by the addition of ASO. These findings support the further development of PrP-lowering ASOs and of CSF PrP as a pharmacodynamic biomarker.

Antisense oligonucleotides extend survival of prion-infected mice.

Prion disease is a fatal, incurable neurodegenerative disease of humans and other mammals caused by conversion of cellular prion protein (PrP; PrPC) into a self-propagating neurotoxic conformer (prions; PrPSc). Strong genetic proofs of concept support lowering PrP expression as a therapeutic strategy. Antisense oligonucleotides (ASOs) can provide a practical route to lowering one target mRNA in the brain, but their development for prion disease has been hindered by three unresolved questions from prior work: uncertainty about mechanism of action, unclear potential for efficacy against established prion infection, and poor tolerability of drug delivery by osmotic pumps. Here we test antisense oligonucleotides (ASOs) delivered by bolus intracerebroventricular injection to intracerebrally prion-infected wild-type mice. Prophylactic treatments given every 2-3 months extended survival times 61-98%, and a single injection at 120 days post-infection, near the onset of clinical signs, extended survival 55% (87 days). In contrast, a non-targeting control ASO was ineffective. Thus, PrP lowering is the mechanism of action of ASOs effective against prion disease in vivo, and infrequent, or even single, bolus injections of ASOs can slow prion neuropathogenesis and markedly extend survival, even when initiated near clinical signs. These findings should empower development of PrP-lowering therapy for prion disease.

Conserved Lipid and Small-Molecule Modulation of COQ8 Reveals Regulation of the Ancient Kinase-like UbiB Family.

Human COQ8A (ADCK3) and Saccharomyces cerevisiae Coq8p (collectively COQ8) are UbiB family proteins essential for mitochondrial coenzyme Q (CoQ) biosynthesis. However, the biochemical activity of COQ8 and its direct role in CoQ production remain unclear, in part due to lack of known endogenous regulators of COQ8 function and of effective small molecules for probing its activity in vivo. Here, we demonstrate that COQ8 possesses evolutionarily conserved ATPase activity that is activated by binding to membranes containing cardiolipin and by phenolic compounds that resemble CoQ pathway intermediates. We further create an analog-sensitive version of Coq8p and reveal that acute chemical inhibition of its endogenous activity in yeast is sufficient to cause respiratory deficiency concomitant with CoQ depletion. Collectively, this work defines lipid and small-molecule modulators of an ancient family of atypical kinase-like proteins and establishes a chemical genetic system for further exploring the mechanistic role of COQ8 in CoQ biosynthesis.