Understanding molecular mechanisms underlying the complexity of allosteric regulation in proteins has attracted considerable attention in drug discovery due to the benefits and versatility of allosteric modulators in providing desirable selectivity against protein targets while minimizing toxicity and other side effects. The proliferation of novel computational approaches for predicting ligand–protein interactions and binding using dynamic and network-centric perspectives has led to new insights into allosteric mechanisms and facilitated computer-based discovery of allosteric drugs. Although no absolute method of experimental and in silico allosteric drug/site discovery exists, current methods are still being improved. As such, the critical analysis and integration of established approaches into robust, reproducible, and customizable computational pipelines with experimental feedback could make allosteric drug discovery more efficient and reliable. In this article, we review computational approaches for allosteric drug discovery and discuss how these tools can be utilized to develop consensus workflows for in silico identification of allosteric sites and modulators with some applications to pathogen resistance and precision medicine. The emerging realization that allosteric modulators can exploit distinct regulatory mechanisms and can provide access to targeted modulation of protein activities could open opportunities for probing biological processes and in silico design of drug combinations with improved therapeutic indices and a broad range of activities.

Atovaquone (ATQ) is a drug used to prevent and treat malaria that functions by targeting the Plasmodium falciparum cytochrome b (PfCytb) protein. PfCytb catalyzes the transmembrane electron transfer (ET) pathway which maintains the mitochondrial membrane potential. The ubiquinol substrate binding site of the protein has heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors that act as redox centers to aid in ET. Recent studies investigating ATQ resistance mechanisms have shown that point mutations of PfCytb confer resistance. Thus, understanding the resistance mechanisms at the molecular level via computational approaches incorporating phospholipid bilayer would help in the design of new efficacious drugs that are also capable of bypassing parasite resistance. With this knowledge gap, this article seeks to explore the effect of three drug resistant mutations Y268C, Y268N and Y268S on the PfCytb structure and function in the presence and absence of ATQ. To draw reliable conclusions, 350 ns all-atom membrane (POPC:POPE phospholipid bilayer) molecular dynamics (MD) simulations with derived metal parameters for the holo and ATQ-bound -proteins were performed. Thereafter, simulation outputs were analyzed using dynamic residue network (DRN) analysis. Across the triplicate MD runs, hydrophobic interactions, reported to be crucial in protein function were assessed. In both, the presence and absence of ATQ and a loss of key active site residue interactions were observed as a result of mutations. These active site residues included: Met 133, Trp136, Val140, Thr142, Ile258, Val259, Pro260 and Phe264. These changes to residue interactions are likely to destabilize the overall intra-protein residue communication network where the proteins’ function could be implicated. Protein dynamics of the ATQ-bound mutant complexes showed that they assumed a different pose to the wild-type, resulting in diminished residue interactions in the mutant proteins. In summary, this study presents insights on the possible effect of the mutations on ATQ drug activity causing resistance and describes accurate MD simulations in the presence of the lipid bilayer prior to conducting inhibitory drug discovery for the PfCytb-iron sulphur protein (Cytb-ISP) complex.

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP into dihydroneopterin triphosphate (DHNP). DHNP is the first intermediate of the folate de novo biosynthesis pathway in prokaryotic and lower eukaryotic microorganisms and the tetrahydrobiopterin (BH4) biosynthesis pathway in higher eukaryotes. The de novo folate biosynthesis provides essential cofactors for DNA replication, cell division, and synthesis of key amino acids in rapidly replicating pathogen cells, such as Plasmodium falciparum(P. falciparum), a causative agent of malaria. In eukaryotes, the product of the BH4 biosynthesis pathway is essential for the production of nitric oxide and several neurotransmitter precursors. An increased copy number of the malaria parasite P. falciparum GCH1 gene has been reported to influence antimalarial antifolate drug resistance evolution, whereas mutations in the human GCH1 are associated with neuropathic and inflammatory pain disorders. Thus, GCH1 stands as an important and attractive drug target for developing therapeutics. The GCH1 intrinsic dynamics that modulate its activity remains unclear, and key sites that exert allosteric effects across the structure are yet to be elucidated. This study employed the anisotropic network model to analyze the intrinsic motions of the GCH1 structure alone and in complex with its regulatory partner protein. We showed that the GCH1 tunnel-gating mechanism is regulated by a global shear motion and an outward expansion of the central five-helix bundle. We further identified hotspot residues within sites of structural significance for the GCH1 intrinsic allosteric modulation. The obtained results can provide a solid starting point to design novel antineuropathic treatments for humans and novel antimalarial drugs against the malaria parasite P. falciparum GCH1 enzyme.

The folate biosynthesis pathway is an essential pathway for cell growth and survival. Folate derivatives serve as a source of the one-carbon units in several intracellular metabolic reactions. Rapidly dividing cells rely heavily on the availability of folate derivatives for their proliferation. As a result, drugs targeting this pathway have shown to be effective against tumor cells and pathogens, but drug resistance against the available antifolate drugs emerged quickly. Therefore, there is a need to develop new treatment strategies and identify alternative metabolic targets. The two de novo folate biosynthesis pathway enzymes, GTP cyclohydrolase I (GCH1) and 6-pyruvoyl tetrahydropterin synthase (PTPS), can provide an alternative strategy to overcome the drug resistance that emerged in the two primary targeted enzymes dihydrofolate reductase and dihydropteroate synthase. Both GCH1 and PTPS enzymes contain Zn²⁺ ions in their active sites, and to accurately study their dynamic behaviors using all-atom molecular dynamics (MD) simulations, appropriate parameters that can describe their metal sites should be developed and validated. In this study, force field parameters of the GCH1 and PTPS metal centers were generated using quantum mechanics (QM) calculations and then validated through MD simulations to ensure their accuracy in describing and maintaining the Zn²⁺ ion coordination environment. The derived force field parameters will provide accurate and reliable MD simulations involving these two enzymes for future in-silico identification of drug candidates against the GCH1 and PTPS enzymes. Communicated by Ramaswamy H. Sarma

The folate de novo synthesis pathway is a well-established drug target in the treatment of many infectious diseases. Antimalarial antifolate drugs have proven to be effective against malaria, however, rapid resistance has emerged on the two primary targeted enzymes: dihydrofolate reductase and dihydroptoreate synthase. The need to identify alternative antifolate drugs and novel metabolic targets is of imminent importance. The 6-pyruvol tetrahydropterin synthase (PTPS) enzyme belongs to the tunneling fold protein superfamily which is characterized by a distinct central tunnel/cavity. The enzyme catalyzes the second reaction step of the parasite’s folate de novo synthesis pathway and is responsible for the conversion of 7,8-dihydroneopterin to 6-pyruvoyl-tetrahydropterin. In this study, we examine the structural dynamics of Plasmodium falciparum PTPS using the anisotropic network model, to elucidate the collective motions that drive the function of the enzyme and identify potential sites for allosteric modulation of its binding properties. Based on our modal analysis, we identified key sites in the N-terminal domains and central helices which control the accessibility to the active site. Notably, the N-terminal domains were shown to regulate the open-to-closed transition of the tunnel, via a distinctive wringing motion that deformed the core of the protein. We, further, combined the dynamic analysis with motif discovery which revealed highly conserved motifs that are unique to the Plasmodium species and are located in the N-terminal domains and central helices. This provides essential structural information for the efficient design of drugs such as allosteric modulators that would have high specificity and low toxicity as they do not target the PTPS active site that is highly conserved in humans.

Neutralizing antibody responses correlate with protection from SARS-CoV-2 infection, yet higher neutralizing responses associate with more severe disease. Whether people without severe disease can also develop strong neutralizing responses to infection, and the pathways involved, is less clear. We performed a proteomic analysis on sera from 71 individuals infected with ancestral SARS-CoV-2, enrolled during the first South African infection wave. We determined disease severity by whether participants required supplemental oxygen and measured neutralizing antibody levels at convalescence. High neutralizing antibodies were associated with high disease severity, yet 40% of participants with lower disease severity had neutralizing antibody levels comparable to those with severe disease. We found 130 differentially expressed proteins between high and low neutralizers and 40 between people with high versus low disease severity. Five proteins overlapped, including furin, a protease which enhances SARS-CoV-2 infection. High neutralizers with non-severe disease had similar levels of differentially expressed neutralization response proteins to high neutralizers with severe disease, yet similar levels of differentially expressed disease severity proteins to participants with non-severe disease. Furthermore, we could reasonably predict who developed a strong neutralizing response based on a single protein, HSPA8, involved in clathrin pit uncoating. These results indicate that a strong antibody response does not always require severe disease and may involve different pathways.