Structure-Guided, Single-Point Modifications in the Phosphinic Dipeptide Structure Yield Highly Potent and Selective Inhibitors of Neutral Aminopeptidases

Seven crystal structures of alanyl aminopeptidase from <i>Neisseria meningitides</i> (the etiological agent of meningitis, <i>Nm</i>APN) complexed with organophosphorus compounds were resolved to determine the optimal inhibitor–enzyme interactions. The enantiomeric phosphonic acid analogs of Leu and hPhe, which correspond to the P1 amino acid residues of well-processed substrates, were used to assess the impact of the absolute configuration and the stereospecific hydrogen bond network formed between the aminophosphonate polar head and the active site residues on the binding affinity. For the hPhe analog, an imperfect stereochemical complementarity could be overcome by incorporating an appropriate P1 side chain. The constitution of P1′-extended structures was rationally designed and the lead, phosphinic dipeptide hPhePψ­[CH₂]­Phe, was modified in a single position. Introducing a heteroatom/heteroatom-based fragment to either the P1 or P1′ residue required new synthetic pathways. The compounds in the refined structure were low nanomolar and subnanomolar inhibitors of <i>N. meningitides</i>, porcine and human APNs, and the reference leucine aminopeptidase (LAP). The unnatural phosphinic dipeptide analogs exhibited a high affinity for monozinc APNs associated with a reasonable selectivity versus dizinc LAP. Another set of crystal structures containing the <i>Nm</i>APN dipeptide ligand were used to verify and to confirm the predicted binding modes; furthermore, novel contacts, which were promising for inhibitor development, were identified, including a π–π stacking interaction between a pyridine ring and Tyr372

<i>Bacillus anthracis</i> Inosine 5′-Monophosphate Dehydrogenase in Action: The First Bacterial Series of Structures of Phosphate Ion‑, Substrate‑, and Product-Bound Complexes

Inosine 5′-monophosphate dehydrogenase (IMPDH) catalyzes the first unique step of the GMP branch of the purine nucleotide biosynthetic pathway. This enzyme is found in organisms of all three kingdoms. IMPDH inhibitors have broad clinical applications in cancer treatment, as antiviral drugs and as immunosuppressants, and have also displayed antibiotic activity. We have determined three crystal structures of <i>Bacillus anthracis</i> IMPDH, in a phosphate ion-bound (termed “apo”) form and in complex with its substrate, inosine 5′-monophosphate (IMP), and product, xanthosine 5′-monophosphate (XMP). This is the first example of a bacterial IMPDH in more than one state from the same organism. Furthermore, for the first time for a prokaryotic enzyme, the entire active site flap, containing the conserved Arg-Tyr dyad, is clearly visible in the structure of the apoenzyme. Kinetic parameters for the enzymatic reaction were also determined, and the inhibitory effect of XMP and mycophenolic acid (MPA) has been studied. In addition, the inhibitory potential of two known <i>Cryptosporidium parvum</i> IMPDH inhibitors was examined for the <i>B. anthracis</i> enzyme and compared with those of three bacterial IMPDHs from <i>Campylobacter jejuni</i>, <i>Clostridium perfringens</i>, and <i>Vibrio cholerae</i>. The structures contribute to the characterization of the active site and design of inhibitors that specifically target <i>B. anthracis</i> and other microbial IMPDH enzymes