molecules Review Reviewing HIV-1 Gag Mutations in Protease Inhibitors Resistance: Insights for Possible Novel Gag Inhibitor Designs Chinh Tran-To Su 1, Darius Wen-Shuo Koh 1 and Samuel Ken-En Gan 1,2,* 1 Antibody & Product Development Lab, Bioinformatics Institute, A*STAR, Singapore 138671, Singapore 2 p53 Laboratory, A*STAR, Singapore 138648, Singapore * Correspondence: [email protected]; Tel.: +65-6478-8417; Fax: +65-6478-9047 Academic Editor: Keykavous Parang Received: 23 August 2019; Accepted: 4 September 2019; Published: 6 September 2019 Abstract: HIV protease inhibitors against the viral protease are often hampered by drug resistance mutations in protease and in the viral substrate Gag. To overcome this drug resistance and inhibit viral maturation, targeting Gag alongside protease rather than targeting protease alone may be more efficient. In order to successfully inhibit Gag, understanding of its drug resistance mutations and the elicited structural changes on protease binding needs to be investigated. While mutations on Gag have already been mapped to protease inhibitor resistance, there remain many mutations, particularly the non-cleavage mutations, that are not characterized. Through structural studies to unravel how Gag mutations contributes to protease drug resistance synergistically, it is thus possible to glean insights to design novel Gag inhibitors. In this review, we discuss the structural role of both novel and previously reported Gag mutations in PI resistance, and how new Gag inhibitors can be designed. Keywords: HIV-1 Gag; Gag inhibitors; protease; protease inhibitors; drug resistance mutations; drug design 1. Introduction Many anti-HIV drugs interfere directly with the viral life cycle by targeting key viral enzymes [1], e.g., reverse transcriptase inhibitors [2,3], integrase inhibitors [4,5], and protease inhibitors [6,7]. While such efforts are already hampered by the emergence of drug resistance mutations in the enzymes (e.g., in [8]), the scenario further worsens when viral enzyme substrates, such as Gag (HIV protease substrate), are found to synergistically contribute to drug resistance. Gag and protease play key roles in the viral maturation process [9] where the immature HIV virion matures into the infectious virion after budding from the infected cell for the next replication cycle. Proteolysis of Gag by protease occurs during the early stage of this maturation (Figure1A), in which the intact full length Gag precursor polyprotein is cleaved by the viral protease into functional subunits [9]. To inhibit this proteolysis, protease inhibitors (PIs) block protease activity in a competitive manner with Gag for protease binding [10]. PI-resistant mutations have been reported on Protease [11–13] and Gag [14–18] alone, or concurrently on both Protease and Gag [17,19–22], revealing an enzyme-substrate synergy to overcome PIs [23] (Figure1B). Expectedly, Gag cleavage site mutations contribute directly to PI resistance [ 24], while non- cleavage site mutations contribute to drug resistance by compensating for the loss of viral fitness [22,25,26] that resulted when protease accumulates drug resistant mutations reducing its proteolytic functions. As Gag is a larger protein than protease, and mutations (both cleavage and non-cleavage) can contribute to PI resistance, there is thus a need to study the mechanisms to how these mutations work Molecules 2019, 24, 3243; doi:10.3390/molecules24183243 www.mdpi.com/journal/molecules Molecules 2018, 23, x FOR PEER REVIEW 2 of 14 MoleculesAs 2019Gag, 24is, a 3243 larger protein than protease, and mutations (both cleavage and non-cleavage)2 ofcan 13 contribute to PI resistance, there is thus a need to study the mechanisms to how these mutations work in synergy with protease. Such studies will unravel potential weak points to which Gag can be in synergy with protease. Such studies will unravel potential weak points to which Gag can be targeted targeted against, opening more opportunities in drug design. against, opening more opportunities in drug design. FigureFigure 1. 1. AnAn overview overview of of the the Gag Gag and and Protease Protease relationship. relationship. (A) (A) A A schematic schematic of of the the early early stage stage of of viral viral maturationmaturation where where HIV-1 HIV-1 Protease cleaves Gag Gag into into th thee functional functional subunits: subunits: Ma Matrixtrix (MA), (MA), capsid capsid (CA), (CA), nucleocapsidnucleocapsid (NC), (NC), p6, p6, and and two two spacer spacer peptides peptides p1 p1 and and p2. p2. (B) (B) To To inhibit inhibit viral viral maturation, maturation, protease protease inhibitorsinhibitors (PIs(PIs iningreen green) are) are used used to competitively to competitively inhibit inhibit protease protease binding binding of Gag. PIof resistantGag. PI mutationsresistant mutationsare denoted are by denoted colored by stars, colored where stars, those where in the those protease in the catalytic protease site catalytic are in blue site, while are in those blue, inwhile Gag thoseare red infor Gag cleavage are red sites,for cleavage and purple sites,for and non-cleavage purple for non-cleavage sites. sites. 2.2. Possible Possible Targets Targets in in Gag Gag TheThe Gag Gag polyprotein polyprotein consists consists of of components components matrix matrix (MA), (MA), capsid capsid (CA), (CA), nucleocapsid nucleocapsid (NC), (NC), p6, p6, andand two two spacer spacer peptides p1 and p2. The The MA MA subuni subunit,t, located located at at the the N-terminus, N-terminus, is is essential essential for for targetingtargeting Gag Gag to to the the cell cell membrane, membrane, while while the the CA CA forms forms a a shell shell to to protect protect the the viral viral RNA RNA genome genome and and otherother core proteinsproteins duringduring maturation. maturation. The The NC NC is is responsible responsibl fore for RNA RNA packing packing and and encapsidation encapsidation [27] [27]while while the twothe two spacer spacer peptides peptides p1 and p1 and p2 regulate p2 regula thete ratethe rate and and the sequentialthe sequential cleavage cleavage process process of Gag of Gagby protease by protease [28]. [28]. This This process process of viral of assemblyviral assembly is complemented is complemented by viral by buddingviral budding moderated moderated by the bysmall the Proline-richsmall Proline-rich p6. Mutations p6. Mutations at either at either the N-terminal the N-terminal or C-terminal or C-terminal of these of these core proteinscore proteins were werereported reported to block to viralblock assembly viral assembly and impair and Gagimpair binding Gag tobinding plasma to membrane, plasma membrane, thereby inhibiting thereby inhibitingviral budding viral [ 27budding]. [27]. SinceSince the the Gag Gag cleavage cleavage sites sites do do not not share share a a consen consensussus sequence sequence (Figure (Figure 22),), the recognition ofof the cleavagecleavage sites sites by by protease protease is is like likelyly to to be be based based on on their their asymmetric asymmetric three-dimensional three-dimensional structures structures [29] [29] thatthat would would fit fit into into the the substrate-binding substrate-binding pocket pocket of of protease protease [30]. [30]. The The cleavage cleavage of of these these scissile scissile bonds bonds (seven-residue(seven-residue peptide peptide sequences sequences unique unique for for each each cleavage cleavage site) site) are are highly highly regulated regulated and and occur occur at at differingdiffering rates [24,28,31]. [24,28,31]. The The first first cleavage cleavage occurs occurs at at the the site site betw betweeneen the the p2 p2 peptide peptide and and NC NC domain domain (Figure(Figure 22),), followedfollowed byby thethe MAMA fromfrom CA–p2CA–p2 atat aa raterate thatthat isis ~14-fold~14-fold slowerslower thanthan thatthat ofof thethe firstfirst cleavage,cleavage, before before proceeding proceeding to to release release p6 p6 from from the the NC-p1 domain (at a rate ~9-fold slower slower than than the the Molecules 2019, 24, 3243 3 of 13 Molecules 2018, 23, x FOR PEER REVIEW 3 of 14 firstfirst cleavage). cleavage). At At the the last step, the two spacerspacer peptidespeptides p1p1 and and p2 p2 are are cleaved cleaved from from NC-p1 NC-p1 and and CA–p2 CA– p2at ratesat rates ~350-fold ~350-fold and and ~400-fold, ~400-fold, respectively, respectively, slower slower than than the the initial initial cleavage cleavage [24 [24,28,30,31].,28,30,31]. FigureFigure 2. 2. TheThe sequential sequential Gag Gag proteolysis proteolysis by by Protease. Protease. The The cleavage cleavage sites sites are are marked marked by by the the 7-residues, 7-residues, alongalong with thethe estimated estimated cleavage cleavage rates rates [28 ][28] marked mark byed arrows. by arrows. For easy For comparison, easy comparison, the initial the cleavage initial cleavagesite rate issite set rate to the is valueset to of the 1, whilevalue theof 1, other while cleavage the other site valuescleavage depict site thevalues reduced depict normalized the reduced rate. normalizedThe cleavage rate. site The sequences cleavage are site colored sequences based are on colored their physicochemical based on their properties,physicochemical e.g., hydrophobic properties, e.g.,(black hydrophobic), charged (positive: (black), chargedblue, negative: (positive:red blue), polar, negative: (other red colors),), polar and (other varied colors), in text and sizes varied based in text