Pathogen-specific peptide to block virulence-related signaling in candida albicans

Abstract

In the present invention, by combining biophysical techniques with AI-based predictive modelling, the structural features of a unique Ras-family protein, Candida albicans Ras-like protein 1 (CaRas1) and of its guanine nucleotide exchange factor (GEF) CaCdc25, both isolated and complexed, are disclosed. Based on the structural information, synthetic peptide sequences capable of inhibiting the activity of CaCdc25 are disclosed. The present invention also refers to compositions comprising the said peptide sequences or some of its variants, each also comprising aminoacid modifications, used alone or in combinations. In another embodiment, the present invention relates to pharmaceutical preparations of medicaments comprising the said compositions. Yet another aspect of the present invention refers to the said sequences, compositions and pharmaceutical formulations for use in a treatment for Candida spp., as well as other fungal infections with agents from the Candida Lodderomyces clade and more broadly, the Sacharomycetales kindom, through specific block of an important virulence-related signaling pathway.

Description

[0001] DESCRIPTION PATHOGEN-SPECIFIC PEPTIDE TO BLOCK VIRULENCE-RELATED SIGNALING IN CANDIDA ALBICANS Technical field of the invention The present invention relates to the technical field of human health; preparations for medical purposes; in particular medicinal preparations containing peptide active ingredients. State of the art Candida albicans, part of the commensal microbiota of most individuals, is an important opportunistic human fungal pathogen and can cause severe infections in immunocompromised patients (1). C. albicans belongs to the CTG-clade of Ascomycetes, which includes a high number of opportunistic pathogenic species that use an alternative genetic code and translate the CUG codon as a serine instead of leucine (2,3). The remarkable adaptability of C. albicans to highly diverse host niches (4) is underscored by its phenotypic plasticity, i.e., the discrete phenotypes adopted in response to varying environmental cues (5). The World Health Organization (WHO), in a recent report (fungal priority pathogens list, WHO-FPPL) classified C. albicans as a first priority pathogen and member of the critical group (6), highlighting the emergency in the discovery and development of novel drugs to battle this important human pathogen. The commensal-to-pathogen transition of C. albicans is associated with its ability to interconvert between yeast and hyphal morphologies (7–9). The activation of hyphae- specific genes is directly mediated by the cyclic adenosine monophosphate (cAMP) and the mitogen-activated protein kinase (MAPK) signaling pathways, which are regulated by the Ras-like protein 1 (CaRas1) and the cell division control protein 25 (CaCdc25) (10,11). CaRas1 is composed of a highly conserved GTPase domain (G-domain) followed by a hypervariable region. The long hypervariable region of CaRas1 represents a major difference between yeast (approximately 120 residues) and human (approximately 20 residues) Ras proteins. Further, the presence of low complexity segments in the hypervariable region of CaRas1, including polyglutamine (PolyQ) repeats and a Q / N-rich stretch, also present in other members of the CTG-clade, makes it unique among Ras-family proteins. The hypervariable region of CaRas1 also contains a C-terminal CCaaX membrane association motif, whose two cysteine residues have been found to be farnesylated and palmitoylated (12), in agreement with the localization of CaRas1 to membranes, as observed for many Ras GTPases. Interestingly, cleavage of CaRas1 at its hypervariable region, removing the last 67 amino acid residues, has been proposed as a mechanism modulating CaRas1 signaling and, consequently, hyphal growth (13). In yeast cells, CaRas1 is generally in an inactivated, GDP-bound form (CaRas1-GDP). CaCdc25 activates CaRas1, promoting its conversion to CaRas1-GTP and switching on the signaling pathway associated with the hyphae-specific genes (11,14,15). The activation of CaRas1 by CaCdc25 is the primary mechanism by which D-glucose induces morphogenesis (11,15,16). In fact, deletion of either CaRas1 or CaCdc25 results in hyphal defects (17). CaCdc25 (~150 kDa) is a Ras-guanine nucleotide exchange factor (GEF) that contains a C-terminal catalytic region, consisting of the tandem Ras-exchange motif domain-catalytic domain (REM-CAT), which catalyzes the exchange of CaRas1-bound GDP to GTP. Despite its biological relevance, no experimental structural information is available for the full-length CaRas1 and the relative arrangement of the G-domain and the unique low complexity hypervariable region, which is predicted to be partially disordered. Also, the elucidation of the molecular basis for the activation of CaRas1 by CaCdc25 would provide crucial information for the design of new drugs to fight this fungal human pathogen. Summary of the Invention Here, we disclose that CaRas1 has an elongated shape and limited conformational flexibility, and that its hypervariable region contains helical structural elements, likely forming an intramolecular coiled-coil. Functional assays revealed that CaRas1 activation by CaCdc25 is highly efficient, with up to 2000-fold higher activity levels than reported for human GEFs. The crystal structure of the catalytic region of CaCdc25 reveals an active conformation for the α-helical hairpin, which is critical for CaRas1 activation, unveiling a specific region exclusive to CTG- clade species, where negatively charged substitutions reduce its activity. Structural studies on CaRas1 / CaCdc25 complexes also revealed that the surface of interaction between the two proteins is clearly distinct from that of homologous human complexes. Using a combination of AI-based predictive modelling and activity assays, we disclose inhibitory synthetic peptide sequences for a specific inhibition of the CaRas1-activation via targeting its GEF. This, together with the unique pathogen-structural features of CaRas1 and CaCdc25 described here, disclose a set of novel peptides, compositions and pharmaceutical formulations comprising said peptides, to specifically block this important virulence- related signaling pathway, for use in a treatment for Candida albicans. Detailed description of the Invention An important feature associated with Candida albicans pathogenicity is its ability to switch between yeast and hyphal forms, a process in which the Ras-like protein 1 (CaRas1) plays a key role. CaRas1 is activated by the guanine nucleotide exchange factor (GEF) CaCdc25, triggering hyphal growth-related signaling pathways through its highly conserved GTP-binding domain (G- domain). An important function in hyphal growth has also been proposed for the long hypervariable region downstream the G-domain of CaRas1, whose unusual content of polyglutamine stretches and Q / N repeats make CaRas1 unique within Ras-family proteins. Despite its biological importance, both the structure of CaRas1 and the molecular basis of its activation by CaCdc25 remain unexplored. CaRas1 is composed by a highly conserved G-domain, followed by an exclusive hypervariable region that contains low complexity sequence segments, including PolyQ repeats and a Q / N-rich stretch (Fig. 1A), a unique feature conserved in Ras-family proteins from various of the CTG-clade opportunistic fungal pathogens. The structural properties of the hypervariable region within CaRas1 and its relative position with respect to the globular G- domain were evaluated using SAXS (Figs. 1A-F, Supplemental Table S1) and two CaRas1 constructs, the full-length protein (CaRas1- FL, 1-290) and the isolated G-domain (1-166). Both CaRas1 constructs were monodisperse and monomeric in solution (Fig. 1C). The distance distribution function (P(r)) for the G-domain has a bell shape characteristic of globular particles with a peak at approximately 23 Å and a maximum dimension (Dmax) of approximately 45 Å (Fig. 1D), in good agreement with a homology model for the G- domain (Figs. 1A, B) that could be easily docked in a low- resolution envelope obtained by ab initio methods from the experimental SAXS data (Fig. 1A). Interestingly, the high resolution of the SAXS data allowed to distinguish between closely related homology models and suggests that the highly dynamic G- domain switch II region displays an α-helical structure in CaRas1. When the hypervariable region of CaRas1 is present, the P(r) function displays a second peak at approximately 52 Å (Fig. 1D), suggesting a certain degree of rigidity and the presence of structured elements in that region. In agreement, the low- resolution envelope for CaRas1-FL, which is an average of 15 independent ab initio models, displays an elongated shape (Fig. 1A). The homology model for the G-domain could be docked in one end of this envelope, allowing the assignment of an adjacent volume to the hypervariable region (Fig. 1A). The differences between CaRas1-FL and its isolated G-domain are further highlighted in the dimensionless Kratky plot of the scattering data, which displays a bell-shaped peak with a maximum at 1.1 for the G-domain, indicating that it is a compact and spherical particle (Fig. 1E). When the hypervariable region is present, the overall shape of this plot is preserved, however the position and amplitude of its maximum deviates largely from the value expected for spherical particles, in line with the highly anisometric shape of CaRas1-FL (Fig. 1E). The overall shapes of the bimodal P(r) function and the dimensionless Kratky plot for CaRas1-FL, are characteristic of a protein composed by relatively rigid two-bodies, suggesting that the hypervariable region is not entirely disordered. In order to better understand the structure and flexibility of the hypervariable region, an ensemble fitting method was employed. A pool of 10,000 CaRas1-FL structures was generated, assuming full flexibility of the hypervariable region and sampling exhaustively its potential orientation. The minimal ensemble that better approximated the experimental SAXS data was mainly populated by conformations with a narrower size distribution than the entire size range of the pool (Fig. 1F), further underscoring the limited flexibility of the hypervariable region and in line with the calculated Rsigma value of 0.61 for this analysis, as values < 1.0 for this parameter are indicative of limited flexibility in the system. In agreement, circular dichroism (CD) analysis of the secondary structure of three CaRas1 truncation constructs with variable lengths of the hypervariable region (Fig. 1A) revealed an increase in α-helical content when the hypervariable region was present (Fig. 1G). Importantly, the CD spectrum of CaRas1-FL displayed minima at 220 nm and 209 nm, with a 220 nm / 209 nm ratio of 1.00, supporting the presence of a coiled-coil structure in the hypervariable region. In the presence of 50% (v / v) trifluoroethanol (TFE), a strong α-helix stabilizer that disrupts coiled-coil formation, the 220 nm / 209 nm ratio shifted to 0.88, which is suggestive of a coiled-coil to isolated helices transition. With the support of these experimental data, we explored further the predicted structure of full-length CaRas1 using the artificial intelligence (AI)-based network AlphaFold2 (Figs. 2A-C). We generated five AlphaFold2 models, which predicted with high confidence (per-residue local Distance Diference Test, lDDT > 70) an extension of the terminal helix of the G-domain through the first polyQ region until near the end of the Q / N stretch (N212). Interestingly, in some of the models obtained, AlphaFold2 predicted an helical component in the second polyQ region and positioned it close to the first polyQ region, in line with the coiled-coil structure observed by CD. Importantly, the calculated SAXS profile of this model is quite similar to that obtained experimentally (Fig. 2C). Since the C-terminal part of the hypervariable region (residues 213-290) was predicted with low confidence (lDDT < 60), the flexibility of this region was further validated by SAXS (Figs. 2D, E). Indeed, ensemble optimization methods (EOM) analysis of the subset of models that better fit the experimental SAXS profile underscored the preference for a compact arrangement. The crystal structure of the catalytic region of the CaRas1 guanine nucleotide exchange factor CaCdc25, which catalyzes the exchange of the bound nucleotide and therefore the conversion of CaRas1- GDP to CaRas1-GTP, was determined at 2.45 Å-resolution (Figs. 3A- F). The protein crystallized in the orthorhombic space group P212121 with two molecules of CaCdc25 in the asymmetric unit (AU) that are very similar. The complete monomers superpose with a root mean square deviation (rmsd) of 1.0 Å for 384 aligned C- atoms. A structural homology search with DALI (27) showed that, despite low amino acid sequence conservation, the catalytic region of CaCdc25 is structurally quite similar to human GEFs containing Cdc25 homology domains and that act on the Ras family (four matches with Z score > 20), and consists of two well-differentiated domains: the REM domain (residues 883-1035, β-strands β1 and β2 and α- helices α1-α10) and the CAT domain (residues 1039-1306, β-strands A and α-helices αA-αQ) (Fig. 3A). In particular, the individual structural elements for the CAT domain are more conserved in the five molecules (rmsd 3.7-4.7 Å for 286 aligned C^ atoms) than those for the REM domain (rmsd 6.3-7.9 Å for 179 aligned C- atoms). The α-helical hairpin of the CAT domains is a key element for the Ras nucleotide exchange in GEFs. In CaCdc25, the α-helical hairpin is formed by the two antiparallel helices αM and αO (residues 1222- 1265) (Fig. 3B), which protrude from the main body of the CAT domain. The relative orientation of the α-helical hairpin is critical for the activity of GEFs. In CaCdc25, the hairpin assumes an orientation similar to that observed in the structure of the active state of the human HRas GEF Sos (Fig. 3B), suggesting that the CAT domain of CaCdc25 is also in the active conformation in the crystal structure. The active state-like orientation of the ^- helical hairpin of CaCdc25 is stabilized mostly by hydrophobic interactions with three regions (Fig. 3C): 1) the REM domain, where α-helical hairpin residues L1240, V1247 and F1249 occupy three hydrophobic grooves formed by K909, L912 and M937 (pocket 1), F926, V929 and F930 (pocket 2), and L897, K905 and G906 (pocket 3) (Fig. 3D), 2) a region called flap 1 (residues 1080-1115) that encompasses helix αD and part of helices αC and αE, and that stabilizes helix αM of the hairpin (Fig. 3E) and 3) the region called flap 2 (residues 1195-1215), which includes part of helix αL (Fig. 3F). In addition to these hydrophobic interactions, typical of the GEF family, the α-helical hairpin of CaCdc25 is further stabilized by three unique polar contacts, which are not observed in the structures of the most similar GEFs: R1245 interacts with E895 and E910 from the REM domain (Fig. 3D); D1238 interacts with K1100 from the flap 1 region; and E1105 from flap 1 interacts with S992 from the REM domain (Fig. 3E). Importantly, a multiple amino acid sequence alignment with human GEFs revealed only partial conservation of the residues mediating the activation of Ras in CaCdc25. The two important residues located in the α- helical hairpin that impede nucleotide binding in the human GEF Sos1, L938 and E942, are replaced in CaCdc25 by T1230 and H1234, respectively. Ultimately, the solution structure of the catalytic region of CaCdc25 is in excellent agreement with the crystallographic model (Fig. 3G), which unsurprisingly docks well into the ab initio molecular envelope derived from the SAXS data (Fig. 3H). The identification of the CaCdc25 / CaRas1 binding interface raised the possibility of exploring this site to specifically inhibit the GEF activity of CaCdc25. In the AlphaFold2 model of full-length CaCdc25 (AF-P43069-F1), a region upstream the REM-CAT tandem occupies the G-domain binding site region. To further explore this finding, we generated several AlphaFold2 models for CaCdc25 segment 837-1306, which comprises the hypothetical inhibitory and catalytic regions (Fig. 4A). In the five models generated, the hypothetical inhibitory region, containing two α-helices (predicted lDDT of ~60-70), localizes to the CaRas1 binding site. In order to evaluate the inhibitory role of this segment, the effect of adding a synthetic peptide comprising sequence of SEQ. ID nr.1 to the nucleotide exchange activity assays is assessed. As a result, a dose-dependent reduction of the activity of CaCdc25 is observed (Fig. 4B), with an estimated EC50 value of 49 + / - 8 µM (Fig. 4C). In the AlphaFold2-derived models of the CaCdc25 (837- 1306) / CaRas1 complex, displacement of segment 837-858 from the G- domain binding site and loss of the helical secondary structure are observed (Fig. 4D). The sequence is only significantly conserved in Cdc25 proteins from species belonging to the Saccharomycetales kingdom, predominantly in proteins from the Candida / Lodderomyces clade (Fig. 5). This sequence may comprise other sequence variants at each position which are also sequences peptides able to inhibit the GEF activity of CaCdc25 (Figure 6, Table 1). Table 1 Sequence of the peptide able to inhibit the GEF activity of CaCdc25 and variants. 130 KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKALTIINYATRVMQMNFDVQLLLVE 98 G G G G Q CQ 66 G G G G Q Q S 33 Q Q Q Q 0 Q Q Q 0 Q Q Q Q Q 538 Q Q Q Q 606 Q S G Q Q 6 Q S Q Q Q S Q Q 80 Q S Q Q 88 Q Q 96 Q Q 0 Q Q 08 Q 50 Q Q 8 GG S S S S G Q Q 86 GG S S S S G Q Q 35 GG S S S S G Q Q GG S S S S G Q Q Both SEQ.ID nr. 1 and the variant pepetide sequences may further comprise modifications, selected from the following list: N terminus Modification: H (free amine) Ac (Acetylation) Fmoc CBZ Bz Bz(4-F) Bz(4-NO2) Pyr D-Pyr LA- Mpa- Mal-b-Ala Mal-Acp Aoa N terminus Fatylation For (Formylation) 2-Br-Ac 2-Cl-Ac 2-I-Ac OH-Ac- But- Suc MeOSuc Iba Hex- 5-heptenoic acid 5-Hexynoic acid heptanedioic acid Oct- Dec- Sebacic acid Lau- Myr- Pal- Ste- Octanedioic acid Oleic Acid- C terminus Modification -OH (free acid) -NH2 (Amidation) Cysteamide, -Cya -AMC -OMe -OEt -OBzl -OtBu -NHMe -NHEt -TBzl p-Nitroanilide D Form Amino Acid {D-Ala} {D-Arg} {D-Asp} {D-Asn} {D-Cys} {D-Glu} {D-Gln} {D-His} {D-Allo-Ile} {D-Ile} {D-Leu} {D-Lys} {D-Met} {D-Pro} {D-Phe} {D-Ser} {D-Tyr} {D-Thr} {D-Trp} {D-Val} Unusual Amino Acid {Beta-Asp} {D-Beta-Asp} {Gamma-Glu} {D-Gamma-Glu} Cys(Cam) D-Cys(Cam) {Cys(Acm)} {Cys(tBu)} {Met(O)} {D-Met(O)} {Met(O)2} {D-Met(O)2} {Lys(Ac)} {Ac-Lys} {Lys(Dde)} {Tle} {D-Ser(octanoic acid)} 2-Thi 3-Thi {Aib} {Abu} {D-Abu} {Hyp} {Phg} {D-Phg} {Nva} {D-Nva} {Norleucine} {D-Nle} {Cit} {D-Cit} {Orn} {D-Orn} {Pen} {D-Pen} {Cpg} {Cha} {D-Cha} {Chg} {D-Chg} {Dab} {Dap} Pra D-Pra Allo-Thr D-Allo-Thr {D-1-Nal} {L-1-Nal} {D-2-Nal} {L-2-Nal} {D-2-Pal} {L-2-Pal} {D-3-Pal} {L-3-Pal} {D-4-Pal} {L-4-Pal} {Oic} {Tic} {D-Tic} X (20 kinds amino acids mixture with equal mol) Dye and Fluorescent Labeling Biotin- -Lys(Biotin)- -Lys(Biotin) Biotin-Ahx- FITC- -Lys(FITC)- -Lys(FITC) FITC-Ahx- (FITC: Fluorescein Isothiocyanate) 5-FAM- 5,6-FAM 6'FAM -Lys(5-FAM)- Lys(5,6-FAM) -Lys(5-FAM) 5-FAM-Ahx- Dansyl- -Lys(Dansyl)- -Lys(Dansyl) Dansyl-Ahx- 5-TAMRA- 5(6)-TAMTA- -Lys(TAMRA)- -Lys(5-TAMRA) {5-TAMRA-Acp} {Lys(Dnp)} {D-Lys(Dnp)} {Dab(Dnp)} Dap(Dnp) MCA- -Lys(MCA)- -Lys(MCA) Rhodamine B- Lys(Rhodamine B) Phe / Tyr Analogs Amino Acid {D-2-Cl-Phe} {L-2-Cl-Phe} {D-3-Cl-Phe} {L-3-Cl-Phe} {D-4-Cl-Phe} {L-4-Cl-Phe} {D-3,4-DiCl-Phe} {L-3,4-DiCl-Phe} {D-4-Br-Phe} {L-4-Br-Phe} {D-3-F-Phe} {L-3-F-Phe} {D-4-F-Phe} {L-4-F-Phe} {D-4-NO2-Phe} {L-4-NO2-Phe} {D-4-I-Phe} {L-4-I-Phe} {D-3-CN-Phe} {L-3-CN-Phe} {D-4-CN-Phe} {L-4-CN-Phe} {D-2-Me-Phe} {L-2-Me-Phe} {D-4-Me-Phe} {L-4-Me-Phe} {D-4-NH2-Phe} {L-4-NH2-Phe} {D-3-Cl-Tyr} {L-3-Cl-Tyr} {D-3,5-DiCl-Tyr} {L-3,5-DiCl-Tyr} {D-3,5-DiBr-Tyr} {L-3,5-DiBr-Tyr} {D-3-I-Tyr} {L-3-I-Tyr} {D-3,5-DiI-Tyr} {L-3,5-DiI-Tyr} {D-3-NO2-Tyr} {L-3-NO2-Tyr} {D-3,5-DiNO2-Tyr} {L-3,5-DiNO2-Tyr} {L-3-F-Tyr} Homo Amino Acid {Har} {D-Har} {HomoCit} {D-HomoCit} {HomoLeu} {HomoPro} {D-HomoPro} {beta-HomoIle} {beta-HomoLeu} {beta-HomoMet} {beta-HomoPro} {beta-HomoVal} {Abz} o-Abz- (o-aminobenzoic acid) {Tyr(3-NO2)} {Glu(EDANS)} {DABCYL} {Lys(DABCYL)} {Lys(Abz)} MAPS and Carrier Complex {Symmetric 2 Branches} {Symmetric 4 Branches} {Symmetric 8 Branches} Lys(Maleimide) Lys(Mpa) Lys(Pra) Lys(Suc) Lys(glutaryl) Lys(pGlu) Lys(For) Lys(2-Br-Ac) Lys(Butanoyl) Lys(Crotonyl) {Lys(octenyl)} lys(Ma) Lys(Pal) Lys(Oleic Acid) Lys(Acryl) Lys(alkine) Lys(Alloc) Lys(Aoa) Lys(cyclopropanecarboxyl) {Lys(3,5-diiodo-4-hydroxybenzoyl)} Lys(Methacryl) Lys(propargyl) Lys(propionyl) Lys(Pyruvoyl) {BSA-Peptide N terminus} {BSA-peptide C terminus} {BSA-Peptide via Cys} {KLH-Peptide N terminus} {KLH-Peptide C terminus} {KLH-peptide via Cys} {OVA-Peptide N terminus} {OVA-peptide C terminus} {OVA-peptide via Cys} Atom Linker {betaAla} {Ava} {Ahx} {8-Aoc} {AEA} {Ado} {ANP Linker} Methyl Amino Acids {Lys(Me)} {Lys(Me2)} {Lys(Me3)} N-Methyl amino acid {N-Me-Ala} {N-Me-Phe} {N-Me-Leu} {N-Me-Ile} {N-Me-Val} {N-Me-Met} {N-Me-Nle} {N-Me-Nva} {Sar} {N-Me-Ser} {N-Me-Tyr} {N-Me-Thr} {N-Me-Asp} {N-Me-Glu} N-Me-beta-Ala Cyclic Peptide {Mono Disulfide bridge} {Double Disulfide bridge} {Triple Disulfide bridge} {Random Disulfide bridge} {Same Seq. Inter-Disulfide bridge } {Different Inter-Disulfide bridge} {Amide cyclic (end)} Phosphorylation {pSer} {pTyr} {pThr} {D-pSer} {D-pTyr} {D-pThr} {Di-sites in sequence} {Tri-sites in sequence} PEG {Mini-PEG} {Mini-PEG2} {Mini-PEG3} {PEG4} {PEG-6} {PEG8} {PEG-11} {PEG-12} Isotope Labeling H2 (deuterium) N15 C13 13C15N-K (C-terminal) 13C15N-R (C-terminal) 13C15N-A (C-terminal) 13C15N-I (C-terminal) 13C15N-L (C-terminal) 13C15N-F (C-terminal) 13C15N-P (C-terminal) 13C15N-V (C-terminal) Stapled Peptides Single stapled S5 and S5 Single stapled S5 and R8 Single stapled R8 and R8 Double stapled S5 and S5 Double stapled S5 and R8 Double stapled R8 and R8 Glycopeptides (Glycosylated Peptides) N-linked glycopeptide synthesis O-linked glycopeptide synthesis C-linked glycopeptide synthesis S- linked glycopeptide synthesis Glycopeptides containing monosaccharides, such as Ser / Thr (GlcNAc), Ser / Thr (GalNAc), Asn (GlcNAc), Ser (Xyl), Thr (Man), etc. Glycopeptides containing oligosaccharides, such as Ser / Thr (Gal- GalNAc), Ser / Thr (Neu-Gal- GalNAc), Asn (Fuc-GlcNAc), etc. Misc FMK, CMK N6-Octanoyl Photocleavable peptides propargyl-gly Lys(Myr) pNA Lys(N3) In conclusion, synthetic peptide sequences capable of inhibiting the activity of CaCdc25 are disclosed. Compositions comprising the said peptide sequences or some of its variants, each also comprising aminoacid modifications, may be used alone or in combinations. Sequences, compositions and pharmaceutical formulations comprising them may be advantageously used in a treatment for Candida spp., as well as other fungal infections with agents from the Candida Lodderomyces clade and more broadly, the Sacharomycetales kindom, through specific block of an important virulence-related signaling pathway. Brief description of the Figures Figure 1. The hypervariable region of CaRas1 displays an unexpected architecture. A) Domain organization scheme for CaRas1, highlighting the G-domain (residues 1-166) and the hypervariable region (residues 167-290). Important structural elements of the G- domain - nucleotide exchange P-loop, Switches I and II and NKxD motif - and the polyQ and Q / N-rich segments within the hypervariable region are labeled. Schemes of the different constructs used in this work are provided in the dotted box. The SAXS-derived molecular envelopes for the CaRas1 G-domain and for full-length CaRas1 are shown as semi-transparent surfaces, below and to the right of the domain organization scheme, respectively. A cartoon representation (orange) of the G-domain model is docked into the envelopes. Below the envelope of full-length CaRas1, predicted secondary structural elements for the hypervariable region are represented, suggesting that the polyQ and Q / N-rich regions are mainly helical. B) Experimental SAXS profiles extrapolated to infinite dilution of CaRas1-FL (dark-grey) and G- domain (orange). Curves are offset on the log scale for readability; the same data color scheme is used in panels C–G. The scattering calculated for the atomic model of the G-domain shown in panel A is represented as a black dashed line. C) Guinier plots of the scattering data shown in B. D) P(r) functions determined from the scattering data shown in B. The maximum for a second peak in CaRas1-FL is indicated by a grey arrow. E) Dimensionless Kratky plots. The crosshair indicates the expected position of the theoretical maximum of the plot for spherical compact particles (qRg approximately 1.732 and (qRg)2I(q) / I(0) approximately 1.104). F) Ensemble optimization methods (EOM) analysis of the flexibility of the hypervariable region. Frequency of size distributions in a pool of 10,000 models (black dashed line) with random orientations of the hypervariable region, and in the selected ensemble that fits the SAXS data of CaRas1-FL (blue line). G) Circular dichroism spectra of CaRas1 G-domain, CaRas1-213 (green) and CaRas1-FL. The negative band at 209 nm (red arrows) observed when the hypervariable region is present in CaRas1, together with that at 220 nm are spectral signatures for α-helical content. Figure 2. Structure of full-length CaRas1 and flexibility analysis of the hypervariable region segment 213-290. A) The five top- ranking structural models predicted by AlphaFold2 for full-length CaRas1. The best model (ranked 1) is docked into the low-resolution experimental envelope. The two polyQ regions are highlighted in red. B) Per-residue local Distance Diference Test (lDDT) of the five predicted structures. The segment 213-290 was predicted with low confidence (lDDT < 60). C) SAXS experimental data of full- length CaRas1 (gray dots) superposed with the scattering profile calculated for the best predicted model (dashed black line). D) Representation of the pool of structures generated to evaluate by EOM analysis the flexibility of the CaRas1 region 213-290 (green). The pool consisted of 10,000 models, in which both the G-domain (orange) and the 167-212 region (green) were fixed, whereas the part of the hypervariable region comprising residues 213-290 was considered fully flexible (for clarity only 50 models are represented). E) EOM analysis of the flexibility of the 213-290 region (hypervariable region). Frequency of radius of gyration (Rg) distributions in a pool of 10,000 models (gray dashed line) with random orientations of the hypervariable region, and in the selected ensemble that fits the experimental SAXS data of CaRas1- FL. Default parameters were employed using native-like models, allowing constant subtraction (0.149) and curve repetition (both the minimum number of curves per ensemble and the number of obtained representative structures, was five). The values for Rflex(random) / Rsigma of ~ 73.56% (~ 89.13%) / 0.91 indicate limited flexibility. Figure 3. Structure of the catalytic region of CaRas1-guanine nucleotide exchange factor CaCdc25. A) Domain organization diagram (top) and cartoon representation (bottom) of the crystal structure of the catalytic region of CaCdc25 at 2.45 Å resolution. The REM (dark blue) and CAT (light blue) domains are labeled and the N- and C- termini are indicated in filled circles. B) Structural superposition of the main body of the CAT domain of CaCdc25 with the active (PDB entry 1NVW (68)) and inactive (PDB entry 2II0 (83)) forms of Sos showing that the ^-helical hairpin in the CaCdc25 structure is poised for activity. C) Overview of the three main interacting regions (numbered in orange circles) that maintain the ^-helical hairpin in an activated position, detailed in panels D- F. D) Interaction of the loop connecting the two main helices of the ^-helical hairpin (main interacting residues shown as sticks) with the REM domain (transparent surface). E) Interaction of the ^-helical hairpin with the flap1 region (main interacting residues shown as sticks) and with the REM domain (transparent surface). F) Interaction of the ^-helical hairpin with the flap2 region (main interacting residues shown as sticks). G) Experimental SAXS profile of the catalytic region of CaCdc25, extrapolated to infinite dilution (upper plot). The scattering calculated for the atomic structure of the tandem REM-CAT of CaCdc25, shown in A, is represented as a black dashed line. Error-weighted residual difference plot delta / σ = [Iexp(q) - cImod(q)] / delta (q) versus q (lower plot). H) Crystal structure of CaCdc25 (cartoon representation) docked into the SAXS-derived molecular envelope (translucent surface), which is the average of 15 independent bead models. Two orthogonal views are shown. Figure 4. A synthetic peptide reduces the nucleotide exchange activity of CaCdc25 supporting a novel auto-inhibited conformation in the Cdc25 GEFs family. A) Domain organization diagram (top) and cartoon representation (middle) of the AlphaFold2-predicted model for a segment of CaCdc25 comprising the REM (dark blue, residues 883-1305) and CAT domains (light blue, residues 1039-1306), and a hypothetical inhibitory region formed by two ^-helices (magenta, residues 837-858) linked to the REM domain by a linker (gray, residues 859-882). The location of the G-domain binding site in CaCdc25 is highlighted. The sequence of the synthetic peptide, tested in the nucleotide exchange assays, is shown at the bottom. B) Nucleotide exchange reactions of CaRas1-mant-dGDP (200 nM) catalyzed by CaCdc25 (50 nM) in the presence of the synthetic peptide 837-858 (1-80 ^M concentration range). Dashed lines are the double exponential decay models fitted to obtain Kapp. C) Dose- dependent effect of peptide 837-858 on the GEF activity of CaCdc25 (50 nM). The curve (black line) is the fitted Hill model. The half maximal effective concentration (EC50) is shown. D) Schematic representations for the AlphaFold2-predicted structures of the isolated segment 837-1306 of CaCdc25, which represents a hypothetical auto-inhibited conformation, and in complex with CaRas1, where the region 837-858 is displaced from the G-domain binding site of CaCdc25. Figure 5. Full or partial conservation of the hypothetical auto- inhibitory region of CaCdc25 in species belonging to the Saccharomycetales kindgdom. Multiple sequence alignment of CaCdc25 (residues 837-858) with sequences of other species producing significant alignments according to BLAST (the 34 with the highest score are shown). Strictly conserved alignment positions are shown in inverted type on a green background. Figure 6. Sequence of the peptide able to inhibit the GEF activity of CaCdc25 and variants. The interaction energy (deltaG) between the peptide TIINYATRVMQDNFDVQLLLVE and the catalytic region of CaCdc25 was calculated by using the structural model showed in Fig. 6 and the FoldX Suite. The effect of 19 point mutations in each position of the peptide was also evaluated (in terms of ^^G = deltaGMUT - deltaGWT). Modifications estimated to be not significant or stabilizing (deltadeltaG changes less than 0.46 kcal / mol) are highlighted in square labels. Both the wild type and variants sequences are independently N-terminus fused to five cell-penetrating peptides, whose sequences are shown at the left and underlined. The annexed Table 1 shows an overall list of the peptide sequences.

[0002] Sequence List: SEQ. ID nr. 1: TIINYATRVMQDNFDVQLLLVE

[0003] References 1. Beck-Sagué C, Jarvis WR. Secular trends in the epidemiology of nosocomial fungal infections in the United States, 1980-1990. National Nosocomial Infections Surveillance System. J Infect Dis. 1993 May;167(5):1247–51. 2. Turner SA, Butler G. The Candida pathogenic species complex. Cold Spring Harb Perspect Med. 2014 Sep 2;4(9):a019778. 3. Rossignol T, Lechat P, Cuomo C, Zeng Q, Moszer I, d’Enfert C. CandidaDB: a multi-genome database for Candida species and related Saccharomycotina. Nucleic Acids Res. 2008 Jan;36(Database issue):D557-561. 4. Nikou SA, Kichik N, Brown R, Ponde NO, Ho J, Naglik JR, et al. Candida albicans Interactions with Mucosal Surfaces during Health and Disease. Pathogens. 2019 Apr 22;8(2):53. 5. Scaduto CM, Bennett RJ. Candida albicans the chameleon: transitions and interactions between multiple phenotypic states confer phenotypic plasticity. Curr Opin Microbiol. 2015 Aug;26:102–8. 6. WHO fungal priority pathogens list to guide research, development and public health action. Geneva: World Health Organization; 2022. https: / / www.who.int / publications-detail- redirect / 9789240060241. [Internet]. Available from: https: / / www.who.int / publications-detail-redirect / 9789240060241 7. Peters BM, Palmer GE, Nash AK, Lilly EA, Fidel PL, Noverr MC. Fungal morphogenetic pathways are required for the hallmark inflammatory response during Candida albicans vaginitis. Infect Immun. 2014 Feb;82(2):532–43. 8. Lo HJ, Köhler JR, DiDomenico B, Loebenberg D, Cacciapuoti A, Fink GR. Nonfilamentous C. albicans mutants are avirulent. Cell. 1997 Sep 5;90(5):939–49. 9. Mitchell AP. Dimorphism and virulence in Candida albicans. Curr Opin Microbiol. 1998 Dec;1(6):687–92. 10. Leberer E, Harcus D, Dignard D, Johnson L, Ushinsky S, Thomas DY, et al. Ras links cellular morphogenesis to virulence by regulation of the MAP kinase and cAMP signalling pathways in the pathogenic fungus Candida albicans. Mol Microbiol. 2001 Nov;42(3):673–87. 11. Pentland DR, Piper-Brown E, Mühlschlegel FA, Gourlay CW. Ras signalling in pathogenic yeasts. Microb Cell. 2017;5(2):63–73. 12. Piispanen AE, Bonnefoi O, Carden S, Deveau A, Bassilana M, Hogan DA. Roles of Ras1 membrane localization during Candida albicans hyphal growth and farnesol response. Eukaryot Cell. 2011 Nov;10(11):1473–84. 13. Piispanen AE, Grahl N, Hollomon JM, Hogan DA. Regulated proteolysis of Candida albicans Ras1 is involved in morphogenesis and quorum sensing regulation. Mol Microbiol. 2013;89(1):166–78. 14. Lai CC, Boguski M, Broek D, Powers S. Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins. Mol Cell Biol. 1993 Mar;13(3):1345–52. 15. Maidan MM, De Rop L, Serneels J, Exler S, Rupp S, Tournu H, et al. The G protein-coupled receptor Gpr1 and the Gα protein Gpa2 act through the cAMP-protein kinase A pathway to induce morphogenesis in Candida albicans. Mol Biol Cell. 2005 Apr 1;16(4):1971–86. 16. Rolland F, De Winde JH, Lemaire K, Boles E, Thevelein JM, Winderickx J. Glucose-induced cAMP signalling in yeast requires both a G-protein coupled receptor system for extracellular glucose detection and a separable hexose kinase- dependent sensing process. Mol Microbiol. 2000 Oct;38(2):348–58. Lisbon, 27thFebruary 2024

Claims

CLAIMS 1. Synthetic peptide sequences capable of inhibiting the activity of CaCdc25 characterized by comprising SEQ. ID nr.

1.

2. Peptide sequences according to claim 1, the said peptide sequence comprising variants selected from the list consisting of sequences described in Table 1., and combinations thereof.

3. Pharmaceutical compositions comprising peptide sequences as described in the preceding claims.

4. Peptide sequences, compositions and pharmaceutical formulations according to the preceding claims for use in a treatment for Candida spp., as well as other fungal infections with agents from the Candida Lodderomyces clade and more broadly, the Sacharomycetales kindom, through specific block of an important virulence-related signaling pathway. Lisbon, 27thFebruary 2024 1

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