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Many bacterial pathogens, including Staphylococcus aureus, require inosine 5'-monophosphate dehydrogenase (IMPDH) for infection, making this enzyme a promising new target for antibiotics. Although potent selective inhibitors of bacterial IMPDHs have been reported, relatively few have displayed antibacterial activity. Here we use structure-informed design to obtain inhibitors of S. aureus IMPDH (SaIMPDH) that have potent antibacterial activity (minimal inhibitory concentrations less than 2 μM) and low cytotoxicity in mammalian cells. The physicochemical properties of the most active compounds were within typical Lipinski/Veber space, suggesting that polarity is not a general requirement for achieving antibacterial activity. Five compounds failed to display activity in mouse models of septicemia and abscess infection. Inhibitor-resistant S. aureus strains readily emerged in vitro. Resistance resulted from substitutions in the cofactor/inhibitor binding site of SaIMPDH, confirming on-target antibacterial activity. These mutations decreased the binding of all inhibitors tested, but also decreased catalytic activity. Nonetheless, the resistant strains had comparable virulence to wild-type bacteria. Surprisingly, strains expressing catalytically inactive SaIMPDH displayed only a mild virulence defect. Collectively these observations question the vulnerability of the enzymatic activity of SaIMPDH as a target for the treatment of S. aureus infections, suggesting other functions of this protein may be responsible for its role in infection.

The mathematics and geometry of the "kobs" method under the tight-binding experimental conditions, when inhibitor depletion is significant, has not been fully explored in the existing biochemical kinetic literature. It is shown here that under tight-binding conditions a plot of the pseudo-first order rate constant against the inhibitor concentration is always nonlinear and concave upward, as opposed to either linear or hyperbolic (concave downward) in the absence of inhibitor depletion. If and when the apparent inhibition constant is lower than the active enzyme concentration, the plot has a distinct local minimum occurring at an inhibitor concentration that is equal to the enzyme concentration minus the inhibition constant. The slope of the plot at inhibitor concentrations significantly higher than the enzyme concentration is equal to the second order bimolecular association rate constant. The intercept on the vertical axis is equal to the sum of the dissociation rate constant of the enzyme–inhibitor complex, plus the product of the enzyme concentration multiplied by the association rate constant. Most importantly, we show here that specifically under tight-binding experimental conditions the “kobs” method only applies to the one-step binding model, without a possible involvement of a transient enzyme–inhibitor complex. Thus, as a matter of principle, under tight binding this method cannot be used to discriminate between the one-step and two-step inhibition mechanisms, nor can it be used to determine the dissociation equilibrium of a transient complex even if such a complex is in fact present.

Covalent (irreversible) enzyme inhibitors are an important group of actual or potential therapeutics. For example, Aspirin is an irreversible inhibitor of the cyclooxygenase enzyme. Evaluating covalent inhibitors in the drug discovery is exceptionally challenging, because their overall inhibitory potency consists of two separate but intertwined contributions: (1) initial binding affinity and (2) chemical reactivity. It is especially difficult to reliably asses the kinetic mechanism of inhibition. This paper describes an objective statistical approach that can be used to decide between two alternate kinetic mechanisms of covalent enzyme inhibition, from kinetic experiments based on the standard "kobs" method [Copeland (2013) "Evaluation of Enzyme Inhibitors in Drug Discovery", section 9.1]. The two alternatives are either a two-step kinetic mechanism, which involves a reversibly formed noncovalent intermediate, or a one-step kinetic mechanism, proceeding in a single bimolecular step. The proposed statistical toolkit uses four independent methods to arrive at a reliable mechanistic conclusion. The results are illustrated by using recently published experimental data on the inhibition of two different protein kinases by the experimental drugs ibrutinib (PCI-32765) and acalabrutinib [Hopper et al. (2020) J. Pharm. Exp. Therap. 372, 331–338].

This report describes an algebraic formula to calculate the optimal duration of the pre-incubation phase in enzyme-inhibition experiments, based on the assumed range of expected values for the dissociation equilibrium constant of the enzyme–inhibitor complex and for the bimolecular association rate constant. Three typical experimental scenarios are treated, namely, (1) single-point primary screening at relatively high inhibitor concentrations; (2) dose-response secondary screening of relatively weakly bound inhibitors; (3) dose-response screening of tightly-bound inhibitors.

The trp RNA-binding attenuation protein (TRAP) assembles into an 11-fold symmetric ring that regulates transcription and translation of trp-mRNA in bacilli via heterotropic allosteric activation by the amino acid tryptophan (Trp). Whereas nuclear magnetic resonance studies have revealed that Trp-induced activation coincides with both microsecond to millisecond rigidification and local structural changes in TRAP, the pathway of binding of the 11 Trp ligands to the TRAP ring remains unclear. Moreover, because each of 11 bound Trp molecules is completely surrounded by protein, its release requires flexibility of Trp-bound (holo) TRAP. Here, we used stopped-flow fluorescence to study the kinetics of Trp binding by Bacillus stearothermophilus TRAP over a range of temperatures and observed well-separated kinetic steps. These data were analyzed using nonlinear least-squares fitting of several two- and three-step models. We found that a model with two binding steps best describes the data, although the structural...

There are two classes of KD08P synthases characterized respectively by the presence or absence of a metal in the active site. The nonmetallo KD08PS from Escherichia coli and the metallo KD08PS from Aquifex aeolicus are the best characterized members of each class. All amino acid residues that make important contacts with the substrates are conserved in both enzymes with the exception of Pro-10, Cys-11, Ser-235, and Gln-237 of the A. aeolicus enzyme, which correspond respectively to Met-25, Asn-26, Pro-252, and Ala-254 in the E. coli enzyme. Interconversion between the two forms of KD08P synthases can be achieved by substituting the metal-coordinating cysteine of metallo synthases with the corresponding asparagine of nonmetallo synthases, and vice versa. In this report we describe the structural changes elicited by the Cl IN mutation and by three combinations of mutations (P10M/C1 IN, Cl 1N/S235P/Q237A, and P10M/C11N/5235P/Q237A) situated along possible evolutionary paths connecting the A. aeolicus and the E. coli enzyme. All four mutants are not capable of binding metal and lack the structural asymmetry among subunits with regard to substrate binding and conformation of the L7 loop, which is typical of A. aeolicus wild-type KD08PS but is absent in the E. coli enzyme. Despite the lack of the active site metal, the mutant enzymes display levels of activity ranging from 46% to 24% of the wild type. With the sole exception of the quadruple mutant, metal loss does not affect the thermal stability of KD08PS. The free energy of unfolding in water is also either unchanged or even increased in the mutant enzymes, suggesting that the primary role of the active site metal in A. aeolicus KD08PS is not to increase the enzyme stability. In all four mutants A5P binding displaces a water molecule located on the si side of PEP. In particular, in the double and triple mutant, A5P binds with the aldehyde carbonyl in hydrogen bond distance of Asn-11, while in the wild type this functional group points away from Cys-11. This alternative conformation of A5P is likely to have functional significance as it resembles the conformation of the acyclic reaction intermediate, which is observed here for the first time in some of the active sites of the triple mutant. The direct visualization of this intermediate by X-ray crystallography confirms earlier mechanistic models of KD08P synthesis. In particular, the configuration of the C2 chiral center of the intermediate supports a model of the reaction in nonmetallo KD08PS, in which water attacks an oxocarbenium ion or PEP from the si side of C2. Several explanations are offered to reconcile this observation with the fact that no water molecule is observed at this position in the mutant enzymes in the presence of both PEP and A5P. Significant differences were observed between the wild-type and the mutant enzymes in the K m values for PEP and A5P and in the K d values for inorganic phosphate and R5P. These differences may reflect an evolutionary adaptation of metallo and nonmetallo KD08PS's to the cellular concentrations of these metabolites in their respective hosts.

We have discovered that 17β-[N,N-(diethyl)carbamoyl]-6-azaandrost-4-en-3-one is a time-dependent inhibitor of type II 5α-reductase, as is the drug finasteride. Unlike finasteride, the 6-aza-steroid is not a time-dependent inhibitor of type I 5α-reductase. Finasteride inhibition of type II enzyme proceeds in a two-step mechanism. At pH 6 and 37 °C, an initial finasteride−reductase complex is formed with a Kiapp of 11.9 ± 4.1 nM. In a second step, an irreversible complex is formed with a rate constant of inactivation of 0.09 ± 0.01 s-1. In contrast, the 6-aza-steroid is a reversible inhibitor. From the results of a simplified mathematical analysis, based on the rapid equilibrium approximation, the inhibitor and the enzyme form an initial complex with a Ki of 6.8 ± 0.2 nM. The reversible formation of a final complex, with an overall Ki of 0.07 ± 0.02 nM, is characterized by a first-order isomerization rate constant 0.0035 ± 0.0001 s-1 for the forward step and 0.00025 ± 0.00006 s-1 for the backward step. All ...

The cyclic undecapeptide cyclosporin A (CsA) is a slow-binding inhibitor of the peptidylprolyl cis-trans isomerase (PPIase) cyclophiiin. Both the initial inhibitory activity and the subsequent time-dependent inhibition are sensitive to the solvent system (DMSO, THF, LiCl-THF) in which CsA is dissolved prior to the assay. NMR experiments demonstrate that in tetrahydrofuran the MeLeu'-MeLeu10 peptide bond has a cis conformation (Kessler, H.; et al. Biochem. Pharmacol. 1990, 40, 169-173). The cis conformer is inactive as a PPIase inhibitor. The same peptide bond adopts a trans conformation when lithium chloride is present as an additive in THF or when CsA is bound to cyclophiiin (Fesik, S. W.; et al. Science 1990, 250, 1406-1409). The trans conformer is a tight-binding inhibitor of PPIase activity (K1 = 20 nM), and the inhibition increases over time (K1 = 7 nM after 30 min). Detailed kinetic analysis of this transition indicates the presence of at least two interconverting forms of unbound CsA, and a slow structural change in the enzyme-inhibitor complex. The kinetic and structural data taken together suggest that the sequence MeLeu9-rraMj-MeLeu10-MeVal" is responsible for efficient binding in the active site of cyclophiiin, while the corresponding cis conformer is not recognized at all. The interconversion between the two conformers is kinetically expressed in the time-dependent binding of the drug.

The kinetic properties and substrate specificity of two well-characterized peptidyl prolyl cis-trans isomerases (PPIases), cyclophilin and the FK-506 binding protein (FKBP), have been previously examined (Fischer, G., Bang, H., Berger, E., & Schellenberger, A. (1984) Biochim. Biophys. Acta 791,8747; Harrison, R. K., & Stein, R. L. (1990) Biochemistry 29, 1684-1689; Albers, M. W., Walsh, C. T., & Schreiber, S. L. (1 990) J. Org. Chem. 55,4984-49861. The chymotrypsin-coupled enzymatic assay employed in these studies suffers from two serious shortcomings. Due to the low equilibrium population of the X-cis-Pro- Phe-pNA isomer (the PPIase substrate), in conjunction with the low solubility of p-nitroaniline generated by chymotrypsin hydrolysis, substrate concentrations in the saturating region are not experimentally attainable. Secondly, the uncatalyzed cis-trans isomerization obscures the interpretation of the initial velocity. As a result of these limitations, the steady-state kinetic parameters (K,, kat) have not been determined. Here we introduce an improved version of the spectrophotometric assay and report for the first time the Michaelis constants and turnover numbers for both PPIases with established substrates. The improvements in the experimental conditions originate in a medium-induced increase in the equilibrium population of the cis X-Pro conformer and in conducting the assay at 0 OC to suppress the uncatalyzed thermal isomerization. In addition, we present a rigorous mathematical model of the spectrophotometric progress curves that accounts for the contributions of the residual background rate. For Suc-Ala-Ala-cis-Pro-Phe-pNA with bovine cyclophilin, K, = 0.98 f 0.14 mM and k,,, = 13200 f 880 s-l; for recombinant human cyclophilin, K,,, = 0.87 f 0.084 mM and kat = 12700 f 550 s-l. The kinetic parameters for Suc-Ala-Leu-cis-Pro-Phe-pNA with FKBP are K, = 0.520 f 0.08 mM and kat = 344 f 26 s-'. We also demonstrate that ((Boc)DabI8-CsA, a cyclosporin A analogue, is a tight-binding, slow-binding inhibitor of cyclophilin and that another cyclosporin A analogue, (MeSBth)'-CsA, is a competitive inhibitor of the same enzyme.

Figure 3. Top: One-dimensional 13C-{ !HI N O E difference experiment as measured overnight on a vancomycin/Ac-D-Ala-o-Ala complex at 50.3 MHz. A recycle delay of 4 s was inserted to allow a complete decay of heteronuclear NOE built up during a 0.5-s (20 dB below 0.2 W decoupler power) presaturation of the 1-NH signal a t 11.6 ppm. Bottom: Reference "C N M R spectrum of the complex. The strong sharp signal a t 174 ppm corresponds to C-1 in the free state, while the smaller broad peak a t 175.3 ppm represents the C-1 atom in bonded Ac-D-Ala-o-Ala.