The ectodomain of HIV-1 gp41 mediates the fusion of viral and host cellular membranes. The peptide-based drug Enfuvirtide1 is precedent that antagonists of this fusion activity may act as anti HIV-agents. Here, NMR screening was used to discover non-peptide leads against this target and resulted in the discovery of a new benzamide 1 series. This series is non-peptide, low molecular weight, and analogs have activity in a cell fusion assay with EC50 values ranging 3–41μM. Structural work on the gp41/benzamide 1 complex was determined by NMR spectroscopy using a designed model peptide system that mimics an open pocket of the fusogenic form of the protein.

IV. Effects of Exper imenta l Errors o n the Resul t s from a C o m p l e t e Re laxat ion R a t e Matr ix Analys is of the N O E S Y Data 31 A. NOESY simulations . . . .... . . . . . . )\ . . . 31 B. Models . . . . . . . . . . . , . . . . . . . . . . . . . . 31 1. Noise-free data . . . . .... . . . 32 2. Random noise . . . . . . . . . . . 33 3. Peak overlap and incomplete NOESY data . . . . . . . . . . . . . . . . . . . . . . . . . 34

Potent inhibitors of 7,8-dihydroneopterin aldolase (DHNA; EC 4.1.2.25) have been discovered using CrystaLEAD X-ray crystallographic high-throughput screening followed by structure-directed optimization. Screening of a 10 000 compound random library provided several low affinity leads and their corresponding X-ray crystal structures bound to the enzyme. The presence of a common structural feature in each of the leads suggested a strategy for the construction of a directed library of approximately 1000 compounds that were screened for inhibitory activity in a traditional enzyme assay. Several lead compounds with IC(50) values of about 1 microM against DHNA were identified, and crystal structures of their enzyme-bound complexes were obtained by cocrystallization. Structure-directed optimization of one of the leads thus identified afforded potent inhibitors with submicromolar IC(50) values.

Non-peptidic small molecule SMAC mimetics were designed and synthesized that bind to the BIR3 domain of XIAP using structure-based design. Substituted five-membered heterocycles such as thiazoles and imidazoles were identified that serve as replacements for peptide fragments of the lead.

The NMR-based discovery of biaryl hydroxamate inhibitors of the matrix metalloproteinase stromelysin (MMP-3) has been previously described (Hajduk et al. J. Am. Chem. Soc. 1997, 119, 5818-5827). While potent in vitro, these inhibitors exhibited no in vivo activity due, at least in part, to the poor pharmacokinetic properties of the alkylhydroxamate moiety. To circumvent this liability, NMR-based screening was implemented to identify alternative zinc-chelating groups. Using this technique, 1-naphthyl hydroxamate was found to bind tightly to the protein (K(D) = 50 microM) and was identified as a candidate for incorporation into the lead series. On the basis of NMR-derived structural information, the naphthyl hydroxamate and biaryl fragments were linked together to yield inhibitors of this enzyme that exhibited improved bioavailability. These studies demonstrate that the NMR-based screening of fragments can be effectively applied to improve the physicochemical or pharmacokinetic profile of lead compounds.

The inhibitor-of-apoptosis proteins (IAPs)1 regulate programmed cell death by inhibiting members of the caspase family of enzymes2,3,4,5. Recently, a mammalian protein called Smac6 (also named DIABLO7) was identified that binds to the IAPs and promotes caspase activation. Although undefined in the X-ray structure, the amino-terminal residues of Smac are critical for its function8,9. To understand the structural basis for molecular recognition between Smac and the IAPs, we determined the solution structure of the BIR3 domain of X-linked IAP (XIAP) complexed with a functionally active nine-residue peptide derived from the N terminus of Smac. The peptide binds across the third β-strand of the BIR3 domain in an extended conformation with only the first four residues contacting the protein. The complex is stabilized by four intermolecular hydrogen bonds, an electrostatic interaction involving the N terminus of the peptide, and several hydrophobic interactions. This structural information, along with the binding data from BIR3 and Smac peptide mutants reported here, should aid in the design of small molecules that may be used for the treatment of cancers that overexpress IAPs10,11,12.

An approach is described for rapidly determining protein structures by NMR that utilizes proteins containing 13C-methyl labeled Val, Leu, and Ile (δ1) and protonated Phe and Tyr in a deuterated background. Using this strategy, the key NOEs that define the hydrophobic core and overall fold of the protein are easily obtained. NMR data are acquired using cryogenic probe technology which markedly reduces the spectrometer time needed for data acquisition. The approach is demonstrated by determining the overall fold of the antiapoptotic protein, Bcl-xL, from data collected in only 4 days. Refinement of the Bcl-xL structure to a backbone rmsd of 0.95 Å was accomplished with data collected in an additional 3 days. A distance analysis of 180 different proteins and structure calculations using simulated data suggests that our method will allow the global folds of a wide variety of proteins to be determined.

The three‐dimensional structure of the anti‐apoptotic protein Bcl‐X L complexed to a 25‐residue peptide from the death promoting region of Bad was determined using NMR spectroscopy. Although the overall structure is similar to Bcl‐X L bound to a 16‐residue peptide from the Bak protein (Sattler et al., 1997), the Bad peptide forms additional interactions with Bcl‐X L . However, based upon site‐directed mutagenesis experiments, these additional contacts do not account for the increased affinity of the Bad 25‐mer for Bcl‐X L compared to the Bad 16‐mer. Rather, the increased helix propensity of the Bad 25‐mer is primarily responsible for its greater affinity for Bcl‐X L . Based on this observation, a pair of 16‐residue peptides were designed and synthesized that were predicted to have a high helix propensity while maintaining the interactions important for complexation with Bcl‐X L . Both peptides showed an increase in helix propensity compared to the wild‐type and exhibited an enhanced affinity for Bcl‐X L .