Substitution of 2-oxoglutarate alters reaction outcomes of the Pseudomonas savastanoi ethylene-forming enzyme.

In seeding plants, biosynthesis of the phytohormone ethylene, which regulates processes including fruit ripening and senescence, is catalyzed by 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase. The plant pathogen Pseudomonas savastanoi (previously classified as: Pseudomonas syringae) employs a different type of ethylene-forming enzyme (psEFE), though from the same structural superfamily as ACC oxidase, to catalyze ethylene formation from 2-oxoglutarate (2OG) in an arginine dependent manner. psEFE also catalyzes the more typical oxidation of arginine to give L-Δ1-pyrroline-5-carboxylate (P5C), a reaction coupled to oxidative decarboxylation of 2OG giving succinate and CO2. We report on the effects of C3 and/or C4 substituted 2OG derivatives on the reaction modes of psEFE. 1H NMR assays, including using the pure shift method, reveal that, within our limits of detection, none of the tested 2OG derivatives is converted to an alkene; some are converted to the corresponding ß-hydroxypropionate or succinate derivatives, with only the latter being coupled to arginine oxidation. The NMR results reveal that the nature of 2OG derivatization can affect the outcome of the bifurcating reaction, with some 2OG derivatives exclusively favoring the arginine oxidation pathway. Given that some of the tested 2OG derivatives are natural products, the results are of potential biological relevance. There are also opportunities for therapeutic or biocatalytic regulation of the outcomes of reactions catalyzed by 2OG-dependent oxygenases by the use of 2OG derivatives.

Fixing the Achilles Heel of Pfizer's Paxlovid for COVID-19 Treatment.

Nirmatrelvir (PF-07321332), a first-in-class inhibitor of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) main protease (Mpro), was developed by Pfizer under intense pressure during the pandemic to treat COVID-19. A weakness of nirmatrelvir is its limited metabolic stability, which led to the development of a combination therapy (paxlovid), involving coadministration of nirmatrelvir with the cytochrome P450 inhibitor ritonavir. However, limitations in tolerability of the ritonavir component reduce the scope of paxlovid. In response to these limitations, researchers at Pfizer have now developed the second-generation Mpro inhibitor PF-07817883 (ibuzatrelvir). Structurally related to nirmatrelvir, including with the presence of a trifluoromethyl group, albeit located differently, ibuzatrelvir manifests enhanced oral bioavailability, so it does not require coadministration with ritonavir. The development of ibuzatrelvir is an important milestone, because it is expected to enhance the treatment of COVID-19 without the drawbacks associated with ritonavir. Given the success of paxlovid in treating COVID-19, it is likely that ibuzatrelvir will be granted approval as an improved drug for treatment of COVID-19 infections, so complementing vaccination efforts and improving pandemic preparedness. The development of nirmatrelvir and ibuzatrelvir dramatically highlights the power of appropriately resourced modern medicinal chemistry to very rapidly enable the development of breakthrough medicines. Consideration of how analogous approaches can be used to develop similarly breakthrough medicines for infectious diseases such as tuberculosis and malaria is worthwhile.

Thiophene-fused γ-lactams inhibit the SARS-CoV-2 main protease via reversible covalent acylation.

Enzyme inhibitors working by O-acylation of nucleophilic serine residues are of immense medicinal importance, as exemplified by the ß-lactam antibiotics. By contrast, inhibition of nucleophilic cysteine enzymes by S-acylation has not been widely exploited for medicinal applications. The SARS-CoV-2 main protease (Mpro) is a nucleophilic cysteine protease and a validated therapeutic target for COVID-19 treatment using small-molecule inhibitors. The clinically used Mpro inhibitors nirmatrelvir and simnotrelvir work via reversible covalent reaction of their electrophilic nitrile with the Mpro nucleophilic cysteine (Cys145). We report combined structure activity relationship and mass spectrometric studies revealing that appropriately functionalized γ-lactams can potently inhibit Mpro by reversible covalent reaction with Cys145 of Mpro. The results suggest that γ-lactams have potential as electrophilic warheads for development of covalently reacting small-molecule inhibitors of Mpro and, by implication, other nucleophilic cysteine enzymes.

Studies on the selectivity of the SARS-CoV-2 papain-like protease reveal the importance of the P2' proline of the viral polyprotein.

The SARS-CoV-2 papain-like protease (PLpro) is an antiviral drug target that catalyzes the hydrolysis of the viral polyproteins pp1a/1ab, so releasing the non-structural proteins (nsps) 1-3 that are essential for the coronavirus lifecycle. The LXGG↓X motif in pp1a/1ab is crucial for recognition and cleavage by PLpro. We describe molecular dynamics, docking, and quantum mechanics/molecular mechanics (QM/MM) calculations to investigate how oligopeptide substrates derived from the viral polyprotein bind to PLpro. The results reveal how the substrate sequence affects the efficiency of PLpro-catalyzed hydrolysis. In particular, a proline at the P2' position promotes catalysis, as validated by residue substitutions and mass spectrometry-based analyses. Analysis of PLpro catalyzed hydrolysis of LXGG motif-containing oligopeptides derived from human proteins suggests that factors beyond the LXGG motif and the presence of a proline residue at P2' contribute to catalytic efficiency, possibly reflecting the promiscuity of PLpro. The results will help in identifying PLpro substrates and guiding inhibitor design.

Cyclic ß2,3-amino acids improve the serum stability of macrocyclic peptide inhibitors targeting the SARS-CoV-2 main protease.

Due to their constrained conformations, cyclic ß2,3-amino acids (cßAA) are key building blocks that can fold peptides into compact and rigid structures, improving peptidase resistance and binding affinity to target proteins, due to their constrained conformations. Although the translation efficiency of cßAAs is generally low, our engineered tRNA, referred to as tRNAPro1E2, enabled efficient incorporation of cßAAs into peptide libraries using the flexible in vitro translation (FIT) system. Here we report on the design and application of a macrocyclic peptide library incorporating 3 kinds of cßAAs: (1R,2S)-2-aminocyclopentane carboxylic acid (ß1), (1S,2S)-2-aminocyclohexane carboxylic acid (ß2), and (1R,2R)-2-aminocyclopentane carboxylic acid. This library was applied to an in vitro selection against the SARS-CoV-2 main protease (Mpro). The resultant peptides, BM3 and BM7, bearing one ß2 and two ß1, exhibited potent inhibitory activities with IC50 values of 40 and 20 nM, respectively. BM3 and BM7 also showed remarkable serum stability with half-lives of 48 and >168 h, respectively. Notably, BM3A and BM7A, wherein the cßAAs were substituted with alanine, lost their inhibitory activities against Mpro and displayed substantially shorter serum half-lives. This observation underscores the significant contribution of cßAA to the activity and stability of peptides. Overall, our results highlight the potential of cßAA in generating potent and highly stable macrocyclic peptides with drug-like properties.

The selective prolyl hydroxylase inhibitor IOX5 stabilizes HIF-1α and compromises development and progression of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a largely incurable disease, for which new treatments are urgently needed. While leukemogenesis occurs in the hypoxic bone marrow, the therapeutic tractability of the hypoxia-inducible factor (HIF) system remains undefined. Given that inactivation of HIF-1α/HIF-2α promotes AML, a possible clinical strategy is to target the HIF-prolyl hydroxylases (PHDs), which promote HIF-1α/HIF-2α degradation. Here, we reveal that genetic inactivation of Phd1/Phd2 hinders AML initiation and progression, without impacting normal hematopoiesis. We investigated clinically used PHD inhibitors and a new selective PHD inhibitor (IOX5), to stabilize HIF-α in AML cells. PHD inhibition compromises AML in a HIF-1α-dependent manner to disable pro-leukemogenic pathways, re-program metabolism and induce apoptosis, in part via upregulation of BNIP3. Notably, concurrent inhibition of BCL-2 by venetoclax potentiates the anti-leukemic effect of PHD inhibition. Thus, PHD inhibition, with consequent HIF-1α stabilization, is a promising nontoxic strategy for AML, including in combination with venetoclax.

Human Oxygenase Variants Employing a Single Protein FeII Ligand Are Catalytically Active.

Aspartate/asparagine-ß-hydroxylase (AspH) is a human 2-oxoglutarate (2OG) and FeII oxygenase that catalyses C3 hydroxylations of aspartate/asparagine residues of epidermal growth factor-like domains (EGFDs). Unusually, AspH employs two histidine residues to chelate FeII rather than the typical triad of two histidine and one glutamate/aspartate residue. We report kinetic, inhibition, and crystallographic studies concerning human AspH variants in which either of its FeII binding histidine residues are substituted for alanine. Both the H725A and, in particular, the H679A AspH variants retain substantial catalytic activity. Crystal structures clearly reveal metal-ligation by only a single protein histidine ligand. The results have implications for the functional assignment of 2OG oxygenases and for the design of non-protein biomimetic catalysts.