A B S T R A C TVenezuelan equine encephalitis virus (VEEV) is a new world alphavirus which can be involved in several central nervous system disorders such as encephalitis and meningitis. The VEEV genome codes for 4 non-structural proteins (nsP), of which nsP3 contains a Macro domain. Macro domains (MD) can be found as stand-alone proteins or embedded within larger proteins in viruses, bacteria and eukaryotes. Their most common feature is the binding of ADP-ribose (ADPr), while several macro domains act as ribosylation writers, erasers or readers. Alphavirus MD erase ribosylation but their precise contribution in viral replication is still under investigation. NMR-driven titration experiments of ADPr in solution with the VEEV macro domain (in apo-and complex state) show that it adopts a suitable conformation for ADPr binding. Specific experiments indicate that the flexibility of the loops β5-α3 and α3-β6 is critical for formation of the complex and assists a wrapping mechanism for ADPr binding. Furthermore, along with this sequence of events, the VEEV MD undergoes a conformational exchange process between the apo state and a low-populated "dark" conformational state.

Macro domains consist of 130-190 amino acid residues and appear to be highly conserved in all kingdoms of life. Intense research on this field has shown that macro domains bind ADP-ribose and other similar molecules, but their exact function still remains intangible. Macro domains are highly conserved in the Alphavirus genus and the Venezuelan equine encephalitis virus (VEEV) is a member of this genus that causes fatal encephalitis to equines and humans. In this study we report the high yield recombinant expression and preliminary solution NMR study of the macro domain of VEEV. An almost complete sequence-specific assignment of its (1)H, (15)N and (13)C resonances was obtained and its secondary structure predicted by TALOS+. The protein shows a unique mixed α/β-fold.

The soluble guanylate cyclase (sGC) is the physiological sensor for nitric oxide and alterations of its function are actively implicated in a wide variety of pathophysiological conditions. Intense research efforts over the past 20 years have provided significant information on its regulation, culminating in the rational development of approved drugs or investigational lead molecules, which target and interact with sGC through novel mechanisms. However, there are numerous questions that remain unanswered. Ongoing investigations, with the critical aid of structural chemistry studies, try to further elucidate the enzyme’s structural characteristics that define the association of “stimulators” or “activators” of sGC in the presence or absence of the heme moiety, respectively, as well as the precise conformational attributes that will allow the design of more innovative and effective drugs. This review relates the progress achieved, particularly in the past 10 years, in understanding the function of this enzyme, and focusses on a) the rationale and results of its therapeutic targeting in disease situations, depending on the state of enzyme (oxidized or not, heme-carrying or not) and b) the most recent structural studies, which should permit improved design of future therapeutic molecules that aim to directly upregulate the activity of sGC.

Soluble guanylate cyclase (sGC) is the main receptor of nitric oxide (NO) and by converting GTP to cGMP regulates numerous biological processes. The β1 subunit of the most abundant, α1β1 heterodimer, harbors an N-terminal domain called H-NOX, responsible for heme and NO binding and thus sGC activation. Dysfunction of the NO/sGC/cGMP axis is causally associated with pathological states such as heart failure and pulmonary hypertension. Enhancement of sGC enzymatic function can be effected by a class of drugs called sGC “stimulators,” which depend on reduced heme and synergize with low NO concentrations. Until recently, our knowledge about the binding mode of stimulators relied on low resolution cryo-EM structures of human sGC in complex with known stimulators, while information about the mode of synergy with NO is still limited. Herein, we couple NMR spectroscopy using the H-NOX domain of the Nostoc sp. cyanobacterium with cGMP determinations in aortic smooth muscle cells (A7r5) to study the impact of the redox state of the heme on the binding of the sGC stimulator BAY 41-2272 to the Ns H-NOX domain and on the catalytic function of the sGC. BAY 41-2272 binds on the surface of H-NOX with low affinity and this binding is enhanced by low NO concentrations. Subsequent titration of the heme oxidant ODQ, fails to modify the conformation of H-NOX or elicit loss of the heme, despite its oxidation. Treatment of A7r5 cells with ODQ following the addition of BAY 41-2272 and an NO donor can still inhibit cGMP synthesis. Overall, we describe an analysis in real time of the interaction of the sGC stimulator, BAY 41-2272, with the Ns H-NOX, map the amino acids that mediate this interaction and provide evidence to explain the characteristic synergy of BAY 41-2272 with NO. We also propose that ODQ can still oxidize the heme in the H-NOX/NO complex and inhibit sGC activity, even though the heme remains associated with H-NOX. These data provide a more-in-depth understanding of the molecular mode of action of sGC stimulators and can lead to an optimized design and development of novel sGC agonists.

In the field of computational drug design, the identification and characterisation of the biological target of interest is a major step. Despite the growing number of such resolved protein structures every year, there are still many drug targets, especially membrane proteins, for which structural determination is very difficult. In these cases, experimental knowledge on already determined bioactive molecules may be used successfully for computational ligand‐based drug‐design methods. However, for the past three decades, most drug discovery efforts have been driven by the structure of the target biomolecule. Advances in structural biology methods have provided structural information of many molecules, giving rise to the structure‐based drug‐design process as a powerful tool for drug discovery in research academia and pharmaceutical industry. Both fields in the in silico drug‐design field are commonly used, each one depending on background experimental information and relevant computational methodologies. Key Concepts The main computer‐aided drug‐design approaches are either ligand or structure based. Main ligand‐based methods for identifying bioactive compounds are chemical similarity, pharmacophore mapping and QSAR. Advances in structural biology methodologies have greatly assisted structure‐based drug design. Structure‐based methods in computational drug design are mostly molecular docking, molecular dynamics, fragment‐based drug‐design and pharmacophore modelling. Docking and pharmacophore methodologies are most commonly used for virtual screening in drug design.