Key features of inhibitor binding to the human mitochondrial pyruvate carrier hetero-dimer

The mitochondrial pyruvate carrier (MPC) has emerged as a promising drug target for metabolic disorders, including non-alcoholic steatohepatitis and diabetes, metabolically dependent cancers and neurodegenerative diseases. Human MPC is a protein complex, but the composition of its active form is debated and the mechanisms of transport and inhibition are not resolved. We have recombinantly expressed and purified the human hetero-complex MPC1L/MPC2 and demonstrate that it is a functional hetero-dimer, like the yeast MPC hetero-dimers. Unlike the latter, human MPC1L/MPC2 binds the known inhibitors with high potencies. We identify the essential chemical features shared between these structurally diverse inhibitors and demonstrate that high affinity binding is not attributed to covalent bond formation with MPC cysteines, as previously thought. We also identify 14 new inhibitors of MPC, one outperforming the most potent compound UK5099 by tenfold. Two of them are the commonly prescribed drugs entacapone and nitrofurantoin, suggesting possible off-target mechanisms associated with their adverse effects. This work advances our understanding of MPC inhibition and will accelerate the development of clinically relevant MPC modulators.

Perspective on the Structural Basis for Human Aldo-Keto Reductase 1B10 Inhibition

Human aldo-keto reductase 1B10 (AKR1B10) is overexpressed in many cancer types and is involved in chemoresistance. This makes AKR1B10 to be an interesting drug target and thus many enzyme inhibitors have been investigated. High-resolution crystallographic structures of AKR1B10 with various reversible inhibitors were deeply analyzed and compared to those of analogous complexes with aldose reductase (AR). In both enzymes, the active site included an anion-binding pocket and, in some cases, inhibitor binding caused the opening of a transient specificity pocket. Different structural conformers were revealed upon inhibitor binding, emphasizing the importance of the highly variable loops, which participate in the transient opening of additional binding subpockets. Two key differences between AKR1B10 and AR were observed regarding the role of external loops in inhibitor binding. The first corresponded to the alternative conformation of Trp112 (Trp111 in AR). The second difference dealt with loop A mobility, which defined a larger and more loosely packed subpocket in AKR1B10. From this analysis, the general features that a selective AKR1B10 inhibitor should comply with are the following: an anchoring moiety to the anion-binding pocket, keeping Trp112 in its native conformation (AKR1B10-like), and not opening the specificity pocket in AR.

Inhibition and reaction mechanism of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase: A potential target for novel drug design

<p>Tuberculosis (TB), which is estimated to affect 2 billion individuals worldwide, is an infection predominately caused by Mycobacterium tuberculosis(M. tuberculosis). Of particular concern is the increasing prevalence of TB, which is becoming resistant to the treatments currently available. Anthranilate phosphoribosyltransferase (AnPRT) catalyses the formation of N-(5’-phosphoribosyl)anthranilate (PRA) from 5-phospho-α-ribose-1-diphosphate (PRPP) and anthranilate and plays an important role in the synthesis of an essential amino acid in M.tuberculosis. A strain with a genetic knockout of the trpD gene, which encodes for the AnPRT enzyme, was unable to cause disease, even in immune-deficient mice. Therefore, this enzyme is a potential drug target for the development of new treatments against TB and other infectious diseases. This research explores the synthesis of different substrates and potential transition state analogues in order to understand catalysis and inhibition of AnPRT enzymes to aid novel drug design. The first part of this study utilises “bianthranilate-like” phosphonate inhibitors that display effective inhibition of the AnPRT enzyme, with the lowest Ki value being 1.3 μM. It was found strong enzymatic inhibition increases with an increased length of the phosphonate linker that occupies multiple anthranilate binding sites within the anthranilate binding channel of the enzyme. Crystal studies of the enzyme in complex with the inhibitors were carried out in order to expose the binding interactions. The second part of this study investigates several new compounds that target the active site of M. tuberculosis AnPRT, based on a virtual screening approach. This approach identified a strong AnPRT inhibitor, which displays an apparent Ki value of 7.0 ± 0.4 μM with respect to both substrates. This study also exposed a conformational change at the active site of the enzyme that occurs on inhibitor binding. The observed conformational changes of the enzyme active site diminish the binding of the substrate PRPP. These pieces of information provide future inhibitor design strategies to aid the development of novel anti-TB agents that target the AnPRT enzyme. To elucidate the reaction mechanism of M. tuberculosis AnPRT, the third part of this study explores the substrate binding sites in detail. This study uses structural analysis, complemented by differential scanning fluorimetry (DSF) and isothermal titration calorimetry (ITC), to reveal detailed information of the substrate and inhibitor binding sites. The final part of this thesis presents the synthesis of various PRPP analogues and potential transition state mimics that were designed based on the likely reaction mechanism of the enzyme. This set of inhibitors includes a number of iminoribitol analogues that were developed to capture the geometry of the flattened ribose ring and include a nitrogen atom within the ring to mimic the positive charge characteristics that are expected in the oxocarbenium-ion-like transition state predicted for M. tuberculosis AnPRT. Additionally, we were able to solve the structure of M. tuberculosis AnPRT in complex with one of the potential transition state mimics, which was observed to bind at the active site of the enzyme. This structure provides new insight into the catalytic mechanism of the enzyme and creates an opportunity to develop more specific inhibitors against the M. tuberculosis AnPRT enzyme.</p>

Inhibition and reaction mechanism of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase: A potential target for novel drug design

<p>Tuberculosis (TB), which is estimated to affect 2 billion individuals worldwide, is an infection predominately caused by Mycobacterium tuberculosis(M. tuberculosis). Of particular concern is the increasing prevalence of TB, which is becoming resistant to the treatments currently available. Anthranilate phosphoribosyltransferase (AnPRT) catalyses the formation of N-(5’-phosphoribosyl)anthranilate (PRA) from 5-phospho-α-ribose-1-diphosphate (PRPP) and anthranilate and plays an important role in the synthesis of an essential amino acid in M.tuberculosis. A strain with a genetic knockout of the trpD gene, which encodes for the AnPRT enzyme, was unable to cause disease, even in immune-deficient mice. Therefore, this enzyme is a potential drug target for the development of new treatments against TB and other infectious diseases. This research explores the synthesis of different substrates and potential transition state analogues in order to understand catalysis and inhibition of AnPRT enzymes to aid novel drug design. The first part of this study utilises “bianthranilate-like” phosphonate inhibitors that display effective inhibition of the AnPRT enzyme, with the lowest Ki value being 1.3 μM. It was found strong enzymatic inhibition increases with an increased length of the phosphonate linker that occupies multiple anthranilate binding sites within the anthranilate binding channel of the enzyme. Crystal studies of the enzyme in complex with the inhibitors were carried out in order to expose the binding interactions. The second part of this study investigates several new compounds that target the active site of M. tuberculosis AnPRT, based on a virtual screening approach. This approach identified a strong AnPRT inhibitor, which displays an apparent Ki value of 7.0 ± 0.4 μM with respect to both substrates. This study also exposed a conformational change at the active site of the enzyme that occurs on inhibitor binding. The observed conformational changes of the enzyme active site diminish the binding of the substrate PRPP. These pieces of information provide future inhibitor design strategies to aid the development of novel anti-TB agents that target the AnPRT enzyme. To elucidate the reaction mechanism of M. tuberculosis AnPRT, the third part of this study explores the substrate binding sites in detail. This study uses structural analysis, complemented by differential scanning fluorimetry (DSF) and isothermal titration calorimetry (ITC), to reveal detailed information of the substrate and inhibitor binding sites. The final part of this thesis presents the synthesis of various PRPP analogues and potential transition state mimics that were designed based on the likely reaction mechanism of the enzyme. This set of inhibitors includes a number of iminoribitol analogues that were developed to capture the geometry of the flattened ribose ring and include a nitrogen atom within the ring to mimic the positive charge characteristics that are expected in the oxocarbenium-ion-like transition state predicted for M. tuberculosis AnPRT. Additionally, we were able to solve the structure of M. tuberculosis AnPRT in complex with one of the potential transition state mimics, which was observed to bind at the active site of the enzyme. This structure provides new insight into the catalytic mechanism of the enzyme and creates an opportunity to develop more specific inhibitors against the M. tuberculosis AnPRT enzyme.</p>

The Crystal Structure of Nα-p-tosyl-lysyl Chloromethylketone-Bound Oligopeptidase B from Serratia Proteamaculans Revealed a New Type of Inhibitor Binding

A covalent serine protease inhibitor—Na-p-tosyl-lysyl chloromethylketone (TCK) is a modified lysine residue tosylated at the N-terminus and chloromethylated at the C-terminus, one molecule of which is capable of forming two covalent bonds with both Ser and His catalytic residues, was co-crystallized with modified oligopeptidase B (OpB) from Serratia proteomaculans (PSPmod). The kinetics study, which preceded crystallization, shows that the stoichiometry of TCK-dependent inhibition of PSPmod was 1:2 (protein:inhibitor). The crystal structure of the PSPmod-TCK complex, solved at a resolution of 2.3 Å, confirmed a new type of inhibitor binding. Two TCK molecules were bound to one enzyme molecule: one with the catalytic Ser, the other with the catalytic His. Due to this mode of binding, the intermediate state of PSPmod and the disturbed conformation of the catalytic triad were preserved in the PSPmod-TCK complex. Nevertheless, the analysis of the amino acid surroundings of the inhibitor molecule bound to the catalytic Ser and its comparison with that of antipain-bound OpB from Trypanosoma brucei provided an insight in the structure of the PSPmod substrate-binding pocket. Supposedly, the new type of binding is typical for the interaction of chloromethylketone derivatives with two-domain OpBs. In the open conformational state that these enzymes are assumed in solution, the disordered configuration of the catalytic triad prevents simultaneous interaction of one inhibitor molecule with two catalytic residues.

Structural basis of main proteases of coronavirus bound to drug candidate PF-07321332

The high mutation rate of COVID-19 and the prevalence of multiple variants strongly support the need for pharmacological options to complement vaccine strategies. One region that appears highly conserved among different genus of coronaviruses is the substrate binding site of the main protease (Mpro or 3CLpro), making it an attractive target for the development of broad-spectrum drugs for multiple coronaviruses. PF-07321332 developed by Pfizer is the first orally administered inhibitor targeting the main protease of SARS-CoV-2, which also has shown potency against other coronaviruses. Here we report three crystal structures of main protease of SARS-CoV-2, SARS-CoV and MERS-CoV bound to the inhibitor PF-07321332. The structures reveal a ligand-binding site that is conserved among SARS-CoV-2, SARS-CoV and MERS-CoV, providing insights into the mechanism of inhibition of viral replication. The long and narrow cavity in the cleft between domains I and II of main protease harbors multiple inhibitor binding sites, where PF-07321332 occupies subsites S1, S2 and S4 and appears more restricted compared with other inhibitors. A detailed analysis of these structures illuminated key structural determinants essential for inhibition and elucidated the binding mode of action of main proteases from different coronaviruses. Given the importance of main protease for the treatment of SARS-CoV-2 infection, insights derived from this study should accelerate the design of safer and more effective antivirals.

Dynamic Allostery Highlights the Evolutionary Differences between the CoV-1 and CoV-2 Main Proteases

ABSTRACTThe SARS-CoV-2 coronavirus has become one of the most immediate and widely-studied systems since its identification and subsequent global outbreak from 2019-2020. In an effort to understand the biophysical changes as a result of mutations, the mechanics of multiple different proteins within the SARS-CoV-2 virus have been studied and compared with SARS-CoV-1. Focusing on the main protease (mPro), we first explored the long range dynamic-relationship, particularly in cross-chain dynamics, using the Dynamic Coupling Index (DCI) to investigate the dynamic coupling between the catalytic site residues and the rest of the protein, both inter and intra chain for the CoV-1 and CoV-2 mPro. We found that there is significant cross-chain coupling between these active sites and distal residues in the CoV-2 mPro but it was missing in CoV-1. The enhanced long distance interactions, particularly between the two chains, suggest subsequently enhanced cooperativity for CoV-2. A further comparative analysis of the dynamic flexibility using the Dynamic Flexibility Index (DFI) between the CoV-1 and CoV-2 mPros shows that the inhibitor binding near active sites induces change in flexibility to a distal region of the protein, opposite in behavior between the two systems; this region becomes more flexible upon inhibitor binding in CoV-1 while it becomes less flexible in the CoV-2 mPro. Upon inspection, we show that, on average, the dynamic flexibility of the sites substituted from CoV-1 to CoV-2 changes significantly less than the average calculated across all residues within the structure, indicating that the differences in behaviors between the two systems is likely the result of allosteric influence, where the new substitutions in COV-2 induce flexibility and dynamical changes elsewhere in the structure.SIGNIFICANCEHere we have conducted a comparative analysis between the SARS-CoV-1 and SARS-CoV-2 mPro systems to shed mechanistic insight on the biophysical changes associated with the mutations between these two enzymes. Our work shows that the CoV-2 mPro system exhibits enhanced cross-chain communication between catalytic site residues and the rest of the structure. Further, both dynamic coupling and dynamic flexibility analyses indicates that, largely, the dynamic changes as evaluated by DCI and DFI occur at sites other than the mutation sites themselves, indicating that the functional differences between these two proteins are a result of dynamic allostery