Novel cytomegalovirus-inhibitory compounds of the class pyrrolopyridines show a complex pattern of target binding that suggests an unusual mechanism of antiviral activity.

Human cytomegalovirus (HCMV) is a major human pathogen with seropositivity rates in the adult population ranging between 40% and 95%. HCMV infection is associated with severe pathology, such as life-threatening courses of infection in immunocompromised individuals and neonates. Current standard therapy with valganciclovir has the disadvantage of adverse side effects and viral drug resistance. A novel anti-HCMV drug, letermovir, has been approved recently, so that improved therapy options are available. Nevertheless, even more so far unexploited classes of compounds and molecular modes of action will be required for a next generation of antiherpesviral treatment strategies. In this study, we focused on the analysis of the antiviral potency of a novel class of compounds, i.e. pyrrolopyridine analogs, and identified both hit compounds and their target protein candidates. In essence, we provide novel evidence as follows: (i) screening hit SC88941 is highly active in inhibiting HCMV replication in primary human fibroblasts with an EC50 value of 0.20 ±â€¯0.01 µM in the absence of cytotoxicity, (ii) inhibition occurs at the early-late stage of viral protein production and shows reinforcing effects upon LMV cotreatment, (iii) among the viruses analyzed, antiviral activity was most pronounced against ß-herpesviruses (HCMV, HHV-6A) and intermediate against adenovirus (HAdV-2), (iv) induction of SC88941 resistance was not detectable, thus differed from the induction of ganciclovir resistance, (v) a linker-coupled model compound was used for mass spectrometry-based target identification, thus yielding several drug-binding target proteins and (vi) a first confocal imaging approach used for addressing intracellular effects of SC88941 indicated qualitative and quantitative alteration of viral protein expression and localization. Thus, our findings suggest a multifaceted pattern of compound-target binding in connection with an unusual mode of action, opening up further opportunities of antiviral drug development.

Inhibition of human mu-calpain by conformationally constrained calpastatin peptides.

The 27-mer peptide CP1B-[1-27] derived from exon 1B of calpastatin stands out among the known inhibitors for mu- and m-calpain due to its high potency and selectivity. By systematical truncation, a 20-mer peptide, CP1B-[4-23], was identified as the core sequence required to maintain the affinity/selectivity profile of CP1B-[1-27]. Starting with this peptide, the turn-like region Glu(10)(i)-Leu(11)(i+1)-Gly(12)(i+2)-Lys(13)(i+3) was investigated. Sequence alignment of subdomains 1B, 2B, 3B and 4B from different mammalians revealed that the amino acid residues in position i+1 and i+2 are almost invariably flanked by oppositely charged residues, pointing towards a turn-like conformation stabilized by salt bridge/H-bond interaction. Accordingly, using different combinations of acidic and basic residues in position i and i+3, a series of conformationally constrained variants of CP1B-[4-23] were synthesized by macrolactamization utilizing the side chain functionalities of these residues. With the combination of Glu(i)/Dab(i+3), the maximum of conformational rigidity without substantial loss in affinity/selectivity was reached. These results clearly demonstrate that the linear peptide chain corresponding to subdomain 1B reverses its direction in the region Glu(10)-Lys(13) upon binding to mu-calpain, and thereby adopts a loop-like rather than a tight turn conformation at this site.

Enantioselective synthesis of non-natural aromatic alpha-amino acids.

We present two complementary methods for the stereoselective synthesis of non-natural alpha-amino acids with aromatic or heteroaromatic side chains. One approach is based on the chemical transformation of methionine, whereas the other applies the stereoselective Myers alkylation of glycine. The resulting product types differ in the linker length between glycine and the aromatic substituent. Since methionine and pseudoephedrine are available in both absolute configurations, R- or S-configured enantiopure amino acids with either C(2) or C(3) linkers can be obtained on gram scales. In each case the key step of the synthesis is hydroboration of the unsaturated building blocks 9 and 17, followed by palladium-catalyzed Suzuki cross-coupling with aryl halides. Attention must in certain cases be paid to the stereochemical integrity when basic Suzuki conditions are applied. Our initial difficulties are reported as well as the final "racemization-proof" procedures. The protecting groups chosen for the alpha-amino acids should be compatible with solid-phase peptide synthesis. This was confirmed by the successful synthesis of a series of tripeptides.