Leigh syndrome is a fatal pediatric neurodegenerative disease caused by mitochondrial dysfunction, which can be modeled in the Ndufs4 KO mouse with mitochondrial respiratory chain complex I (CI) deficiency. This study explores NV354, a prodrug of succinate with enhanced oral bioavailability and brain uptake, as a potential therapy to counteract this devastating condition. NV354 modulated whole-body respiration and metabolic flexibility, prevented late-stage motor dysfunction, delayed clinical ataxia scores, and improved body weight development, but had otherwise minimal effect on neurobehavior and lifespan of the animals. The succinate prodrug prevented development of the brain stem lesions pathognomonic for Leigh syndrome, attenuated neuronal loss in the brainstem, diminished activation of astrocytes, blocked hypertrophic microglial accumulation, and reduced reactive oxygen species (ROS) levels in the brain. NV354 also partially alleviated motor symptoms and metabolic decompensation in a rat model of Parkinson disease induced by the CI inhibitor rotenone. In conclusion, the succinate prodrug NV354 shows promise as a potential treatment of mitochondrial CI-related neurodegeneration.

The design of functional enzymes holds promise for transformative solutions across various domains but presents significant challenges. Inspired by the success of language models in generating nature-like proteins, we explored the potential of an enzyme-specific language model in designing catalytically active artificial enzymes. Here, we introduce ZymCTRL ('enzyme control'), a conditional language model trained on the enzyme sequence space, capable of generating enzymes based on user-defined specifications. Experimental validation at diverse data regimes and for different enzyme families demonstrated ZymCTRL's ability to generate active enzymes across various sequence identity ranges. Specifically, we describe the design of carbonic anhydrases and lactate dehydrogenases in zero-shot, without requiring further training of the model, and showcasing activity at sequence identities below 40% compared to natural proteins. Biophysical analysis confirmed the globularity and well-folded nature of the generated sequences. Furthermore, fine-tuning the model enabled the generation of lactate dehydrogenases more likely to pass in silico filters and with activity comparable to their natural counterparts. Two of the artificial lactate dehydrogenases were scaled up and successfully lyophilised, maintaining activity and demonstrating preliminary conversion in one-pot enzymatic cascades under extreme conditions. Our findings open a new door towards the rapid and cost-effective design of artificial proficient enzymes. The model and training data are freely available to the community. ### Competing Interest Statement G.M., G.A., P.L. are employees of Basecamp Research. N.F. is an advisor with Basecamp Research. S.F. is an employee of Johnson Matthey. I.N., L.M., and S.M. are employees of Isomerase Therapeutics. R.I and U.E declare no competing interest.

Hepatocellular carcinoma (HCC), the most common primary liver cancer, is usually diagnosed in its late state. Tyrosine kinase inhibitors such as sorafenib and regorafenib are one of the few treatment options approved for advanced HCC and only prolong the patient’s life expectancy by a few months. Therefore, there is a need for novel effective treatments. Cyclophilins are intracellular proteins that catalyze the cis/trans isomerization of peptide bonds at proline residues. Cyclophilins are known to be overexpressed in HCC, affecting therapy resistance and cell proliferation. In the present study, we explored the potential of cyclophilin inhibitors as new therapeutic options for HCC in vitro and in vivo. Our results showed that the novel cyclophilin inhibitor, NV651, was able to significantly decrease proliferation in a diverse set of HCC cell lines. The exposure of HCC cells to NV651 caused an accumulation of cells during mitosis and consequent accumulation in the G2/M phase of the cell cycle. NV651 reduced tumor growth in vivo using an HCC xenograft model without affecting the body weights of the animals. The safety aspects of NV651 were also confirmed in primary human hepatocytes without any cytotoxic effects. Based on the results obtained in this study, we propose NV651 as a potential treatment strategy for HCC.

Hepatic fibrosis can result as a pathological response to nonalcoholic steatohepatitis (NASH). Cirrhosis, the late stage of fibrosis, has been linked to poor survival and an increased risk of developing hepatocellular carcinoma, with limited treatment options available. Therefore, there is an unmet need for novel effective antifibrotic compounds. Cyclophilins are peptidyl-prolyl cis-trans isomerases that facilitate protein folding and conformational changes affecting the function of the targeted proteins. Due to their activity, cyclophilins have been presented as key factors in several stages of the fibrotic process. In this study, we investigated the antifibrotic effects of NV556, a novel potent sanglifehrin-based cyclophilin inhibitor, in vitro and in vivo. NV556 potential antifibrotic effect was evaluated in two well-established animal models of NASH, STAM, and methionine-choline-deficient (MCD) mice, as well as in an in vitro 3D human liver ECM culture of LX2 cells, a human hepatic stellate cell line. We demonstrate that NV556 decreased liver fibrosis in both STAM and MCD in vivo models and decreased collagen production in TGFβ1-activated hepatic stellate cells in vitro. Taken together, these results present NV556 as a potential candidate for the treatment of liver fibrosis.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.

Mitochondrial complex I (CI) deficiency is the most prevalent defect in the respiratory chain in paediatric mitochondrial disease. This heterogeneous group of diseases includes serious or fatal neurological presentations such as Leigh syndrome and there are very limited evidence-based treatment options available. Here we describe that cell membrane-permeable prodrugs of the complex II substrate succinate increase ATP-linked mitochondrial respiration in CI-deficient human blood cells, fibroblasts and heart fibres. Lactate accumulation in platelets due to rotenone-induced CI inhibition is reversed and rotenone-induced increase in lactate:pyruvate ratio in white blood cells is alleviated. Metabolomic analyses demonstrate delivery and metabolism of [(13)C]succinate. In Leigh syndrome patient fibroblasts, with a recessive NDUFS2 mutation, respiration and spare respiratory capacity are increased by prodrug administration. We conclude that prodrug-delivered succinate bypasses CI and supports electron transport, membrane potential and ATP production. This strategy offers a potential future therapy for metabolic decompensation due to mitochondrial CI dysfunction.

Inhibition of host-encoded targets, such as the cyclophilins, provides an opportunity to generate potent high barrier to resistance antivirals for the treatment of a broad range of viral diseases. However, many host-targeted agents are natural products, which can be difficult to optimize using synthetic chemistry alone. We describe the orthogonal combination of bioengineering and semisynthetic chemistry to optimize the drug-like properties of sanglifehrin A, a known cyclophilin inhibitor of mixed nonribosomal peptide/polyketide origin, to generate the drug candidate NVP018 (formerly BC556). NVP018 is a potent inhibitor of hepatitis B virus, hepatitis C virus (HCV), and HIV-1 replication, shows minimal inhibition of major drug transporters, and has a high barrier to generation of both HCV and HIV-1 resistance.

Institut für Organische Chemie, Leibniz U 30167 Hannover, Germany. E-mail: frank.h 762-3011; Tel: +49 511-762-4603 Department of Biochemistry, University of Cambridge CB2 1QW, UK Institut für Pharmazeutische Wissenscha Albertstr. 25, 79104 Freiburg im Breisgau, G Isomerase Therapeutics, Chesterford Resea Department of Molecular Microbiology, Joh † Electronic supplementary information (E characterizytion data and spectra of synth for enzyme assays and characterization da See DOI: 10.1039/c4sc00883a Cite this: Chem. Sci., 2014, 5, 3563

Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.