Hybrid QM/MM free energy evaluation of drug-resistant mutational effect on binding of an inhibitor Indinavir to HIV protease

Human immunodeficiency virus 1 (HIV-1) protease is a homo-dimeric aspartic protease essential for replication of HIV. The HIV-1 protease is a target protein in drug discovery for antiretroviral therapy, and various inhibitor molecules of transition state analog were developed. However, serious drug-resistant mutants have emerged. For understanding molecular mechanism of the drug-resistance, accurate examination of the impacts of the mutations on ligand binding as well as enzymatic activity is necessary. Here, we present a molecular simulation study on the ligand binding of Indinavir, a potent transition state analog inhibitor, to the native protein and a V82T/I84V drug-resistant mutant of HIV-1 protease. We employed a hybrid ab initio quantum mechanical/molecular mechanical (QM/MM) free energy optimization technique which combines highly accurate QM description of the ligand molecule and its interaction with statistically ample conformational sampling of MM protein environment by long-time molecular dynamics simulations. Through free energy calculations of protonation states of catalytic groups at the binding pocket and of ligand binding affinity changes upon the mutations, we successfully reproduced the experimentally observed significant reduction of the binding affinity upon the drug-resistant mutations and elucidated the underlying molecular mechanism. The present study opens the way for understanding the molecular mechanism of drug-resistance through direct quantitative comparison of ligand binding and enzymatic reaction with the same accuracy.

Hybrid QM/MM free energy evaluation of drug-resistant mutational effect on binding of an inhibitor Indinavir to HIV protease

Human immunodeficiency virus 1 (HIV-1) protease is a homo-dimeric aspartic protease essential for replication of HIV. The HIV-1 protease is a target protein in drug discovery for antiretroviral therapy, and various inhibitor molecules of transition state analog were developed. However, serious drug-resistant mutants have emerged. For understanding molecular mechanism of the drug-resistance, accurate examination of the impacts of the mutations on ligand binding as well as enzymatic activity is necessary. Here, we present a molecular simulation study on the ligand binding of Indinavir, a potent transition state analog inhibitor, to the native protein and a V82T/I84V drug-resistant mutant of HIV-1 protease. We employed a hybrid ab initio quantum mechanical/molecular mechanical (QM/MM) free energy optimization technique which combines highly accurate QM description of the ligand molecule and its interaction with statistically ample conformational sampling of MM protein environment by long-time molecular dynamics simulations. Through free energy calculations of protonation states of catalytic groups at the binding pocket and of ligand binding affinity changes upon the mutations, we successfully reproduced the experimentally observed significant reduction of the binding affinity upon the drug-resistant mutations and elucidated the underlying molecular mechanism. The present study opens the way for understanding the molecular mechanism of drug-resistance through direct quantitative comparison of ligand binding and enzymatic reaction with the same accuracy.

Structural Comparisons of Cefotaximase (CTX-M-ase) Sub Family 1

The cefotaximase or CTX-M, family of serine-β-lactamases represents a significant clinical concern due to the ability for these enzymes to confer resistance to a broad array of β-lactam antibiotics an inhibitors. This behavior lends CTX-M-ases to be classified as extended spectrum β-lactamases (ESBL). Across the family of CTX-M-ases most closely related to CTX-M-1, the structures of CTX-M-15 with a library of different ligands have been solved and serve as the basis of comparison within this review. Herein we focus on the structural changes apparent in structures of CTX-M-15 in complex with diazabicyclooctane (DABCO) and boronic acid transition state analog inhibitors. Interactions between a positive surface patch near the active site and complementary functional groups of the bound inhibitor play key roles in the dictating the conformations of active site residues. The insights provided by analyzing structures of CTX-M-15 in complex with DABCO and boronic acid transition state analog inhibitors and analyzing existing structures of CTX-M-64 offer opportunities to move closer to making predictions as to how CTX-M-ases may interact with potential drug candidates, setting the stage for the further development of new antibiotics and β-lactamase inhibitors.

Crystal Structures of Streptomyces Coelicolor Methylmalonyl-CoA Epimerase with Substrate or Transition State Analog Contradicts a Simple General Acid-Base Catalytic Mechanism

Crystal structures of Streptomyces coelicolor methylmalonyl-CoA epimerase in the holo-form, with substrate or the putative transition state analog, 2-nitroproionyl-CoA. The proposed catalytic mechanism is general acid-base catalysis. The proposed catalytic residues are too far from the substrate or analog, unless conformational changes take place or some other mechanism is used. <br>

Crystal Structures of Streptomyces Coelicolor Methylmalonyl-CoA Epimerase with Substrate or Transition State Analog Contradicts a Simple General Acid-Base Catalytic Mechanism

Crystal structures of Streptomyces coelicolor methylmalonyl-CoA epimerase in the holo-form, with substrate or the putative transition state analog, 2-nitroproionyl-CoA. The proposed catalytic mechanism is general acid-base catalysis. The proposed catalytic residues are too far from the substrate or analog, unless conformational changes take place or some other mechanism is used. <br>

Mechanisms of feedback inhibition and sequential firing of active sites in plant aspartate transcarbamoylase

Aspartate transcarbamoylase (ATC), an essential enzyme for de novo pyrimidine biosynthesis, is uniquely regulated in plants by feedback inhibition of uridine 5-monophosphate (UMP). Despite its importance in plant growth, the structure of this UMP-controlled ATC and the regulatory mechanism remain unknown. Here, we report the crystal structures of Arabidopsis ATC trimer free and bound to UMP, complexed to a transition-state analog or bearing a mutation that turns the enzyme insensitive to UMP. We found that UMP binds and blocks the ATC active site, directly competing with the binding of the substrates. We also prove that UMP recognition relies on a loop exclusively conserved in plants that is also responsible for the sequential firing of the active sites. In this work, we describe unique regulatory and catalytic properties of plant ATCs that could be exploited to modulate de novo pyrimidine synthesis and plant growth.

Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2

SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme’s active site. Our findings provide new insights for the development of uracil scaffold-based drugs.

Structural Insights into Inhibition of the Acinetobacter-Derived Cephalosporinase ADC-7 by Ceftazidime and Its Boronic Acid Transition State Analog

ABSTRACT Extended-spectrum class C β-lactamases have evolved to rapidly inactivate expanded-spectrum cephalosporins, a class of antibiotics designed to be resistant to hydrolysis by β-lactamase enzymes. To better understand the mechanism by which Acinetobacter-derived cephalosporinase-7 (ADC-7), a chromosomal AmpC enzyme, hydrolyzes these molecules, we determined the X-ray crystal structure of ADC-7 in an acyl-enzyme complex with the cephalosporin ceftazidime (2.40 Å) as well as in complex with a boronic acid transition state analog inhibitor that contains the R1 side chain of ceftazidime (1.67 Å). In the acyl-enzyme complex, the carbonyl oxygen is situated in the oxyanion hole where it makes key stabilizing interactions with the main chain nitrogens of Ser64 and Ser315. The boronic acid O1 hydroxyl group is similarly positioned in this area. Conserved residues Gln120 and Asn152 form hydrogen bonds with the amide group of the R1 side chain in both complexes. These complexes represent two steps in the hydrolysis of expanded-spectrum cephalosporins by ADC-7 and offer insight into the inhibition of ADC-7 by ceftazidime through displacement of the deacylating water molecule as well as blocking its trajectory to the acyl carbonyl carbon. In addition, the transition state analog inhibitor, LP06, was shown to bind with high affinity to ADC-7 (Ki, 50 nM) and was able to restore ceftazidime susceptibility, offering the potential for optimization efforts of this type of inhibitor.

N-Benzyl Residues as the P1′ Substituents in Phosphorus-Containing Extended Transition State Analog Inhibitors of Metalloaminopeptidases

Peptidyl enzyme inhibitors containing an internal aminomethylphosphinic bond system (P(O)(OH)-CH2-NH) can be termed extended transition state analogs by similarity to the corresponding phosphonamidates (P(O)(OH)-NH). Phosphonamidate pseudopeptides are broadly recognized as competitive mechanism-based inhibitors of metalloenzymes, mainly hydrolases. Their practical use is, however, limited by hydrolytic instability, which is particularly restricting for dipeptide analogs. Extension of phosphonamidates by addition of the methylene group produces a P-C-N system fully resistant in water conditions. In the current work, we present a versatile synthetic approach to such modified dipeptides, based on the three-component phospha-Mannich condensation of phosphinic acids, formaldehyde, and N-benzylglycines. The last-mentioned component allowed for simple and versatile introduction of functionalized P1′ residues located on the tertiary amino group. The products demonstrated moderate inhibitory activity towards porcine and plant metalloaminopeptidases, while selected derivatives appeared very potent with human alanyl aminopeptidase (Ki = 102 nM for 6a). Analysis of ligand-protein complexes obtained by molecular modelling revealed canonical modes of interactions for mono-metallic alanyl aminopeptidases, and distorted modes for di-metallic leucine aminopeptidases (with C-terminal carboxylate, not phosphinate, involved in metal coordination). In general, the method can be dedicated to examine P1′-S1′ complementarity in searching for non-evident structures of specific residues as the key fragments of perspective ligands.