A Kinetic Alignment of Orthologous Inosine-5′-monophosphate Dehydrogenases

IMP dehydrogenase (IMPDH) catalyzes two very different chemical transformations, a dehydrogenase reaction and a hydrolysis reaction. The enzyme toggles between the open conformation required for the dehydrogenase reaction and the closed conformation of the hydrolase reaction by moving a mobile flap into the NAD site. Despite these multiple functional constraints, the residues of the flap and NAD site are highly diverged, and the equilibrium between open and closed conformations (<i>K</i>_<i>c</i>) varies widely. In order to understand how differences in the dynamic properties of the flap influence the catalytic cycle, we have delineated the kinetic mechanism of IMPDH from the pathogenic protozoan parasite <i>Cryptosporidium parvum</i> (<i>Cp</i>IMPDH), which was obtained from a bacterial source through horizontal gene transfer, and its host counterpart, human IMPDH type 2 (hIMPDH2). Interestingly, the intrinsic binding energy of NAD⁺ differentially distributes across the dinucleotide binding sites of these two enzymes as well as in the previously characterized IMPDH from <i>Tritrichomonas foetus</i> (<i>Tf</i>IMPDH). Both the dehydrogenase and hydrolase reactions display significant differences in the host and parasite enzymes, in keeping with the phylogenetic and structural divergence of their active sites. Despite large differences in <i>K</i>_<i>c</i>, the catalytic power of both the dehydrogenase and hydrolase conformations are similar in <i>Cp</i>IMPDH and <i>Tf</i>IMPDH. This observation suggests that the closure of the flap simply sets the stage for catalysis rather than plays a more active role in the chemical transformation. This work provides the essential mechanistic framework for drug discovery

Dynamic Characteristics of Guanosine-5′-monophosphate Reductase Complexes Revealed by High-Resolution ³¹P Field-Cycling NMR Relaxometry

The ability of enzymes to modulate the dynamics of bound substrates and cofactors is a critical feature of catalysis, but the role of dynamics has largely been approached from the perspective of the protein. Here, we use an underappreciated NMR technique, subtesla high-resolution field-cycling ³¹P NMR relaxometry, to interrogate the dynamics of enzyme bound substrates and cofactors in guanosine-5′-monophosphate reductase (GMPR). These experiments reveal distinct binding modes and dynamic profiles associated with the ³¹P nuclei in the Michaelis complexes for the deamination and hydride transfer steps of the catalytic cycle. Importantly, the substrate is constrained and the cofactor is more dynamic in the deamination complex E·GMP·NADP⁺, whereas the substrate is more dynamic and the cofactor is constrained in the hydride transfer complex E·IMP·NADP⁺. The presence of D₂O perturbed the relaxation of the ³¹P nuclei in E·IMP·NADP⁺ but not in E·GMP·NADP⁺, providing further evidence of distinct binding modes with different dynamic properties. dIMP and dGMP are poor substrates, and the dynamics of the cofactor complexes of dGMP/dIMP are disregulated relative to GMP/IMP. The substrate 2’-OH interacts with Asp219, and mutation of Asp219 to Ala decreases the value of <i>V</i>_max by a factor of 30. Counterintuitively, loss of Asp219 makes both substrates and cofactors less dynamic. These observations suggest that the interactions between the substrate 2’-OH and Asp219 coordinate the dynamic properties of the Michaelis complexes, and these dynamics are important for progression through the catalytic cycle

Inhibition of Inosine-5′-monophosphate Dehydrogenase from <i>Bacillus anthracis</i>: Mechanism Revealed by Pre-Steady-State Kinetics

Inosine-5′-monophosphate dehydrogenase (IMPDH) catalyzes the conversion of inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP). The enzyme is an emerging target for antimicrobial therapy. The small molecule inhibitor <b>A110</b> has been identified as a potent and selective inhibitor of IMPDHs from a variety of pathogenic microorganisms. A recent X-ray crystallographic study reported that the inhibitor binds to the NAD⁺ cofactor site and forms a ternary complex with IMP. Here we report a pre-steady-state stopped-flow kinetic investigation of IMPDH from <i>Bacillus anthracis</i> designed to assess the kinetic significance of the crystallographic results. Stopped-flow kinetic experiments defined nine microscopic rate constants and two equilibrium constants that characterize both the catalytic cycle and details of the inhibition mechanism. In combination with steady-state initial rate studies, the results show that the inhibitor binds with high affinity (<i>K</i>_d ≈ 50 nM) predominantly to the covalent intermediate on the reaction pathway. Only a weak binding interaction (<i>K</i>_d ≈ 1 μM) is observed between the inhibitor and E·IMP. Thus, the E·IMP·<b>A110</b> ternary complex, observed by X-ray crystallography, is largely kinetically irrelevant

Boc₃Arg-Linked Ligands Induce Degradation by Localizing Target Proteins to the 20S Proteasome

Targeted protein degradation is a promising strategy for drug design and functional assessment. Several small molecule approaches have been developed that localize target proteins to ubiquitin ligases, inducing ubiquitination and subsequent degradation by the 26S proteasome. We discovered that the degradation of a target protein can also be induced by a recognition ligand linked to <i>tert</i>-butyl carbamate (Boc₃)-protected arginine (B₃A). Here, we show that this process requires the proteasome but does not involve ubiquitination of the target protein. B₃A does not perturb the structure of the target protein; instead, a B₃A-ligand stabilizes its target protein. B₃A ligands stimulate activity of purified 20S proteasome, demonstrating that the tag binds directly to the 20S proteasome. Moreover, purified 20S proteasome is sufficient to degrade target proteins in the presence of their respective B₃A-linked recognition ligands. These observations suggest a simple model for B₃A-mediated degradation wherein the B₃A tag localizes target proteins directly to the 20S proteasome. Thus, B₃A ligands are the first example of a ubiquitin-free strategy for targeted protein degradation

Selective and Potent Urea Inhibitors of Cryptosporidium parvum Inosine 5′-Monophosphate Dehydrogenase

Cryptosporidium parvum and related species are zoonotic intracellular parasites of the intestine. Cryptosporidium is a leading cause of diarrhea in small children around the world. Infection can cause severe pathology in children and immunocompromised patients. This waterborne parasite is resistant to common methods of water treatment and therefore a prominent threat to drinking and recreation water even in countries with strong water safety systems. The drugs currently used to combat these organisms are ineffective. Genomic analysis revealed that the parasite relies solely on inosine-5′-monophosphate dehydrogenase (IMPDH) for the biosynthesis of guanine nucleotides. Herein, we report a selective urea-based inhibitor of C. parvum IMPDH (<i>Cp</i>IMPDH) identified by high-throughput screening. We performed a SAR study of these inhibitors with some analogues exhibiting high potency (IC₅₀ < 2 nM) against <i>Cp</i>IMPDH, excellent selectivity >1000-fold versus human IMPDH type 2 and good stability in mouse liver microsomes. A subset of inhibitors also displayed potent antiparasitic activity in a Toxoplasma gondii model

Optimization of Benzoxazole-Based Inhibitors of <i>Cryptosporidium parvum</i> Inosine 5′-Monophosphate Dehydrogenase

<i>Cryptosporidium parvum</i> is an enteric protozoan parasite that has emerged as a major cause of diarrhea, malnutrition, and gastroenteritis and poses a potential bioterrorism threat. <i>C. parvum</i> synthesizes guanine nucleotides from host adenosine in a streamlined pathway that relies on inosine 5′-monophosphate dehydrogenase (IMPDH). We have previously identified several parasite-selective <i>C. parvum</i> IMPDH (<i>Cp</i>IMPDH) inhibitors by high-throughput screening. In this paper, we report the structure–activity relationship (SAR) for a series of benzoxazole derivatives with many compounds demonstrating <i>Cp</i>IMPDH IC₅₀ values in the nanomolar range and >500-fold selectivity over human IMPDH (hIMPDH). Unlike previously reported <i>Cp</i>IMPDH inhibitors, these compounds are competitive inhibitors versus NAD⁺. The SAR study reveals that pyridine and other small heteroaromatic substituents are required at the 2-position of the benzoxazole for potent inhibitory activity. In addition, several other SAR conclusions are highlighted with regard to the benzoxazole and the amide portion of the inhibitor, including preferred stereochemistry. An X-ray crystal structure of a representative E·IMP·inhibitor complex is also presented. Overall, the secondary amine derivative <b>15a</b> demonstrated excellent <i>Cp</i>IMPDH inhibitory activity (IC₅₀ = 0.5 ± 0.1 nM) and moderate stability (<i>t</i>_1/2 = 44 min) in mouse liver microsomes. Compound <b>73</b>, the racemic version of <b>15a</b>, also displayed superb antiparasitic activity in a <i>Toxoplasma gondii</i> strain that relies on <i>Cp</i>IMPDH (EC₅₀ = 20 ± 20 nM), and selectivity versus a wild-type <i>T. gondii</i> strain (200-fold). No toxicity was observed (LD₅₀ > 50 μM) against a panel of four mammalian cells lines

Synthesis, in Vitro Evaluation and Cocrystal Structure of 4‑Oxo-[1]benzopyrano[4,3‑<i>c</i>]pyrazole Cryptosporidium parvum Inosine 5′-Monophosphate Dehydrogenase (<i>Cp</i>IMPDH) Inhibitors

Cryptosporidium inosine 5′-monophosphate dehydrogenase (<i>Cp</i>IMPDH) has emerged as a therapeutic target for treating Cryptosporidium parasites because it catalyzes a critical step in guanine nucleotide biosynthesis. A 4-oxo-[1]­benzopyrano­[4,3-<i>c</i>]­pyrazole derivative was identified as a moderately potent (IC₅₀ = 1.5 μM) inhibitor of <i>Cp</i>IMPDH. We report a SAR study for this compound series resulting in <b>8k</b> (IC₅₀ = 20 ± 4 nM). In addition, an X-ray crystal structure of <i>Cp</i>IMPDH·IMP·<b>8k</b> is also presented

Lionello Venturi e le polemiche sull’arte astratta in Italia alla metà del XX secolo.

Storia, critica dell'arte e politica nell'Italia del secondo dopoguerra

<i>Mycobacterium tuberculosis</i> IMPDH in Complexes with Substrates, Products and Antitubercular Compounds

<div><p>Tuberculosis (TB) remains a worldwide problem and the need for new drugs is increasingly more urgent with the emergence of multidrug- and extensively-drug resistant TB. Inosine 5’-monophosphate dehydrogenase 2 (IMPDH2) from <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) is an attractive drug target. The enzyme catalyzes the conversion of inosine 5’-monophosphate into xanthosine 5’-monophosphate with the concomitant reduction of NAD⁺ to NADH. This reaction controls flux into the guanine nucleotide pool. We report seventeen selective IMPDH inhibitors with antitubercular activity. The crystal structures of a deletion mutant of <i>Mtb</i>IMPDH2 in the apo form and in complex with the product XMP and substrate NAD⁺ are determined. We also report the structures of complexes with IMP and three structurally distinct inhibitors, including two with antitubercular activity. These structures will greatly facilitate the development of <i>Mtb</i>IMPDH2-targeted antibiotics.</p></div

Expanding Benzoxazole-Based Inosine 5′-Monophosphate Dehydrogenase (IMPDH) Inhibitor Structure–Activity As Potential Antituberculosis Agents

New drugs and molecular targets are urgently needed to address the emergence and spread of drug-resistant tuberculosis. <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) inosine 5′-monophosphate dehydrogenase 2 (<i>Mtb</i>IMPDH2) is a promising yet controversial potential target. The inhibition of <i>Mtb</i>IMPDH2 blocks the biosynthesis of guanine nucleotides, but high concentrations of guanine can potentially rescue the bacteria. Herein we describe an expansion of the structure–activity relationship (SAR) for the benzoxazole series of <i>Mtb</i>IMPDH2 inhibitors and demonstrate that minimum inhibitory concentrations (MIC) of ≤1 μM can be achieved. The antibacterial activity of the most promising compound, <b>17b</b> (<b>Q151</b>), is derived from the inhibition of <i>Mtb</i>IMPDH2 as demonstrated by conditional knockdown and resistant strains. Importantly, guanine does not change the MIC of <b>17b</b>, alleviating the concern that guanine salvage can protect <i>Mtb</i> in vivo. These findings suggest that <i>Mtb</i>IMPDH2 is a vulnerable target for tuberculosis