Colin W. Levy

Lenacapavir is a first-in-class viral capsid inhibitor, approved for the treatment of HIV, and which has been shown to give almost complete protection against transmission of the virus. However the high costs currently associated with the manufacture of Lenacapavir place restrictions on its wider application, making the development of cheaper synthetic routes a key priority. Herein we report the development of an engineered aminotransferase for the synthesis of the central chiral amine core of Lenacapavir. Due to the sterically demanding nature of the ketone substrate, a substrate walking approach was adopted during directed evolution to unlock desired aminotransferase activity starting from a parent template with no observable activity for the target reaction. Introduction of ten mutations into an aminotransferase from Ruegeria sp. TM1040 led to the development of a robust engineered aminotransferase that allowed production of the target chiral amine product with high conversions (>98%), isolated yields (>90%) and optical purity (>99% e.e.). This engineered enzyme will facilitate the development of a biocatalytic process to a key chiral intermediate of Lenacapavir in order to reduce its manufacturing costs and thereby enable global access to this important therapy.

Mycobacterium tuberculosis ( Mtb ) is the world’s most deadly infectious pathogen and new drugs are urgently required to combat the emergence of multi-(MDR) and extensively-(XDR) drug resistant strains. The bacterium specifically upregulates sterol uptake pathways in infected macrophages and the metabolism of host-derived cholesterol is essential for Mtb’s long-term survival in vivo. Here, we report the development of antitubercular small molecules that inhibit the Mtb cholesterol oxidases CYP125 and CYP142, which catalyze the initial step of cholesterol metabolism. An efficient biophysical fragment screen was used to characterize the structure-activity relationships of CYP125 and CYP142, and identify a non-azole small molecule 1a that can bind to the heme cofactor of both enzymes. A structure-guided fragment-linking strategy was used to optimize the binding affinity of 1a , yielding a potent dual CYP125/142 inhibitor 5m (K _D CYP125/CYP142 = 0.04/0.16 µM). Compound 5m potently inhibits the catalytic activity of CYP125 and CYP142 in vitro (K _I values < 0.1 µM), and rapidly depletes Mtb intracellular ATP (IC ₅₀ = 0.15 µM). The compound has antimicrobial activity against both drug susceptible and MDR Mtb ( MIC ₉₉ values 0.4 - 1.5 µM ) in extracellular assays, and inhibits the growth of Mtb in human macrophages (MIC = 1.7 µM) with good selectivity over mammalian cytotoxicity (LD ₅₀ ≥ 50 µM). The combination of small molecule inhibitors and structural data reported here provide useful tools to study the role of cholesterol metabolism in Mtb and are a promising step towards novel antibiotics targeting bioenergetic pathways, which could be used to help combat MDR-TB.

We have used a combination of computational and structure-based redesign of the low molecular weight protein tyrosine phosphatase, LMW-PTP, to create new activity towards phosphoinositide substrates for which the wild-type enzyme had little or no activity. The redesigned enzymes retain catalytic activity despite residue alterations in the active site, and kinetic experiments confirmed specificity for up to four phosphoinositide substrates. Changes in the shape and overall volume of the active site where critical to facilitate access of the new substrates for catalysis. The kinetics data suggest that both the position and the combination of amino acid mutations are important for specificity towards the phosphoinositide substrates. The introduction of basic residues proved essential to establish new interactions with the multiple phosphate groups in the inositol head, thus promoting catalytically productive complexes. The crystallographic structures of the top-ranking designs confirmed the computational predictions and showed that residue substitutions do not alter the overall folding of the phosphatase or the conformation of the active site P-loop. The engineered LMW-PTP mutants with new activities can be useful reagents in investigating cell signalling pathways and offer the potential for therapeutic applications.

Nature employs a limited number of genetically encoded axial ligands to control diverse heme enzyme activities. Deciphering the functional significance of these ligands requires a quantitative understanding of how their electron donating capabilities modulate the structures and reactivities of the iconic ferryl intermediates compounds I and II. However, probing these relationships experimentally has proven challenging as ligand substitutions accessible via conventional mutagenesis do not allow fine tuning of electron donation and typically abolish catalytic function. Here we exploit engineered translation components to replace the histidine ligand of cytochrome c peroxidase (CcP) by a less electron donating N_δ-methyl histidine (Me-His) with little effect on enzyme structure. The rate of formation (k₁) and the reactivity (k₂) of compound I are unaffected by ligand substitution. In contrast, proton coupled electron transfer to compound II (k₃) is 10-fold slower in CcP Me-His, providing a direct link between electron do-nation and compound II reactivity which can be explained by weaker electron donation from the Me-His ligand (‘the push’) affording an electron deficient ferryl-oxygen with reduced proton affinity (‘the pull’). The deleterious effects of the Me-His ligand can be fully compensated by introducing a W51F mutation, designed to increase ‘the pull’ by removing a hydrogen bond to the ferryl-oxygen. Analogous substitutions in ascorbate peroxidase (APX) lead to similar activity trends to those observed in CcP, suggesting a common mechanistic strategy is employed by enzymes using distinct electron transfer pathways. Our study highlights how non-canonical active site substitutions can be used to directly probe and deconstruct highly evolved bioinorganic mechanisms.

Serine arginine (SR) proteins are a family of mRNA regulatory proteins that can modulate spliceosome association with different splice sites and therefore regulate alternative splicing. Phosphorylation within SR proteins is necessary for splice-site recognition, and this is catalyzed by SR protein kinase 1 (SRPK1). The herpes simplex virus (HSV-1) protein ICP27 has been shown previously to interact with and downregulate SRPK1 activity in vivo ; however, the molecular mechanism for this interaction and inhibition was unknown. Here, we demonstrate that the isolated peptide fragment of ICP27 containing RGG box binds to SRPK1 with high affinity, and competes with a native cellular substrate. Elucidation of the SRPK1-RGG box crystal structure further showed that a short palindromic RGRRRGR sequence binds in the substrate docking groove of SRPK1, mimicking the binding of SR repeats of substrates. These data reveal how the viral protein ICP27 inactivates SRPK1, promoting hypophosphorylation of proteins regulating splicing.

A series of analogues of cyclo(L-tyrosyl-L-tyrosine), the substrate of the Mycobacterium tuberculosis enzyme CYP121, have been synthesized and analyzed by UV-Vis and EPR spectroscopy and by X-ray crystallography. The introduction of iodine substituents onto cyclo(L-tyrosyl-L-tyrosine) results in sub-μM binding affinity for the CYP121 enzyme and a complete shift to the high spin state of the heme Fe^III. The introduction of halogens that are able to interact with heme groups is thus a feasible approach to the development of next-generation, tight binding inhibitors of the CYP121 enzyme, in the search for novel anti-tubercular compounds.

PSD-95 is a member of Membrane Associated Guanylate Kinase class of proteins which form scaffolding interactions with partner proteins including ion and receptor channels. PSD-95 is directly implicated in modulating the electrical responses of excitable cells. The first two PSD-95/Disks Large/Zona Occludens domains of PSD-95 have been shown to be the key component in the formation of channel clusters. We report crystal structures of the dual domain in both in apo and ligand-bound form; thermodynamic analysis of ligand association and Small Angle X-ray Scattering of the dual domain in the absence and presence of ligands. These experiments reveal that the ligated double domain forms a scaffold in the complete sense of the word. The concentration of the components in this study is comparable to those found in compartments of excitable cells such as the postsynaptic density and juxta-paranodes of Ranvier. The properties of the dual domain explain the basis of the scaffolding function of PSD-95, and provide a more detailed understanding of the integration of key components of neuronal specializations involved in nervous signal transmission.