The development of therapies for the treatment of neurological cancer faces a number of major challenges including the synthesis of small molecule agents that can penetrate the blood-brain barrier ...(BBB). Given the likelihood that in many cases drug exposure will be lower in the CNS than in systemic circulation, it follows that strategies should be employed that can sustain target engagement at low drug concentration. Time dependent target occupancy is a function of both the drug and target concentration as well as the thermodynamic and kinetic parameters that describe the binding reaction coordinate, and sustained target occupancy can be achieved through structural modifications that increase target (re)binding and/or that decrease the rate of drug dissociation. The discovery and deployment of compounds with optimized kinetic effects requires information on the structure-kinetic relationships that modulate the kinetics of binding, and the molecular factors that control the translation of drug-target kinetics to time-dependent drug activity in the disease state. This Review first introduces the potential benefits of drug-target kinetics, such as the ability to delineate both thermodynamic and kinetic selectivity, and then describes factors, such as target vulnerability, that impact the utility of kinetic selectivity. The Review concludes with a description of a mechanistic PK/PD model that integrates drug-target kinetics into predictions of drug activity.
Failure due to poor
in vivo efficacy is a primary contributor to attrition during the development of new chemotherapeutics. Lead optimization programs that in their quest for efficacy focus solely on ...improving the affinity of drug–target binding are flawed, since this approach ignores the fluctuations in drug concentration that occur
in vivo. Instead the lifetime of the drug–target complex must also be considered, since drugs only act when they are bound to their targets. Consequently, to improve the correlation between the
in vitro and
in vivo activity of drugs, measurements of drug–target residence time must be incorporated into the drug discovery process.
The bacterial type II fatty acid biosynthesis (FASII) pathway is an essential but unexploited target for drug discovery. In this review we summarize SAR studies on inhibitors of InhA, the enoyl-ACP ...reductase from the FASII pathway in M. tuberculosis. Inhibitor scaffolds that are described include the diaryl ethers, pyrrolidine carboxamides, piperazine indoleformamides, pyrazoles, arylamides, fatty acids and imidazopiperidines, all of which form ternary complexes with InhA and the NAD cofactor, as well as isoniazid and the diazaborines which covalently modify the cofactor. Analysis of the structural data has enabled the development of a common binding mode for the ternary complex inhibitors, which includes a hydrogen bond network, a large hydrophobic pocket and a third ' size-limited' binding area comprised of both polar and non-polar groups. A critical factor in InhA inhibition involves ordering of the substrate binding loop, located close to the active site, and a direct link is proposed between loop ordering and slow onset enzyme inhibition. Slow onset inhibitors have long residence times on the enzyme target, a property that is of critical importance for in vivo activity.
•PK/PD models that integrate drug–target kinetics.•Predictions of drug activity in the non-equilibrium environment of the human body.•Inclusion of the rate of target turnover in modeling and ...generation of target vulnerability functions.
Pharmacokinetic/pharmacodynamic (PK/PD) models predict the effect time course resulting from a drug dose. In this review, we summarize the development of mechanistic PK/PD models that explicitly integrate the kinetics of drug–target interactions into predictions of drug activity. Such mechanistic models are expected to have several advantages over approaches in which concentration and effect are linked using variations of the Hill equation, and where preclinical data are often used as a starting point for modeling drug activity. Instead, explicit use of the full kinetic scheme for drug binding enables time-dependent changes in target occupancy to be calculated using the kinetics of drug–target interactions and drug PK, providing a more precise picture of target engagement and drug action in the non-equilibrium environment of the human body. The mechanistic PK/PD models also generate target vulnerability functions that link target occupancy and effect, and inform on the sensitivity of a target to engagement by a drug. Key factors such as the rate of target turnover can also be integrated into the modeling which, together with target vulnerability, provide additional information on the PK profile required to achieve the desired pharmacological effect and on the utility of kinetic selectivity in developing drugs for specific targets.
The modern age of drug discovery, which had been slowly gathering momentum during the early part of the twentieth century, exploded into life in the 1940s with the isolation of penicillin and ...streptomycin. The immense success of these early drug discovery efforts prompted the general view that many infectious diseases would now be effectively controlled and even eradicated. However this initial optimism was misplaced, and pathogens such as multidrug-resistant Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus present a major current threat to human health. Drug resistance arises through the unrelenting pressure of natural selection, and there is thus a continuing need to identify novel drug targets and develop chemotherapeutics that circumvent existing drug resistance mechanisms. In this Account, we summarize current progress in developing inhibitors of FabI, the NADH-dependent enoyl reductase from the type II bacterial fatty acid biosynthesis pathway (FAS-II), a validated but currently underexploited target for drug discovery. The FabI inhibitors have been divided into two groups, based on whether they form a covalent adduct with the NAD (+) cofactor. Inhibitors that form a covalent adduct include the diazaborines, as well as the front-line tuberculosis drug isoniazid. The NAD adducts formed with these compounds are formally bisubstrate enzyme inhibitors, and we summarize progress in developing novel leads based on these pharmacophores. Inhibitors that do not form covalent adducts form a much larger group, although generally these compounds also require the cofactor to be bound to the enzyme. Using structure-based approaches, we have developed a series of alkyl diphenyl ethers that are nanomolar inhibitors of InhA, the FabI from M. tuberculosis, and that are active against INH-resistant strains of M. tuberculosis. This rational approach to inhibitor development is based on the proposal that high-affinity inhibition of the FabI enzymes is coupled to the ordering of a loop of amino acids close to the active site. Compounds that promote loop ordering are slow onset FabI inhibitors with increased residence time on the enzyme. The diphenyl ether skeleton has also been used as a framework by us and others to develop potent inhibitors of the FabI enzymes from other pathogens such as Escherichia coli, S. aureus, and Plasmodium falciparum. Meanwhile chemical optimization of compounds identified in high-throughput screening programs has resulted in the identification of several classes of heteroaromatic FabI inhibitors with potent activity both in vitro and in vivo. Finally, screening of natural product libraries may provide useful chemical entities for the development of novel agents with low toxicity. While the discovery that not all pathogens contain FabI homologues has led to reduced industrial interest in FabI as a broad spectrum target, there is substantial optimism that FabI inhibitors can be developed for disease-specific applications. In addition, the availability of genome sequencing data, improved methods for target identification and validation, and the development of novel approaches for determining the mode of action of current drugs will all play critical roles in the road ahead and in exploiting other components of the FAS-II pathway.
The accurate and precise determination of binding interactions plays a central role in fields such as drug discovery where structure-activity relationships guide the selection and optimization of ...drug leads. Binding is often assessed by monitoring the response caused by varying one of the binding partners in a functional assay or by using methods where the concentrations of free and/or bound ligand can be directly determined. In addition, there are also many approaches where binding leads to a change in the properties of the binding partner(s) that can be directly quantified such as an alteration in mass or in a spectroscopic signal. The analysis of data resulting from these techniques invariably relies on computer software that enable rapid fitting of the data to nonlinear multiparameter equations. The objective of this Perspective is to serve as a reminder of the basic assumptions that are used in deriving these equations and thus that should be considered during assay design and subsequent data analysis. The result is a set of guidelines for authors considering submitting their work to journals such as ACS Infectious Diseases.
Light activated proteins are at the heart of photobiology and optogenetics, so there is wide interest in understanding the mechanisms coupling optical excitation to protein function. In addition, ...such light activated proteins provide unique insights into the real-time dynamics of protein function. Using pump-probe spectroscopy, the function of a photoactive protein can be initiated by a sub-100 fs pulse of light, allowing subsequent protein dynamics to be probed from femtoseconds to milliseconds and beyond. Among the most interesting photoactive proteins are the blue light using flavin (BLUF) domain proteins, which regulate the response to light of a wide range of bacterial and some euglenoid processes. The photosensing mechanism of BLUF domains has long been a subject of debate. In contrast to other photoactive proteins, the electronic and nuclear structure of the chromophore (flavin) is the same in dark- and light-adapted states. Thus, the driving force for photoactivity is unclear.To address this question requires real-time observation of both chromophore excited state processes and their effect on the structure and dynamics of the surrounding protein matrix. In this Account we describe how time-resolved infrared (IR) experiments, coupled with chemical biology, provide important new insights into the signaling mechanism of BLUF domains. IR measurements are sensitive to changes in both chromophore electronic structure and protein hydrogen bonding interactions. These contributions are resolved by isotope labeling of the chromophore and protein separately. Further, a degree of control over BLUF photochemistry is achieved through mutagenesis, while unnatural amino acid substitution allows us to both fine-tune the photochemistry and time resolve protein dynamics with spatial resolution.Ultrafast studies of BLUF domains reveal non-single-exponential relaxation of the flavin excited state. That relaxation leads within one nanosecond to the original flavin ground state bound in a modified hydrogen-bonding network, as seen in transient and steady-state IR spectroscopy. The change in H-bond configuration arises from formation of an unusual enol (imine) form of a critical glutamine residue. The dynamics observed, complemented by quantum mechanical calculations, suggest a unique sequential electron then double proton transfer reaction as the driving force, followed by rapid reorganization in the binding site and charge recombination. Importantly, studies of several BLUF domains reveal an unexpected diversity in their dynamics, although the underlying structure appears highly conserved. It is suggested that this diversity reflects structural dynamics in the ground state at standard temperature, leading to a distribution of structures and photochemical outcomes. Time resolved IR measurements were extended to the millisecond regime for one BLUF domain, revealing signaling state formation on the microsecond time scale. The mechanism involves reorganization of a β-sheet connected to the chromophore binding pocket via a tryptophan residue. The potential of site-specific labeling amino acids with IR labels as a tool for probing protein structural dynamics was demonstrated.In summary, time-resolved IR studies of BLUF domains (along with related studies at visible wavelengths and quantum and molecular dynamics calculations) have resolved the photoactivation mechanism and real-time dynamics of signaling state formation. These measurements provide new insights into protein structural dynamics and will be important in optimizing the potential of BLUF domains in optobiology.
The rise of multi-drug resistant (MDR) and extensively drug resistant (XDR) tuberculosis around the world, including in industrialized nations, poses a great threat to human health and defines a need ...to develop new, effective and inexpensive anti-tubercular agents. Previously we developed a chemical systems biology approach to identify off-targets of major pharmaceuticals on a proteome-wide scale. In this paper we further demonstrate the value of this approach through the discovery that existing commercially available drugs, prescribed for the treatment of Parkinson's disease, have the potential to treat MDR and XDR tuberculosis. These drugs, entacapone and tolcapone, are predicted to bind to the enzyme InhA and directly inhibit substrate binding. The prediction is validated by in vitro and InhA kinetic assays using tablets of Comtan, whose active component is entacapone. The minimal inhibition concentration (MIC(99)) of entacapone for Mycobacterium tuberculosis (M.tuberculosis) is approximately 260.0 microM, well below the toxicity concentration determined by an in vitro cytotoxicity model using a human neuroblastoma cell line. Moreover, kinetic assays indicate that Comtan inhibits InhA activity by 47.0% at an entacapone concentration of approximately 80 microM. Thus the active component in Comtan represents a promising lead compound for developing a new class of anti-tubercular therapeutics with excellent safety profiles. More generally, the protocol described in this paper can be included in a drug discovery pipeline in an effort to discover novel drug leads with desired safety profiles, and therefore accelerate the development of new drugs.
Isoniazid (INH), a frontline antitubercular drug, inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis, by forming a covalent adduct with the NAD cofactor. Here, we report that the ...INH-NAD adduct is a slow, tight-binding competitive inhibitor of InhA. Demonstration that the adduct binds to WT InhA by a two-step enzyme inhibition mechanism, with initial, weak binding$(K_{-1} = 16 \pm 11\>nM)$followed by slow conversion to a final inhibited complex (EI*) with overall$K_i = 0.75 \pm 0.08\>nM$, reconciles existing contradictory values for the inhibitory potency of INH-NAD for InhA. The first order rate constant for conversion of the initial EI complex to EI* (k2= 0.13 ± 0.01 min-1) is similar to the maximum rate constant observed for InhA inhibition in reaction mixtures containing InhA, INH, NADH, and the INH-activating enzyme KatG (catalase/peroxidase from M. tuberculosis), consistent with an inhibition mechanism in which the adduct forms in solution rather than on the enzyme. Importantly, three mutations that correlate with INH resistance, 121V, 147T, and S94A, have little impact on the inhibition constants. Thus, drug resistance does not result simply from a reduction in affinity of INH-NAD for pure InhA. Instead, we hypothesize that protein-protein interactions within the FASII complex are critical to the mechanism of INH action. Finally, for M161V, an InhA mutation that correlates with resistance to the common biocide triclosan in Mycobacterium smegmatis, binding to form the initial EI complex is significantly weakened, explaining why this mutant inactivates more slowly than WT InhA when incubated with INH, NADH, and KatG.
Methicillin-resistant Staphylococcus aureus (MRSA) infections constitute a serious health threat worldwide, and novel antibiotics are therefore urgently needed. The enoyl-ACP reductase (saFabI) is ...essential for the S. aureus fatty acid biosynthesis and, hence, serves as an attractive drug target. We have obtained a series of snapshots of this enzyme that provide a mechanistic picture of ligand and inhibitor binding, including a dimer-tetramer transition combined with extensive conformational changes. Significantly, our results reveal key differences in ligand binding and recognition compared to orthologous proteins. The remarkable observed protein flexibility rationalizes our finding that saFabI is capable of efficiently reducing branched-chain fatty acid precursors. Importantly, branched-chain fatty acids represent a major fraction of the S. aureus cell membrane and are crucial for its in vivo fitness. Our discovery thus addresses a long-standing controversy regarding the essentiality of the fatty acid biosynthesis pathway in S. aureus rationalizing saFabI as a drug target.
Display omitted
► Insights into saFabI ligand binding including a dimer-tetramer transition ► Identification of a loop motif that determines altered cofactor specificity ► Increased flexibility modulates substrate and inhibitor recognition ► Ability of saFabI to reduce branched-chain fatty acid precursor molecules
PDF
